Detection of Cells, Proteins, and DNA using Tapered

Fiber-Optic Biosensors (TFOBS)

A Thesis
Submitted to the Faculty
Of
Drexel University
by
Angela Leung
in partial fulfillment of the
requirements for the degree
of
Doctor of Philosophy
May 2007

 Copyright 2007
Angela S. Y. Leung. All Rights Reserved.

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ACKNOWLEDGEMENTS
I would like to thank my advisors, Dr. Raj Mutharasan and Dr. P.M. Shankar for
their guidance and mentorship throughout my graduate studies. In addition, I
acknowledge the students in my laboratory for their help: Kishan Rijal, Dave Maraldo,
Gossett Campbell, Sen Xu, Harsh Sharma. Finally, I am grateful for the technical support
provided by Dan ve Luu and Dr. Kevin Scoles throughout this project.

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LIST OF TABLES ..................................................................................................................................... VI

LIST OF FIGURES ..................................................................................................................................VII
ABSTRACT ................................................................................................................................................ XI
1. INTRODUCTION .....................................................................................................................................1
1.1 MOTIVATION AND RATIONALE ..............................................................................................................1
1.2 RESEARCH OBJECTIVES .........................................................................................................................2
1.3 CONTRIBUTIONS ....................................................................................................................................3
2. LITERATURE REVIEW .........................................................................................................................4
2.1 OPTICAL FIBERS ....................................................................................................................................4
2.1.1 Light Propagation in Optical Fibers ............................................................................................5
2.1.2 EFFECTS OF TAPERING THE FIBER ON THE EVANESCENT FIELD ..........................................................6
2.1.3 Effects of Bending the Fiber on the Evanescent Field ..................................................................7
2.1.4 Effects of Launching angle on the Evanescent Field ....................................................................8
2.1.5 Effects of Wavelength on the Evanescent Field ............................................................................9
2.2 TAPERED FIBER GEOMETRIES ................................................................................................................9
2.2.1 Tapered Tips ...............................................................................................................................10
2.1.2 Continuous, Biconical Tapers.....................................................................................................11
2.3 COMMON DETECTION PRINCIPLES IN TAPERED FIBER OPTICAL SENSORS ...........................................11
2.3.1 Changes in output power due to refractive index changes alone................................................12
2.3.2 Evanescent field absorption........................................................................................................13
2.3.3 Evanescent Wave Fluorescence ..................................................................................................15
2.3.4 Surface Plasmon Resonance .......................................................................................................16
2.4 APPLICATIONS OF TAPERED FIBER OPTIC SENSORS .............................................................................18
2.4.1 Cell Concentration......................................................................................................................19
2.4.2 Proteins or biomolecule Concentration ......................................................................................23
2.4.3 DNA Hybridization .....................................................................................................................32
2.5 Conclusions and Future Directions ...............................................................................................34
3. EFFECTS OF GEOMETRY ON TRANSMISSION AND SENSING POTENTIAL OF TAPERED
FIBER SENSORS .......................................................................................................................................44
3.1 INTRODUCTION ....................................................................................................................................44
3.2 PHYSICS OF SENSING ...........................................................................................................................45
3.2.1 Wave propagation in absorption sensors....................................................................................45
3.2.2 Wave Propagation in continuous bi-conical tapered fibers........................................................46
3.3 MATERIALS AND METHODS .................................................................................................................47
3.3.1 Fiber Tapering............................................................................................................................47
3.3.2 Taper Characterization...............................................................................................................47
3.3.3 Numerical Simulation of Light Transmission in a Tapered Fiber ..............................................48
3.4 RESULTS AND DISCUSSION ..................................................................................................................52
3.4.1 Characteristics of Tapers............................................................................................................52
3.4.2 Air-Water Transmission Ratios...................................................................................................54
3.4.3 Response of Fusion Spliced Tapers to Low Concentrations of E. coli........................................55
3.4.4 Response of Heat Drawn Tapers to Low Concentrations of E. coli............................................60
3.4.5 Transmission in tapered fibers: Simulation Results....................................................................64
3.5 CONCLUSIONS .....................................................................................................................................68
4. DETECTION OF E.COLI O157:H7 USING ANTIBODY-IMMOBLIZED TFOBS.......................70
4.1 INTRODUCTION ....................................................................................................................................70
4.2 PHYSICS OF TAPERED FIBER SENSING .................................................................................................72
4.3 MATERIALS AND METHODS .................................................................................................................74

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4.3.1 Fiber tapering .............................................................................................................................74
4.3.2 Experimental arrangement ........................................................................................................77
4.3.3 Antibody immobilization .............................................................................................................78
4.3.4 Pathogen Detection experiments ................................................................................................79
4.3.5 Sample preparation for Electron Microscope.............................................................................80
4.4 RESULTS AND DISCUSSIONS ................................................................................................................81
4.4.1 Tapered Fiber characterization ..................................................................................................81
4.4.2 EC Detection in aqueous samples...............................................................................................82
4.4.3
E. COLI RELEASE .........................................................................................................................83
4.4.4 Effect of EC concentration on sensor response ..........................................................................86
4.4.5 Loss of sensitivity with use.........................................................................................................88
4.4.6 Selectivity of antibody to immobilized fiber ................................................................................89
5.0 REAL-TIME MONITORING OF BOVINE SERUM ALBUMIN AT FEMTOGRAM/ML
LEVELS ON ANTIBODY-IMMOBILIZED TAPERED FIBERS ........................................................97
5.1 INTRODUCTION ....................................................................................................................................97
5.2 PHYSICS OF SENSING IN TAPERED FIBERS ............................................................................................98
5.3 MATERIALS AND METHODS ...............................................................................................................100
5.3.1 Fiber tapering ...........................................................................................................................100
5.3.2 Permanent attachment of the tapered fiber to a fiber holder....................................................100
5.3.3 Permanent connectorization of tapered fibers..........................................................................101
5.3.4 Experimental arrangement ......................................................................................................101
5.3.5 Antibody immobilization ...........................................................................................................102
5.3.6 Absorption Characteristics of BSA and antibody to BSA .........................................................103
5.3.7 Protein attachment and release experiments ............................................................................104
5.3.8 Multiple step attachment experiment ........................................................................................105
5.4 RESULTS AND DISCUSSIONS ..............................................................................................................105
5.4.1 Tapered Fiber characterization ................................................................................................105
5.4.2 Immobilization of Antibody to BSA...........................................................................................106
5.4.3 Absorption characteristics of BSA and antibody to BSA ..........................................................107
5.4.4 Detection of BSA Attachment and Release................................................................................108
5.4.5 Multiple step attachment experiment ........................................................................................109
5.5 KINETICS OF BSA ATTACHMENT TO ANTIBODY-IMMOBILIZED SENSORS ...........................................113
5.6 EFFECT OF CONCENTRATION OF REFRACTIVE INDEX ..........................................................................114
5.7 CONCLUSIONS ...................................................................................................................................115
6.0 MODEL PROTEIN DETECTION USING ANTIBODY-IMMOBILIZED TAPERED FIBER
OPTIC BIOSENSORS (TFOBS) IN A FLOW CELL AT 1310 NM AND 1550 NM..........................117
6.1 INTRODUCTION ..................................................................................................................................117
6.2 PHYSICS OF SENSING IN TAPERED FIBERS ..........................................................................................118
6.3 MATERIALS AND METHODS ...............................................................................................................120
6.3.1 Flow Cell Apparatus.................................................................................................................120
6.3.2 Optical Arrangement ................................................................................................................121
6.3.3 TFOBS Fabrication ..................................................................................................................122
6.3.4 Response of TFOBS to liquid refractive index changes............................................................123
6.3.5 Antibody immobilization procedure..........................................................................................123
6.3.6 Multi-step detection and release experimental procedure ........................................................125
6.4 RESULTS AND DISCUSSIONS ..............................................................................................................126
6.4.1 Optical characterization of TFOBS ..........................................................................................126
6.4.2 Response of TFOBS to antibody immobilization.......................................................................131
6.4.3 Multi-step detection and release of BSA ...................................................................................136
6.4.4 Multi-step detection of BSA and BSA in the presence of OVA..................................................140
7.0 LABEL-FREE DETECTION OF DNA HYBRIDIZATION ..........................................................144
7.1 INTRODUCTION ..................................................................................................................................144
7.2 PHYSICS OF SENSING .........................................................................................................................145

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7.3 Materials and Methods ................................................................................................................147
7.3.1 Flow Cell Apparatus.................................................................................................................147
7.3.2 Optical Arrangement ................................................................................................................148
7.3.3 TFOBS Fabrication ..................................................................................................................148
7.3.4 Reagents....................................................................................................................................149
7.3.5 Probe Preparation ....................................................................................................................149
7.3.6 Experimental apparatus, probe immobilization and hybridization detection ...........................150
7.4 RESULTS AND DISCUSSIONS ..............................................................................................................151
7.4.1 Probe immobilization................................................................................................................151
7.4.2 Hybridization of complementary ssDNA...................................................................................152
7.4.3 Hybridization of single mismatch ssDNA .................................................................................154
7.5 CONCLUSIONS ...................................................................................................................................155
LIST OF REFERENCES .........................................................................................................................156
VITA...........................................................................................................................................................170

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LIST OF TABLES
Table 2-1: TFOBS for pathogen and toxin measurements ............................................... 38
Table 2-2: TFOBS for biochemical measurements .......................................................... 40
Table 2-3: TFOBS for clinical measurements .................................................................. 41
Table 4-1 Transmission of light through six different tapered fibers fabricated using a
fusion splicer. The percentage change in transmission in water compared to that in
air depends on the geometry of the taper. ................................................................. 96
Table 4-2 The rates of attachment (k) of EC (7x107 cells/mL ) calculated from
attachment on six different flame-drawn fiber tapers. .............................................. 96
Table 4-3 Rates of attachment (k) obtained from four use-cycles of EC (7x107 cells/mL)
attachment shown in Figure 4-6................................................................................ 96

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LIST OF FIGURES
Figure 3-1 Top: Micrograph of a typical taper. Bottom: Schematic of a tapered fiber.
Lengths a, b, c, are along the z axis, while d is the diameter of the waist region..... 53
Figure 3-2 Transmission characteristics of two tapers fabricated using heat drawn (Left:
HD-1. Right: HD-2) whose air to water transmission ratio is near unity. Sensitivity
to cell concentrations as high as 7,000 cells/mL showed almost no response over the
entire wavelength investigated. Small changes are observed for higher EC
concentration............................................................................................................. 55
Figure 3-3 Transmission characteristics of three short tapers: FS-158, FS-164 and FS-2.
FS-158 and FS-164 have nearly the same values of a, c and d, while the waist length
(b) is longer in FS 158 compared to FS-164. The taper FS-2 has same value of a,
and twice the length c, and a very long b with an intermediate waist diameter, 6.3
microns...................................................................................................................... 58
Figure 3-4 Transmission characteristics of Fusion Splicer fabricated tapers at 470 nm.
Data were normalized with respect to PBS, and ordinate value is relative
transmission. Most of the fibers show modest response for 100 to 1,000 cells/mL,
but most show good response for 7,000 and 7 million cells/mL. See for example,
FS-2........................................................................................................................... 60
Figure 3-5 Normalized transmission characteristics of heat-drawn tapers. HD-7 and HD14 have an overall length and waist diameter of 3.82 and 3.99 mm, and 14.2 and
20.6 m. HD-14 (bottom panel) is responsive at both low and high cell
concentrations while HD-7 (top panel) shows strong response at low cell
concentration, and is relatively insensitive at higher cell concentration. ................ 62
Figure 3-6 Relative transmission response of heat-drawn tapers at 470 nm when exposed
to dilute ECJ cell suspensions. Transmission was normalized with respect to values
obtained with water at 470 nm for each taper. Tapers such as HD-7 show good
overall sensitivity for low ECJ concentration while tapers such as HD-14 show
sensitivity at higher cell concentration. .................................................................... 63
Figure 3-7 Transmission in water normalized with respect to air as waist diameter is
altered. Panel A. A short symmetric taper: a= 0.425, b=0.325, c=0.500 mm.
Operating wavelength = 470 nm. Panel B. A longer asymmetric taper: a=2.25,
b=0.245, c=4.5 mm. Operating wavelength = 550 nm. Note that larger undulations
in transmission occur at lower wavelength. V-number is higher at 470 nm............ 66
Figure 3-8 Transmission characteristics of a taper (a= 2.25, b=0.25, c=4.5 mm) in water
at 470 nm as a function of change in waist diameter. Transmission is normalized
with respect to transmission in air at the corresponding geometric values. Smaller
starting diameter tapers show large changes in transmission for small (0.01 m)
changes is waist diameter.......................................................................................... 67
Figure 4-1 Some of the fibers discussed in this paper were fabricated using a simple fiber
tapering device. Fibers without sheathing were mounted on a stand and two pieces
of weights were attached to the ends to provide tension required for tapering. The
fibers were then heated with a flame while carefully monitoring the diameter of the
tapers. When the desired diameters were reached, the two ends of the fibers were
clipped, their diameters were measured using a calibrated image processing software
in an optical microscope. .......................................................................................... 76

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Figure 4-2 100X magnification of a tapered fiber. The lighter region in the center of the
fiber is the core and the dark region surrounding the core is the cladding. .............. 77
Figure 4-3 Typical experimental arrangement for detecting pathogens. Light from a LED
is launched from one end of the fiber. The tapered section of the fiber is held in a
plexiglass fiber holder. Light transmitted is detected by a CCD spectrometer at the
distal end, which is read by a computer.................................................................... 78
Figure 4-4 Comparison of transmission spectra in air and in water. Fabricated tapers
were mounted on a fiber holder and transmission through them were measured using
a 470 nm light source. DI water was added to the sample chamber and the resulting
spectrum was compared to the spectrum in air. Panel A: Diameter of the taper was
7.0 m. Overall length of taper was 700 m. Panel B: Diameter of the taper was
7.0 m. Overall length of taper was 880 m. In many tapers upon addition of water
transmission decreased (example: A) while in others transmission increased
(example: B). This behavior is very strongly dependent on taper geometry. ........... 82
Figure 4-5 Detection and release of EC on antibody immobilized tapered fiber. EC
detection and release experiment was carried out using a 8.8 m diameter tapered
fiber. The antibody immobilized taper was soaked in PBS before antigen containing
solution was injected into the analyte chamber for attachment. After complete
attachment, release buffer (glycine-HCl/ethylene glycol buffer, pH 1.7) was injected
into the chamber to release attached antigen. Transmitted light was measured using
a CCD detector. AU is arbitrary unit of light............................................................ 84
Figure 4-6 SEM of EC attached to the antibody immobilized tapered fiber. Panel A:
Image of a tapered fiber with EC cells and their fragments attached to the surface at
time= 60 min. Panel B: At a slightly higher magnification, one can see EC cells
attached to the surface in a different section of the fiber. The fiber was sparsely
populated with EC..................................................................................................... 85
Figure 4-7 Effect of pathogen concentration of sensor response. Panel A: Change in
transmission for the attachment of various concentration of EC for antibody
immobilized taper fiber with waist diameter of 10.2 m. Panel B: Change in
transmission for the attachment of various concentration of EC for antibody
immobilized taper fiber with waist diameter of 12.3 m. The change in transmission
obtained were in the opposite direction compared to panel A. Such a reversal in
transmission pattern is probably due to the difference in actual shape of the taper.
Detector was a PMT (PTI 701)................................................................................ 88
Figure 4-8 Change in Transmission vs. Time for four cycles of sensor use..................... 89
Figure 4-9 Effect of pathogenic and non-pathogenic EC mixture. Percent above refers to
% of cells that are pathogenic. Experiment performed on a 9.5 m diameter tapered
fiber to check selectivity of immobilized antibody to its complementary antigen.
When 0% pathogen (100% wild strain JM101 ) was injected around the taper, there
was no significant change in transmission through the taper over time. When a
solution containing an EC and the wild strain is added to the solution, the cells bind
to the antibody thus resulting in a decrease in transmission through the fiber. As the
concentration of EC is increased to 50% and 70%, there is a greater binding of
pathogen to the antibody on the surface and thus greater change in transmission
occurs. AU is arbitrary unit of light. ......................................................................... 91

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Figure 4-10 Rate analysis of ln ( I * I ) vs Cb*t.......................................................... 94
*

Figure 5-1 (a) Experimental arrangement. The fabricated taper was fixed to the base with
epoxy. The two ends were connectorized with FC adapter. (b) Cross-sectional view
of the fiber holder. The sample chamber has a 200 L volume. The two ports on
the topside are used for sample addition and removal using a standard 200 L
pipetter. ................................................................................................................... 102
Figure 5-2 Micrograph of a typical tapered fiber taken using a light microscope camera.
................................................................................................................................. 106
Figure 5-3 Transmission change in dB vs. time for antibody immobilization in two
separate experiments is plotted. Temperature is held constant at 30 oC 0.5 oC as
indicated. The experiments were performed in an incubator to keep the temperature
stable. ...................................................................................................................... 107
Figure 5-4 BSA attachment and release of 10 pg/mL sample. The attachment response
was obtained when the tapered region was first exposed to 10 pg/mL of BSA. Data
was collected for 30 minutes, rinsed with PBS, and then the tapered region was
exposed to pH2 PBS. The low pH denatures the antibody and changes its
conformation, resulting in the weakening of the bond between the antibody and
BSA, and causes BSA to be released. Consequently, the transmission changes due to
attachment and release are in opposite direction and have approximately the same
magnitude................................................................................................................ 109
Figure 5-5 Attachment of 100 fg/mL of BSA to antibody-immobilized tapered fibers for
three separate experiments. In each experiment, the BSA was exposed to the
antibody-immobilized taper for 40 minutes............................................................ 110
Figure 5-6 Semi-batch staircase experiment showing attachment of BSA from10 fg/mL to
10 pg/mL. Temperature was maintained at 30 oC 0.5 oC using an incubator .
Temperature variation is also shown. The BSA solutions were added sequentially
from lowest to highest upon removal of the previous solution. The dark line with
peaks represents transmission through the fiber. The peaks correspond to time
instants when the samples were introduced. The dotted line at the bottom represents
the trend exhibited by the steady state value of transmission with respect to time. 111
Figure 6-1 Experimental arrangement. The two sources are connected to the input of the
TFOBS by a Y-coupler. The output of the sensor was connected to the spectrum
analyzer, whose input was fed into the computer for storage and analysis. The
sample chamber has a volume of 50 L. The samples are pumped into the chamber
at 0.5 mL/min from the reservoir. Temperature was maintained at 29 0.5 oC by a
water bath................................................................................................................ 121
Figure 6-2 Schematic representation of a tapered fiber. Lengths a, b, c, are along the zaxis, while d is the diameter of the waist region. Length of the waist region is
denoted by b, convergent length is denoted by a, and divergent length is denoted by
c............................................................................................................................... 123
Figure 6-3 a) Non-specific response of a TFOBS. A = 1 g/mL to 1 mg/mL of glucose,
B = 0.01 g/mL of glucose, C = 0.1 g/mL of glucose, D = DI water. There was no
response from 1 g/mL to 1 mg/mL of glucose, but there was a slight response
starting at 0.01 g/mL of glucose. There was a large response at 0.1 g/mL of glucose.
The dimension of this TFOBS in m is a = 360, b = 134, c = 500, d = 8. ............. 131

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Figure 6-4 Transmission of a non-antibody modified TFOBS. There was no difference in
transmission caused by PBS and DI water, but there was a large response caused by
0.09 g/mL glucose. After flowing in 0.09 g/mL glucose, the transmission was
restored to the original value by the introduction of DI water. Further flow of 0.09
g/mL glucose yielded the same response as previous additions. D = DI water, G =
0.09 g/mL glucose. The dimension of this TFOBS in m is a = 360, b = 134, c =
500, d = 8. ............................................................................................................... 131
Figure 6-5 Response of a TFOBS to antibody immobilization. Dimension of the TFOBS
in m is a = 465, b = 296, c = 340, d = 5. 100 g/mL of antibody to BSA was
injected and the transmission was recorded. After 2 hours of antibody
immobilization, hydroxylamine was flowed in for 40 minutes, followed by PBS,
until the transmission reaches steady state. AB = antibody, HA = hydroxylamine. a)
The response from antibody at 1310 nm. b) The response from antibody at 1550 nm.
c) ln (1-theta) vs. time to determine the rate of immobilization of the antibody.... 135
Figure 6-6 The change in transmission of two antibody-immobilized TFOBS. a)
Response at 1310 nm. BSA of 1 pg/mL to 100 pg/mL were attached sequentially to
this fiber, and then pH 2.4 PBS was used to release the protein. A = 1 pg/mL BSA,
B = 10 pg/mL BSA, C = 100 pg/mL BSA, D = pH 2.4 PBS. The dimension of this
TFOBS in m is a = 480, b = 213, c = 500, d = 5. pH 2.4 PBS causes the opposite
change in transmission, with the almost same value as the value prior to any BSA
addition, thus confirming the attachment of BSA in the first place. b) Repsonse at
1550 nm for the same experiment as that done in a). c) ln (1-theta) vs. time plot
used to determine the rate of attachment for 1 pg/mL and 100 pg/mL of BSA. .... 139
Figure 6-7 a) The response at 1310 nm of an antibody-immobilized TFOBS. The
response of 1 pg/mL of BSA in the presence of 1 pg/mL of OVA is shown here. O =
1 g/mL OVA, P = PBS, A = pH 2.4 PBS, M = 1:1/ v:v mixture of 1 pg/mL BSA
and 1 pg/mL OVA. The dimension of this TFOBS in m is a = 400, b = 245, c =
574, d = 5. Response at 1550 nm is similar but not shown because the magnitudes of
the transmission changes are much smaller. b) The same data as a), plotted in terms
of change in transmission compared to the transmission due to PBS prior to protein
addition. 1:1 represents the mixture of 1 pg/mL OVA and BSA. Time scale was
adjusted such that the sample addition for each solution is at time zero. c) ln (1theta) vs. time, plotted to determine the rate of attachment.................................... 142
Figure 7-1: The response of the TFOBS during thiolated ssDNA probe immobilization.
................................................................................................................................. 152
Figure 7-2: Response of the TFOBS at 1310 nm for 750 fM to 750 M of complementary
10-mer ssDNA probe. ............................................................................................. 153
Figure 7-3: Response of TFOBS due to a mismatch sequence and complementary
sequence.................................................................................................................. 155

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Abstract
Detection of Cells, Proteins, and DNA using Tapered
Fiber-Optic Biosensors (TFOBS)
Angela S. Y. Leung
P.M. Shankar, Ph.D.
Raj Mutharasan, Ph.D.

Tapered Fiber Optic Biosensors (TFOBS) have a wide variety of applications

including medical diagnostics, drug screening, pathogen detection, and environmental
contaminants detection. Over the past four years, TFOBS were developed in our
laboratory to detect cells, proteins, and DNA. In particular, the use of TFOBS as
intensity-based label-free sensors, long wavelengths for detection, and the incorporation
of sample flow into detection are distinct achievements of this project.
TFOBS were fabricated with using a fusion splicer and housed in a plexiglass
fiber holders or flow cells. Non-specific response of the TFOBS was measured by
recording the transmission due to the presence of E.coli JM101 cells and glucose
solutions. For detection experiments, antibodies or thiolated DNA probes were
immobilized on the TFOBS surface using silane chemistry or gold coating. Detection was
carried out by recording the transmission while antibody or DNA probe-immobilized
TFOBS were exposed to various concentrations of analytes.
Some of the highlight results of this study include the following: 1) The nonspecific measurement of 100 to 7 million E.coli JM101 cells/mL was possible in the
visible range; 2) at 1310 and 1550 nm, the TFOBS were sensitive to 0.01 to 0.1 g/mL of
glucose, which gives a sensitivity in terms of dB/RI; 3) antibody-immobilized TFOBS
was able to detect down to 70 E.coli O157:H7 cells/mL in the visible range; 4) antibodyxi

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immobilized TFOBS detected down to 10 fg/mL of BSA at 1310 and 1550 nm in
stagnant condition; 5) antibody-immobilized TFOBS detected BSA at 1 pg/mL in flow;
6) ss-DNA probe-immobilized TFOBS detected 1 pM target DNA in flow.

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1. INTRODUCTION

1.1 Motivation and Rationale

TFOBS have been widely investigated for detection because of its application in
diverse fields such as clinical diagnostics, biochemical measurements, detection of
pathogens, and detection of environmental contaminants. Some advantages of TFOBS
include the ability for remote sensing, speedy response, affordability, selectivity,
sensitivity, and requirement for small volume.
To date, most TFOBS have been used with fluorescent labels in order to achieve
pg/mL detection limits. The use of intensity-based TFOBS has rarely been studied
because of the large dependence of transmission on geometry. However, it is important to
realize the attractiveness of label-free detection, which includes more rapid results, lower
costs, and ease of use. Aside from intensity-based detection, the use of IR wavelengths
with TFOBS also has not been studied in detail. The reason for this is that IR
wavelengths are not typically absorbed by biological species. However, it may not be
necessary to use a wavelength which is absorbed because intensity-based TFOBS has the
ability to detect based on refractive index changes alone, provided that these changes
occur close to the surface of the sensing region. Another aspect which has not been
investigated extensively is the use of flow with TFOBS. The use of flow has significant
advantages, such as reducing transmission changes due to mechanical movements,
removal of samples, and effective exposure of the sensing region to analytes.

2
A majority of the work done in this project are related to the detection of proteins.
There is much motivation to detect proteins because they are often markers for diseases,
they are likely to be detected within the evanescent region unlike cells, and there is no
equivalent of PCR in proteins in that they cannot be amplified. Aside from proteins,
E.coli O157:H7 cells were detected in stagnant condition with limited success because
the detection of pathogens remains an important challenge in ensuring food safety.
Finally, preliminary detection of DNA was performed because DNA is used increasingly
for pathogen identification.

1.2 Research Objectives

The main research objectives of this project include:
1) Fabrication of TFOBS.
2) Determine the effect of geometry on the non-specific measurement of E.coli JM101
cells.
3) Functionalize the TFOBS to detect E.coli and BSA in stagnant condition, in the visible
range.
4) Functionalize the TFOBS to detect BSA in stagnant condition, in the IR range.
5) Functionalize the TFOBS to detect BSA in flow condition, in the IR range.
6) Explore functionalization using gold surface and detection of DNA hybridization.

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1.3 Contributions
As a result of this project, several major contributions were made in the optical
sensing field. The first major contribution is the fabrication of TFOBS and determination
of the effect of geometry on the transmission, as well as non-specific measurement of
E.coli JM101 cells in the visible range and glucose in the IR range. The second
contribution is the detection of low concentrations of BSA in stagnant condition in the IR
range. The third contribution is the detection of BSA in flow condition in the RI range.
Recently, work has also been done in detecting hybridization of DNA on a gold coated
TFOBS.

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2. LITERATURE REVIEW

2.1 Optical Fibers

Optical fibers were originally used for telecommunications, and therefore
designed for transmitting light over long distances with little loss in intensity. Typically
optical fibers consist of a cylindrical core and surrounding cladding, both made of silica.
The core is doped with Germanium to make its refractive index is slightly higher than the
cladding refractive index. Light propagates through the fiber by total internal reflection.
Even though light is mostly reflected, some decays exponentially into the cladding.
Because the cladding is much thicker than that core, light propagates over long distances
with little loss in intensity.
While optical fibers were first designed for use in telecommunications, they soon
found other applications such as sensing. Sensing requires modification of the fiber such
that some interaction of light with external environment occurs. Such interactions can be
brought about by altering the geometry of the fiber.
The earliest fiber sensors were made by cladding removal, which results in the
evanescent wave interacting with the external environment. The changes in the
evanescent field are reflected in the overall transmission through the fiber. Aside from
simply exposing the core, there are other methods which can increase the percentage of
the power in the evanescent region. For instance, it was previously found that the
magnitude and penetration of the evanescent field was enhanced by tapering a fiber.1-5
The use of tapered fibers for evanescent sensing has been explored for many applications,
including sensing of temperature, pH, concentration of molecules, and humidity 6.

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2.1.1 Light Propagation in Optical Fibers
Optical field in a fiber consists of two components: the oscillatory portion
propagating in the fiber core, known as the guided field, and an exponentially decaying
portion in the cladding, known as the evanescent field. Light intensity is continuous at the
core-cladding interface. In a uniform-diameter fiber the evanescent field decays to almost
zero within the cladding and cannot interact with the surface of the fiber. The evanescent
field can penetrate to the surroundings once the cladding is removed, but the percentage
of light in the evanescent field is still small. The penetration depth dp gives an indication
to the distance to which the evanescent field extends beyond the core-cladding interface.
Mathematically it is the distance where the evanescent field falls to 1/e of its value at the
core-cladding interface 7.
Thus, starting at the core-cladding interface, the evanescent field can be described
as 7:

E ( x) = E 0 exp
d
p

(2 1)

Where x is the distance away from the fiber core, starting at x=0 at the core-cladding
interface, E0 is the magnitude of the evanescent field at the interface, and dp is the
pentration depth.
Using the uniform decladded fiber approximation, the penetration depth is given
by 7, 8:
dp =

2 n sin 2 ncl2
2
co

(2 2)

6
where is the wavelength of the light source, is the angle of incidence of the light at
the core/cladding interface, nco and ncl are the refractive indices (RI) of the core and
cladding respectively. The RI of the solution is usually lower than that of the original
fiber cladding. Therefore, a decrease in the penetration depth is expected upon sample
addition compared to the original core/cladding fiber configuration. For example, if the
refractive index of the liquid sample is 1.33, and that of the original cladding and core are
1.458 and 1.46 respectively, the penetration depth will change from 0.98 m to 0.13 m
upon adding liquid. Small penetration depth results in an evanescent field that decays
more sharply with distance away from the fiber surface. Although a small penetration
depth may be sufficient for sensing small molecules close to the surface of a fiber, the
ability to sense larger analytes far away from the surface is greatly diminished.
The penetration must be kept in mind when designing fiber optic biosensors. A
design with a large penetration dept would be most suitable as it has the ability to sense
both small and large molecules. Some well known methods to manipulate the fiber
geometrically to increase penetration depth including bending

9-12

, tapering

1, 3, 5, 13, 14

changing the launching angle of the light 8, increasing the wavelength 15.

2.1.2 Effects of Tapering the Fiber on the Evanescent Field

Tapering refers to cladding removal and then tapering the core, or tapering of both
the core and the cladding at the same time, such that the overall diameter of the fiber at
the tapered region is less than the original core diameter.
Tapering not only exposed the evanescent field to the surroundings, but also
increases the evanescent field magnitude and penetration depth, thus increases the

7
potential of the optical fiber as a sensor

1, 3, 5, 13, 14

. In a study by Mignani et al.1, the

optical characteristics of a de-cladded uniform-core fiber were compared to that of a decladded tapered core fiber. While the behavior of both fibers followed the LambertBeers Law of absorption, the percentage of power () in the evanescent field differed.
For a uniform fiber with a 200 m diameter, ranged from 4E-4 to 8E-4. However, once
the core has been tapered, ranged from 6E-3 to 8E-3. Because sensitivity is
proportional to , tapered core fibers are ten times more sensitive. In a study by Villatoro
et al. 5, a parameter used for characterization of tapers is r, the ratio of the power in the
evanescent field to the total power of the guided light. It was found that nano-sized fibers
had an r of 100%, while de-cladded uniform cores or tapered optical fibers with
diameters larger than the wavelength had r less than 20% 5. In another similar study by
Mackenzie et al. 13, it was found that adiabatic tapers as small as 1 m in diameter had an
evanescent optical power intensity that is 100 times than what is achieved with a similar
polished fiber.

2.1.3 Effects of Bending the Fiber on the Evanescent Field

The effect of bending is much like tapering in that it results in loss in light
propagation and increase in evanescent field

9, 10

. Evanescent fields of the higher order

modes penetrate farther beyond the core interface than do lower order modes. Fiber
bending creates higher order modes

10

. One absorption study by Khijwania et al.

demonstrates clearly the difference between a straight probe of 35 cm in length, with 5

cm of cladding removed, to a U probe of similar dimension. It was found that absorption
coefficient was larger in a U probe compared to a straight fiber with the same diameter.

8
In a straight probe, the number of ray reflections as well as the evanescent absorption
coefficient is inversely proportional to the core radius. The number of reflection only
depends on radius because the angle of ray propagation is constant. U probes were found
to have increased sensitivity at the same radius because the ray propagation angle
decreases. When the angle of propagation decreases, penetration depth increases. If the
bending angle is kept constant however, increased diameter in the probe results in
increased absorption as demonstrated by probes of 100 and 300 m in diameter.
However, the sensitivity did not increased indefinitely with smaller bending angles, and
in a follow up study

11

, it was reported that there is an optimum of bending angle.

Sensitivity increases when bending radius decreases up to a certain value, and then starts
to decrease when bending radius decreases. When the bending radius is reduced so much
that the two arms of the U probe are really close together, the evanescent field overlap,
and there is a transfer of power between the arms without propagating through the bent
sensing region. In a study by Littlejohn et al. 10, optical fibers are de-cladded, bent, and
coiled by heat. It was shown that sensors made with more than one bend are more
sensitive. However, the disadvantage of using too many bends is that less light is
transmitted with more bends. Using absorption of water bands, the authors observed
signal enhancements of up to 9 in bent fibers over straight fibers.

2.1.4 Effects of Launching angle on the Evanescent Field

In a single mode fiber, only the lowest order mode is supported by the fiber and
all other modes are lost. However, various modes can be supported in multimode fibers
and therefore light can be launched at different angles. The effect of launch angle on

9
evanescent wave penetration has been studied by Ahmad et al. 8. It was found that by
altering the launch angle, penetration depth can increase by as much as 300% for tapered
fibers of certain geometry.

2.1.5 Effects of Wavelength on the Evanescent Field

According to Eq. 2-2, penetration depth should increase with an increase in

wavelength of the launched light. Some research has been done on the use of near-IR or
IR wavelengths with fibers, mostly as H2 sensors 5. However, the use of long
wavelengths for biosensing was mostly unexplored because most evanescent sensors
depend on absorption of light by biomolecules in the sensing region, and absorption
behavior of biomolecules in the IR region is largely unknown.

2.2 Tapered Fiber Geometries

Although there are several methods to increase the evanescent field of FOBS,
tapering proved to be a useful method in both the laboratory and industrial setting, and
therefore the focus in this laboratory is tapered FOBS (TFOBS). In general, there are two
types of tapered fibers currently used in biosensing: tapered tips and continuous tapered
fibers. A tapered tip is a fiber which gradually decreases in diameter until it becomes a
tiny tip. Either the tip itself or an external fiber can collect the light from the tip for
measurement. A continuous tapered fiber consist of an optical fiber gradually decreasing
in diameter, which then reaches a constant-diameter waist region, and then gradually

10
increases back to the original diameter. Light enters a continuous taper from one end and
is transmitted through the taper to the detector.

2.2.1 Tapered Tips

Tapered tips are used extensively in biochemical and clinical applications

16-19

Tapered tips are usually used with fluorescence, which is discussed in section 2.3.
Tapered tips can be further divided into the most commonly used geometries: stepetched, conical, and combination tapers.
A step-etched tapered tip can be made by immersing a uniform fiber into
hydrofluoric acid. Because hydrofluoric acid etches away glass, this results in a fiber tip
that abruptly decreases in diameter. The disadvantage of using etching is that this type of
fiber loses a lot of power due to V number mismatch and had low sensitivity 20, 21. On the
other hand, the diameter of a conical tapered tip decreases smoothly over the tapered
region 20. If hydrofluoric acid is used for making a conical taper, different sections of the
fiber must be exposed to different etching times, and is usually achieved automatically by
mounting the fiber on a motor. The diameter of a combination tapered tip decrease at one
slope up to a point, and then decrease at a different slope. It provides an even return of
signal along the fiber. Because it is tapered in such a way that the V number is matched,
the combination taper fiber has little loss in signal 20.

11
2.1.2 Continuous, Biconical Tapers

A continuous biconical taper has three regions: a convergent region of decreasing

diameter, a region of almost fixed diameter (waist), and a divergent region of increasing
diameter 22. Sensing typically occurs at the waist region because the evanescent field is at
its maximum in this region. At the waist region, absorption, scattering, fluorescence, and
resonance can occur, and causes the overall transmission of the fiber to be altered. If
fluorescent labels are used, fluorescent signals are also picked up along with the
transmitted and scattered light at the divergent region 23.
Although uniformly de-cladded fibers are technically not of tapered geometry,
they are important for discussion because they are predecessors to TFOBS. The optical
field in de-cladded fibers is similar to that in tapered fibers, and all the optical principles
used with TFOBS are used with uniformly de-cladded fibers as well. Therefore, for the
completeness uniformly-decladded fibers are included as one type of TFOBS on the
discussion on detection principles and studies done to date.

2.3 Common Detection Principles in Tapered Fiber Optical Sensors

Evanescent sensing can be a very flexible method it can be detected alone,

amplified, or detected in conjunction with various optical methods. Oftentimes because
the penetration depth is too small in relation to the target analyte, the use of additional
optical principles is imperative to low limit detection. One can choose appropriate
principles to use with evanescent sensing depending on the analytes and the application
of the sensor. The use of evanescent sensing has been previously investigated with many
different sensing principles, some of which include:

12

Changes in output power due to refractive index changes alone (intensity-based

TFOBS)

Evanescent field absorption (absorption TFOBS)

Fluorescence (fluorescent TFOBS)

Surface Plasmon Resonance (SPR)

2.3.1 Changes in output power due to refractive index changes alone

Intensity-based sensors were the earliest reported and is the focus of our
laboratory 7. They are based on the changes in the magnitude of the evanescent field,
which are reflected by the changes in output power in a de-cladded uniform fiber or
TFOBS. The amount of evanescent field propagating depends on the refractive index of
the analyte, and therefore the output power is a measure of refractive index. Geometric
parameters dominate the performance of intensity-based sensors. Factors which influence
the sensitivity of an intensity-based TFOBS include bending, radius, length, and taper
ratio.
As mentioned in Section 2.0, bending increases the fraction of evanescent field in
the sensing region, and therefore increases sensitivity in intensity-based TFOBS 1, 3, 5, 7, 13,
14

. As for the effect of radius, it was found that smaller diameters are associated with

higher sensitivity. For example, Villatoro et al.

demonstrated that nano fibers has a

percentage of power in evanescent field that approaches 100%. A study by Diaz-Herrera

et al. on a fiber-optic temperature sensor also showed that sensitivity increases with
decreasing waist diameter 3. As for length, it was shown by Chen et al. 2 that longer fibers
can be used to overcome a small dp. In a study by Ahmad et al. 8, a ray model was used to

13
obtain an expression to relate dp to refractive indices, numerical aperture, taper ratio,
length, launch angle, and position along the taper axis. The result was that the longest
taper (10 cm) gave the largest dp. In the shorter tapers the penetration depth depends on
launch angle and there was not a clear trend between the angle and dp. Taper ratio is the
ratio of the waist diameter of a tapered fiber to the total diameter of a uniform fiber, and
is another factor which affects the performance of the sensor. According to Bures et al. 4,
the evanescent wave is negligible when taper ratio is large, whereas when taper ratio is
small, the mode is so highly spread out that it tends towards a free-space wave. Ahmad et
al. 8 concluded that a taper ratio of unity gave the minimum dp, while dp can increase up
to 300% for tapers of certain geometry at a suitable launch angle.

2.3.2 Evanescent field absorption

Evanescent absorption sensors are based on the transmission decrease when

analytes absorb light in the wavelength used for transmission. The transmission depends
on the analytes concentration because absorption is proportional to concentration. In
order to use tapers for absorption measurements, the light source must be at a wavelength
which is absorbed by the sample analyte.
In a uniform-core de-cladded optical fiber, absorbance is governed by the
Lambert-Beers Law:

A = L

(2 5)

where is the absorption coefficient, L is the length of interaction, and is the fraction
of light in the evanescent domain. 1
The absorption coefficient is given by:

14

= C

(2 6)

where is the molar absorptivity, and C is the concentration of the species.

Sensitivity is given by:

S = L + C

(2 7)

If the species is weakly absorbing,

S L

(2 8)

In a uniform-core optical fiber, is quite low. A fiber with a 200 m diameter has an
of only about 10-4 1. Geometric factors which influence the sensitivity of an absorption
TFOBS include radius, taper ratio, length, and bending.
As mentioned previously, Mignani et al. compared tapered core optical fibers to
the uniform core de-clad fibers in the context of an absorption study 1. In the tapered
fiber, radius changes as a function of the distance along the axis of the fiber, meaning

r = r ( z ) , where z is the distance along the axis. The angle of each guided ray, , is also a
variable. The taper is in this case described as a sequence of uniform sensing regions,
such that
L

A = ( z )dz

(2 9)

It was found that reducing the core radius enhances absorbance by increasing and
increasing the integration range of the angle of ray propagation. The average value for
in a taper was found to be 10 times that of a uniform core decladded fiber 1. Radius
affects the number of ray reflections per unit length, and therefore also affects the
sensitivity in an absorption sensor. Khijwania et al.

the number of reflections N

15
increases as radius decreases in straight fibers. As N increases, the evanescent absorption
coefficient also increases. Taper ratio also determines the sensitivity of an absorption
FOBS. Guo et al. found that lower taper ratios resulted in higher absorbance in cladded
multimode tapers

14

. According to Eq. 2-8, sensitivity likely increases when length

increases. Experimentally this was found to be the case by Guo et al.

14

using a

multimode cladded taper. As mentioned above, bending increases the penetration depth
and consequently an increase in absorption occurs. In a U probe, the angle of a guided ray
decreases in the bent region, which causes penetration depth to increase 9. In a
spectroscopic study which compares bent fibers with straight ones, it was concluded that
bending can lead to higher order modes and therefore higher percentage of power in the
evanescent field. Using absorption of water bands, signal enhancements of up to 9 times
can be obtained compared to straight fibers 10.

2.3.3 Evanescent Wave Fluorescence

The two general methods of fluorescent sensing with TFOBS are the sandwich
assay and the direct method. In the sandwich assay, a primary antibody is first covalently
attached to the surface of the fiber. Then, the fiber is exposed to target biomolecule that
are attracted to the antibody. Presence of this biomolecule is then confirmed using a
secondary fluorescent antibody, which fluoresces when excited by the incident light from
the evanescent wave. In the direct method, a fluorescent dye is first attached on the
surface of the taper. When a molecule is near the surface of the taper, the fluorescence
quenching is measured. Hale et al. found that the detection limit using fluorescence with
a fiber loop was in the pg/mL range 12.

16
V-number matching is a unique problem in fluorescence FOBS 20, 21, 24. Anderson
et al. found that the return of fluorescent signal was inefficient in the step-etched design
25

. The inefficiency was due to the V-number mismatch, because mismatch limits signal

coupling. The results from this study suggest that for fluorescence sensing, a step-etched
probe is not ideal.
Other geometric factors which influence the sensitivity of fluorescent TFOBS
include length, angle of incidence, bending, and surface area. The effect of length on
fluorescent FOBS was studied using tapered quartz rods 2. The rods were immersed in a
100 ppm tetracycline solution, and the signal from the evanescent field was reciprocally
detected by a PMT. It was found that a long taper can be used to overcome the low
penetration depth 2. In a multimode fiber, the angle of incidence affects the percentage of
light in the evanescent field. When the angle is optimized for obtaining the maximum
percentage of light in the evanescent field, higher emission signals are observed. Similar
to evanescent absorption and scattering, bending and looping of optical fibers improve
coupling and sensitivity in fluorescence sensing 2. Hale et al. detected human IgG using a
looped fiber in a sandwich assay 12. In terms of surface area, tapered single mode fibers
suffer from having a small surface area and possibly lower source collection efficiency 26.
It was found that with tapers greater than 10 m in diameter, the longer tapers were found
to have 5-10 times greater coupling efficiency 26.

2.3.4 Surface Plasmon Resonance

Surface Plasmon Resonance (SPR) is a variation of evanescent wave sensing

27

The geometry for a SPR sensor is a dielectric where light propagates, coated with a thin

17
metal layer. Instead of having the evanescent field interact directly with the sample or
excite fluorescent molecules, optical energy is transferred to the surface of the metal layer
as packets of electrons called plasmons. SPR is an optical phenomenon where a ppolarized light beam satisfies the resonance condition and excites a charge density
oscillation 100 nm above and below the metal surface known as a surface plasmon wave
(SPW)

27

. When SPR occurs, the intensity of reflected light is greatly reduced. The

resonance condition depends on the incident angle, wavelength, and dielectric functions
of the metal and dielectric. The wavelength or angle at which resonance occurs depends
on the refractive index of the analyte. Shifts in resonance can be monitored by angle,
wavelength, or reflection intensity changes. From the resonance angle or wavelength one
can obtain the RI of the analyte

28

. The most popular and commercially available

configuration for SPR is the Krestchmann configuration, which consists of a metallic

layer deposited directly on the base of a prism. More recently, the tapered fiber became
another popular configuration for SPR 29. Fibers have certain advantages over prisms in
that they are simple, flexible, small, and allows for multichannel and remote sensing 29.
Usually gold or silver is used as the coating for SPR sensors. The use of gold
results in increase in the shift of the resonance parameter with respect to changes in the
refractive index. The use of silver narrows the resonance curve, thus gives a higher
signal-to-noise ratio (SNR). However, unlike gold, the problem with silver is the low
chemical stability 29. Recently it has been suggested that there should be two metal layers,
and that gold should act as the outer layer while silver as the inner layer. The total
thickness of the metal layers were kept constant at 50 nm, and it was found that when the
silver layer increase in thickness, SNR increases and sensitivity decreases

29

. Silver

18
decreases the width of the resonance curve whereas gold increases the separation between
resonance wavelength, thus increases sensitivity 29.
Geometric factors which affect the performance of a SPR sensor include the metal
thickness, length, waist diameter, and launch angle. It was previous stated that the metal
coating thickness must be on the order of the wavelength used

30

. In the study done

Sharma et al., it was concluded that increasing the length of the sensing region increases
the number of reflections, and causes a broader SPR curve and lower SNR 29. Fabrication
and modeling of a uniform-waist single mode SPR sensor was performed by Villatoro et
al. and it was found that thicker waist diameters resulted in sharper resonance peaks,
which increases sensitivity

30

. Sharma et al. also found that increasing the diameter

decreases the number of reflections, which results in sharper resonance peaks 29. Sharma
et al. studied launching of selected guided rays at desired angles, and found that SNR for
all guided ray launching is 1.5 times that of selected rays launching 29.

2.4 Applications of Tapered Fiber Optic Sensors

TFOBS have been widely used for the measurement of cells, proteins, and DNA.
Table 2.1, 2.2, and 2.3 summarizes the biosensing application to date including the
parameter detected, the range of values detected, the matrix in which the analyte was
detected, taper geometry, fiber type, principle behind detection, and references.

19

2.4.1 Cell Concentration

Pathogen Detection

Ferreira et al. developed a step-etched intensity-based evanescent sensor to detect

the growth of Escherichia coli O157:H7

31

. The intensity changes are due to the

evanescent-field coupling interaction attenuation due to the presence of bacteria.

Therefore, power loss is proportional to the intrinsic bulk absorption and scattering,
which depends on the concentration of the bacteria. The sensitivity of this sensor was
0.016/dB/h/No, where No ranges from 10 to 800 and is the initial number of bacteria. In
our laboratory, Maraldo et al. first demonstrated the use of TFOBS to detect the growth
of Escherichia coli JM 101 32. Tapered surface was coated with poly-L-lysine and E.coli
JM 101 expressing green fluorescent protein was immobilized. Growth was monitored by
light transmission through the tapered fiber. The transmission decreased exponentially
with cell growth on the tapered surface. In a follow up study by Rijal et al., Escherichia
coli O157:H7 was covalently bonded to the surface of a TFOBS via an antibody, and
concentrations as low as 70 cells/mL was detected by changes in intensity 22. Compared
to detection in buffer, detection of Escherichia coli O157:H7 in real samples is more
useful and was investigated by DeMarco et al.

33

. Escherichia coli O157:H7 in seeded

ground beef samples was prepared and detected by a sandwich immunoassay using
cyanine 5 dye-labeled polyclonal anti-E. coli O157:H7. Responses were obtained within
20 minutes, and E. coli O157:H7 of 3 to 30 CFU/mL were detected. The response due to
Listeria monocytogenes, Salmonella Typhimurium, or E. coil non O157:H7 were
negligible compared to those observed with a similar concentration of E. coli O157:H7.

20
A similar study was recently conducted by Geng et al., where a sandwich fluorescent
antibody-based FOBS was developed to detect Escherichia coli O157:H7 in ground beef
34

. The sensor detected 10(3) CFU/ml of pure cultured E. coli O157: H7 cells grown in

culture broth. Artificially inoculated E. coli O157: H7 at concentration of 1 CFU/ml in

ground beef was detected after 4 hours of enrichment.
Fluorescence resonance energy transfer (FRET) is the non-radiative energy
transfer from a fluorescent donor molecule to an acceptor molecule due to the dipoledipole interactions. Ko et al. used the FRET principle with a step-etched optical fiber tip
sensor to detect Salmonella typhimurium 35. Antibody to Salmonella was labeled with a
FRET donor fluorophore (Alexa Fluor 546), and Protein G (PG) was labeled with FRET
acceptor fluorophore (Alexa Fluor 594). The antibody was immobilized covalently by
silanization on the tips. The rate of energy transfer is proportional to 1/ro6, where ro is the
distance between the donor and acceptor. PG binds specifically to the Fc portion of the
antibody. When binding of S. typhimurium to the antibody occurs, the conformation of
the antibody changes, which decreases the distance between the donor and acceptor, and
this causes an increase in fluorescence.

The limit of detection was determined by

measuring the lowest concentration that generated a significant change in signal over the
baseline (three times the standard deviation from the baseline). The best packing density
of acceptor and donor was 0.033 mg/mL, which results in a limit of detection of 103
cells/ml with 8.2% change in fluorescence. The fiber probes were shown to work in real
samples as well by detecting S. typhimurium at 105 CFU/g in homogenized pork samples
with a 6.67% change in fluorescence within a 5-min response time. Kramer et al. studied
the detection of Salmonella Typhimurium in sprout rinse water using the sandwich

21
method

36

. Alfalfa seeds contaminated with various concentrations of Salmonella

Typhimurium were sprouted. The sprout rinse water was assayed with the RAPTOR,
an evanescent fluorescence sensor developed by Research International, Monroe,
Washington. Using this method, Salmonella Typhimurium was identified for seeds that
were contaminated with 50 CFU/g. Zhou et al. also used a sandwich immunoassay to
detect Salmonella 37. Tapered fiber tips with different shapes and treatments were studied
and optimized, and Salmonella was detected at 10(4) CFU/mL.
An antibody-based sandwich fluorescence FOBS was developed by Geng et al. to
detect Listeria monocytogenes

38

. This sensor was specific for L. monocytogenes as

shown by the significantly lower signals caused by other Listeria species or

microorganisms. The limit of detection (LOD) was 4.3x10(3) CFU/ml for a pure culture
of L. monocytogenes. In less than 24 h, this method could detect L. monocytogenes in hot
dog or bologna at 10 to 1,000 CFU/g after enrichment. Recently, Kim et al. also detected
L. monocytogenes using the previously mentioned RAPTOR sensor 39. Detection of L.
monocytogenes in hotdog sample achieved a LOD of 5.4 x 10(7) CFU/ml. RAPTOR
detection of L. monocytogenes in phosphate buffered saline (PBS) was performed again
by Nanduri et al. to evaluate the effect of flow on antibody immobilization

40

. It was

found that both the static and the flow through mode method resulted in a LOD of 1 x
10(3) CFU/ml in PBS. However, the effective disassociation constant and the binding
valences for static modes were higher than for flow through method of antibody
immobilization. The flow through mode was chosen to test real samples, and the LOD
was found to be 5 x 10(5) CFU/ml of L. monocytogenes.

22
There are currently few rapid and sensitive methods to detect Bacillus anthracis,
which is a significant threat in national security. Tims et al. addressed this need by
detecting Bacillus anthracis at a cocentration of 3.2 x 10(5) spores/mg in spiked powders
in less than 1 hour 41. The principle of detection is fluorescence sandwich assay used with
a polystyrene tapered fiber.

Clinical Measurements

Although the detection of whole mammalian cells would be useful for clinical
measurements, little work has been completed in this area using TFOBS. The reason is
that mammalian cells are more difficult to culture than bacterial cells, and oftentimes
small chemicals indicative of diseases are easier to detect with TFOBS once isolated
from the cells. However, there were few attempts to detect mammalian cells, and the
methods used are similar to the ones for bacterial cells detection. For example, Haddock
et al. measured the concentration of Chinese Hamster Ovary (CHO) cells using intensitybased T FOBS 42. They found that the sensitivity in tapered fiber is an order of magnitude
higher than that obtained in a cuvette. The detection limit of fiber was 0.1 millions
cells/ml. MIR spectra of mouse liver tissues have been recorded using a chalcogenide
glass fiber in contact with the tissue

43

. The measurements were based on evanescent

wave spectroscopy. The IR light was absorbed by the chemical bond vibrations with
characteristic frequencies, which gives rise to absorption peaks. The results were in
agreement with histological colorations.

23
2.4.2 Proteins or biomolecule Concentration
Biochemical Measurements

Nicotinamide

adenine

dinucleotide

(NADH)

and

nicotinamide

adenine

dinucleotide phosphate (NADPH) at various concentrations were successfully detected

by Haddock et al. using a tapered fiber sensors drawn by a flame

42

. The transmission

depends on the solute absorption, which is proportional to the concentration. An

important parameter in evanescent absorption is the product of the extinction coefficient
and light path, and is used to characterize sensitivity. In a tapered fiber, the interaction
length and extinction coefficient cannot be separated. A comparison was made between
the limit of detection using the taper and cuvette measurements. It was found that the
limit of detection of the taper was 0.2 M of NADH and 0.5 m of NADPH. On the
other hand, the limit of detection for the cuvette was 3 M for both NADH and NADPH.
Chen et al. developed a chemiluminescent-based fiber optic sensor to detect a
multilayer of enzyme alkaline phosphatase 44. The enzyme was immobilized by chemical
crosslinking to the optical fiber surface. Then, chemiluminescence, ellipsometry, and
SPR were used to characterize the structure and activity of the assembly. The
chemiluminescence method was also used for the detection of organophosphorus-based
pesticides at sub-ppm levels. The detection takes advantage of the competition between
the OP-based pesticide and the chemiluminescent substrate for the enzyme, which results
in partial inhibition of the chemiluminescence signal. Both the multilayer thickness and
the RI confirmed with ellipsometry and SPR. Detection at sub-ppm levels was achieved
for paraoxon.

24
Kishen et al. developed a FOBS to monitor mutans streptococci activity in human
saliva

45

. The mutans streptococci mediated reaction with sucrose is monitored using a

photosensitive indicator, which is immobilized within a porous glass coating on the

surface of the de-cladded fiber.
Kapoor et al. used a fluorescent sandwich FOBS to detect trophic factor activated
signaling molecules in cells 46. Quantitative detection of signal transducers and activators
of transcription 3 (STAT3) phosphorylation in neuroblastoma cells was achieved. The
method is two orders of magnitude more sensitive than the Western blotting technique.

Pathogen Toxins Measurements

Staphylococcal enterotoxins are a major cause of food poisoning. Tempelman et

al. quantified Staphyloccoccal Enterotoxin B (SEB) was in a fiber optic biosensor using a
fluorescence sandwich immunoassay 47. Light from a 635 nm diode laser was launched to
the taper to excite the labeled antibody. The fluorescence was measured and gave a
detection limit of 0.5 ng/mL. Shriver-Lake et al. used an array biosensor to detect SEB
staphylococcal enterotoxin B (SEB) in buffer and six different types of food samples 48. It
was found that the LOD was 0.5 ng/ml could of SEB. In addition, Staphylococcus aureus
is the only species which produces protein A, and was detected by Chang et al. using a
fluorescence sandwich FOBS

49

. The LOD was 1 ng/mL. Similar to SEB, Clostridium

botulinum toxin A was detected by a fluorescence sandwich FOBS

50

. Antibody to C.

botulinum was covalently attached to the surface of the tapered fiber, and the toxin was
detected at concentrations of 5 ng/ml.

25
Narang et al. reported an fluorescence TFOBS for the detection of ricin

51

Antibody to ricin was immobilized onto tapered fiber surface using two methods:
silanization and avidin-biotin linkage. Then, ricin was introduced into the vicinity of the
sensor. Finally, a Cy5-labeled secondary antibody was used to complete the sandwich
immunoassay. The avidin-biotin method showed higher sensitivity and had a wider linear
dynamic range. In the avidin-biotin sensor, the response was linear in the concentration
range of 100 pg/mL to 250 ng/mL. The limits of detection for ricin in buffer solution was
100 pg/mL, and in river water it was 1 ng/ml. At concentrations of ricin greater than 50
ng/ml, there was strong interaction of ricin with the avidin due to the lectin activity of
ricin. This interaction was significantly reduced using fibers coated with neutravidin or
by adding galactose to the ricin samples.
James et al. developed a method to detect lipopolysaccharide (LPS) endotoxin,
which is the most powerful immune stimulant and causes sepsis

52

. In the study, LPS

from E.coli was detected at concentrations as low as 10 ng/mL using fluorescence FOBS
based on the competitive assay. Polymyxin B was used as a recognition molecule. It was
covalently immobilized onto the surface of the probe. Fluorescent labeled LPS was
introduced to the fiber and attached to the Polymyxin B. Unlabeled LPS was then
introduced and competed with the labeled LPS for the binding sites on the Polymyxin B.
As the unlabeled LPS concentration increases, fluorescence decreases. The detection
limit of this sensor in buffer and plasma samples was 10 ng/mL.
Thompson et al. developed a evanescent fiber optic immunosensor to detect
fumonisin B-1 (FB1) 53. Antibody to FB1 was covalently bonded to the fiber surface via a
heterobifunctional silane. A competitive assay was used to measure unlabeled FB1.

26
Fluorescein isothiocyanate labeled FB1 was added to the fiber, and then unlabeled FB1
was added. Competition of unlabeled and labeled FB1 and FB1-FITC determines the
fluorescence signal. The fluorescence was inversely proportional to the concentration of
unlabeled FB1. The sensor had a working range of 10 to 1000 ng/mL of FB1. The sensor
response was specific and was not affected by a sample matrix of methanol/waterextracted corn. In a follow up to Thompsons study, Maragos et al. used the fluorescence
sensor to detect fumonisins and aflatoxins in maize

54

. Unlike Thompsons study, here

both competitive and non-competitive assays were used. The competitive assay was used
to measure fumonisin B-1 (FB1) in both spiked and naturally contaminated maize
samples. Antibody to FB1 was covalently bonded to the fiber, and the antibody binding
sites were exposed to the competition between FB1 and FITC-labeled FB1. The
fluorescence signal was inversely proportional to the concentration of FB1. The
immunosensor results for the naturally contaminated samples agreed with detection with
the HPLC method, except when there is a large amount of contaminating fumonisins that
cross-reacted with the immunosensor. The mycotoxin aflatoxin B-1 (AFB(1)) was
detected using a non-competitive assay because the AFB(1) fluoresces. The fluorescence
signal in this case was directly proportional to the concentration of AFB(1). The detection
limit was 2 ng/mL of AFB(1).

Clinical Measurements

Protein-based medical diagnostics are usually immunological assays. TFOBS is

an attractive alternative because of the quick response time and sensitivity. Effort is
focused on detecting clinically-relevant proteins such as cardiac markers and

27
anticoagulants. However, investigators have also detected a variety of model proteins in
order to characterize TFOBS potential for detection. Preejith et al. detected model
proteins using fiber optic evanescent wave spectroscopy

55

. They immobilized a dye,

Comassie Blue, in a porous glass coating, which was coated on the surface of a
multimode optical fiber. Comassie Blue normally absorbs at 467 nm, but it forms a dyeprotein complex with the protein when it is in an acidic environment, and such a complex
absorbs at 590 nm. The concentration of the protein is inversely proportional to the
output power at 590 nm, because increase in protein levels increases the evanescent light
absorption. Calibration curves were obtained for BSA, hemoglobin, ovalbumin, and
cytochrome c in the range of 0 to 20 g/mL. In our laboratory, BSA has recently been
detected at 10 fg/mL using intensity-based TFOBS modified with antibody under
stagnant condition 56. Tromberg et al. detected antibody to IgG on a fluorescence FOBS
tip using a competitive assay

57

. Rabbit IgG was immobilized on the tip of a fiber, and

exposed to fluorescein isothiocyanate (FITC) labeled and unlabeled anti-IgG. The

response was inversely proportional to the amount of unlabeled anti-IgG, because the
unlabeled anti-IgG displaced the labeled one. Using this sensor, 20 fM of unlabeled
antibody was detected in 20 minutes. Hale et al. developed a fluorescence optical fibre
loop sensor to detect antibody to IgG

12

. A bent TFOBS was used with a two-step

sandwich assay. IgG was labeled with the fluorescent dyes fluorescein isothiocyanate or
tetramethyl rhodamine. In the first step of the assay, the tapered fiber was silanized so
that unlabeled IgG attaches to the sensing region covalently. Then, antibody to IgG binds
to the sensing region due to the presence of the attached IgG. Finally, labeled IgG was
introduced, and the argon ion laser enters the evanescent region to excite the fluorescent

28
dye. The fluorescence emitted by the dye was coupled back into the fiber and is a direct
measurement of the IgG concentration. A concentration of 75 pg/mL was detected with
this method.
Deficiency in Protein C (PC), if left untreated, may result in thrombotic
complications, such as major clotting problems, and thus presents an important clinical
challenge. Traditionally, the presence of PC is detected by ELISA, but that method is
time-consuming, expensive, and requires skilled technicians to perform. Spiker et al.
detected PC in real-time using a fluorescent fiber optic sensor 58. Monoclonal antibody to
PC was immobilized on a tapered quartz fiber. The system is probed with a fluorophoretagged secondary antibody against PC. When PC was introduced, the immobilized
antibody, PC, and secondary antibody form a sandwich complex, and fluorescence levels
were detected and corresponded to the concentration of PC. Using this system, the
authors detected 0.1 g/mL in pure buffer. Regeneration of the fiber was achieved using
CaCl2, because in the presence of the Calcium, the configuration of PC changes such that
it can no longer bind to the antibody. Using the Calcium buffer at pH of 9.4, a significant
increase in the fiber liftetime was achieved, averaging in 6.3 assays per fiber. Real-time
detection of PC in plasma is an important challenge in the clinical setting. Convective
flow plays a vital role in the transport of PC in a viscous media such as plasma, as
demonstrated by a study by Tang et al. which concluded that fluorescent sandwich FOBS
can detect PC in plasma at 0.5 g/mL 59.
The cardiac markers myoglobin (MG) and cardiac tropinin I (cTnI) are released
from cardiac muscles when they are damaged, and therefore their concentrations can
predict the occurrence of myocardial infarction. A fiber-optic SPR sensor to detect

29
markers of MG and cTnI at 3 ng /mL was developed by Masson et al.

60

. A direct

fluorescence FOBS was also used to detect myoglobin 61. Cascade Blue-labeled antibody
was entrapped in the sensing element. Fluorescence quenching occurred when myoglobin
bound to labeled antibody. Using this method, myoglobin was detected at 5 nmol/L. A
recent study by Tang et al. developed a fiber-optic multi-analyte system which
simultaneously quantify the above two groups of multi-biomarkers related to
cardiovascular diseases (CVD): anticoagulants (protein C, protein S, antithrombin III,
and plasminogen) for deficiency diagnosis; and cardiac markers (B-type natriuretic
peptide, cardiac troponin I, myoglobin, and C-reactive protein) for coronary heart disease
diagnosis.
An interesting assay was developed by Garden et al. to detect thrombin using
fluorescence

62

. Unlabelled fibrinogen was first bound to the surface of the evanescent

FOBS. Then, coagulation of solution phase fluorescently labeled fibrinogen to unlabelled

fibrinogen bound to the surface was observed. Thrombin concentrations down to 0.01
NIH/mL were detected. Lee et al. detected thrombin using a fluorescence fiber-optic
biosensor immobilized with an antithrombin DNA aptamer receptor 63. The aptamer was
immobilized on the surface of silica microspheres, which were distributed in microwells
on the distal tip of an imaging fiber. A similar type of silica microspheres was prepared
with a different oligonucleotide to measure the non specific binding. The distal end of the
imaging fiber was incubated with fluorescein-labeled thrombin (F-thrombin). Then, nonlabeled thrombin was detected using the competitive method. The imaging fiber was
coupled to a modified epifluorescence microscope system. The limit of detection was 1
nM of non-labeled thrombin.

30
Protein is commonly measured by analytical methods like Gas ChromatographyMass Spectroscopy (GC-MS) and High Performance Liquid Chromatography-Mass
Spectroscopy (HPLC-MS), which requires pre-concentration of samples by orders of
magnitude to achieve detectable amounts 64. Immunoanalytical methods at very low limit
of detection (LOD) and low limit of quantification (LOQ) are becoming increasingly
important for environmental analysis. Based on animal studies, Progesterone was found
to have evidence of carcinogenicity, and can be found in various surface waters, which is
used for drinking water. In a study by Tschmelak et al., a fluorescence optical biosensor
was immobilized with a labeled-antibody and used successfully with evanescent field and
binding inhibition to obtain detection of progesterone at concentrations lower than ng/L
64

.
A fiber tip fluorescence sensor was used to measure adriamycin (ADM) in vivo 16.

A polymeric fluorescent D-70 membrane with pore sizes of 1-2 m was immobilized on
the fiber tip. The signal fluorescence was quenched by ADM in the blood, and the
fluorescence signal was detected by a photomultiplier tube (PMT) at a wavelength of 530
nm. Light was emitted from a xenon lamp and directed into one branch of a bifurcated
optical fiber, and was transmitted to a probe inserted into the blood vessel. This method
has a detection limit of 10 ng/mL.
Another protein, cytochrome c, which is involved in apoptosis, was detected by a
fluorescence nanobiosensor fabricated by Song et al. 65. -Aminolevulinic acid (5-ALA),
a photodynamic therapy (PDT) drug, was activated by a He-Ne laser to induce apoptosis
in MCF-7 human breast carcinoma cells. When mitochondria are damaged by PDT,
cytochrome c is released into the cytoplasm, which means that cytochrome c

31
concentration is indicative of apoptosis. In this study, antibody to cytochrome c is
immobilized on a nanoprobe. The nanoprobe is inserted inside a cell where binding of
cytochrome c to the antibody occurs. Then, enzyme-linked immunosorbent assay
(ELISA) was performed on the nanosensor outside the cell. Results indicate that 5-ALA
PDT-treated cells show a much higher fluorescence intensity, which indicates that the
cytochrome c concentration is much higher in the treated cells.
Yersinia pestis is an etiologic agent of plague. An optical system devised by Cao
et al. was used to detect Yersinia pestis Fraction 1 Antigen 66. Antibody was immobilized
on the core surface, and then antigen was added. Then, tetramethylrhodamine-labeled
anti-plague antibody was added and formed a sandwich fluorescent complex with the
immobilized antibody and antigen. An argon ion laser at 514 nm launched light into a
long-clad fiber and the fluorescence was produced by the immunofluorescent complex
formed in the evanescent wave region. This system detected 50 400 ng/mL of protein in
serum, with a limit of 5 ng/mL. The results were in excellent agreement with results from
ELISA.
Nath et al. developed a fluorescence fiber optic sensor to detect L. donovani
specific antibodies

17

. The sensor was made by decladding an optical fiber so that the

evanescent wave propagated outside the tapered region. Cell surface protein of L.
donovani was immobilized covalently on the sensing region. Then, the sensor was
incubated with patient serum for 10 minutes, followed by incubation with goat antihuman IgG tagged with FTIC. The amount of L. donovani specific antibodies in the
patient serum was proportional to the fluorescence. There were no false positive results
from leprosy, tuberculosis, typhoid, and malaria serum.

32
Cullum et al. detected a metabolite of benzo-a-pyrene in mammary carcinoma
cells using a fluorescence fiber-optic nanosensor tip

19

. The tip was coated with

antibodies and sensing was based on the fluorescence of a secondary antibody. The
sensor was used to detect concentration of benzo-a-pyrene tetrol (BPT), a metabolite of
benzo-a-pyrene, within the cells of two different cell lines, human mammary carcinoma
and rat liver epithelial cells. By plotting the increase in fluorescence from one
concentration to the next, versus the concentration of BPT, and fitting the relationship
with a straight line, one is able to calibrate the sensor and obtain an unknown
concentration by observing the level of fluorescence. This technique is useful for cancer
screening since carcinogens bind to DNA and form substances such as BPT. The
detection limit was found to be 6.4 1.7 E pM of BPT.
Three cytokines related to chronic wound healing are interleukin-1 (IL-1),
interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-alpha)

67

. A fiber-optic SPR

sensor was able to detect these proteins with LOD of 1 ng/mL in buffered saline solution
and spiked cell culture medium (CCM). The sensor surface was modified with antibodies
specific to the cytokine of interest.

2.4.3 DNA Hybridization

Kleinjung et al. detected DNA hybridization by a fluorescence multimode FOBS

with 13mer probes attached to the de-cladded core

68

. The complementary strands were

labeled and detected when introduced to the sensor. It was found that the sensor was
capable of detecting concentrations as low as 3.2 attomoles (70 fM). This sensors

33
performance was selective as it distinguished between matching sequences, single
nucleotide mismatch, and mismatch caused by additional deviations.
Zeng et al. examined the interfacial hybridization kinetics of oligonucleotides
immobilized onto silica using a fluorescence FOBS

69

. Probe DNA dT20 was used as

recognition molecules, while target fluorescein-labeled ncDNA dT20 and fluoresceinlabeled dA20 were detected. The target DNA concentrations were 0.005 to 0.1 M. The
kinetic response of the sensor was shown to follow second order Langmuir model.
Molecular beacons (MB) are oligonucelotide probes that become fluorescent upon
hybridization with target DNA or RNA molecules

70

. Liu et al. immobilized MB on a

fluorescence FOBS and studied the effects of ionic strength and target DNA
concentration on hybridization kinetics of target DNA. It was found that the limit of
detection was 1.1 nM of DNA. The sensor showed selectivity by distinguishing between
100 nM of ncDNA, 100 nM of one-base mistmatch, and 100 nM of cDNA.

Pathogen Detection via DNA

A fiber optic fluorescence biosensor was developed by Almadidy et al. to detect

short sequences of oligonucleotides that indicate the presence of E. coli microbial
contamination

71

. This biosensor detected genomic targets from E. coli at pM levels

within minutes. First, oligonucleotide probes were immobilized to silica surface via a
silane reagent. Then, stepwise synthesis of oligonucleotides by the -cyanoehylphosphoramidite protocol was performed on the surface. Experiments were done with
both fully complementary (cDNA) and non-complementary (ncDNA) 20mers, in addition
to genomic DNA from E.coli. The cDNA and ncDNA were introduced at a concentration

34
of about 1.7 nM, whereas genomic DNA was introduced at 1.7 pM to 170 pM
molecules/L. Fluorescence intercalating dye was used to detect hybridization. Quantities
as low as 100 fM provided signals at three times the standard deviation levels. The fibers
were washed and regenerated using a 90C water and 90% formamide solution in TE
buffer between samples.
Pilevar et al. detected Helicobacter pylori total RNA using a fluorescence FOBS
72

. Probe oligonucleotides were cross-linked to the tapered surface. Real-time

hybridization of IRD 41-labeled oligonucleotide at various concentrations to the surface

bound probes was performed. Light was launched at 785 nm, and the sensor used the
evanescent wave at the tapered region to excite the IRD41. Using 20mers as probes,
complementary oligonucleotides at lower than nM concentration have been detected.
Sandwich assays were performed with Helicobacter pylori total RNA to determine if the
sensor can detect bacterial cells using rRNA as the target, and it was found that this
sensor can detect H. pylori in a sandwich assay at pM concentrations.

2.5 Conclusions and Future Directions

In the past twenty years, the design of FOBS has evolved from the use of simple
de-cladded fibers to gradually tapered geometries with surface modifications. FOBS have
been used for many biological applications such as the detection of pathogens, medical
diagnosis based on protein or cell concentration, and real-time detection of DNA
hybridization.
In terms of detection principles, it appears that intensity-based sensors have been
used to a limited extent in the detection of cells. However, for the protein and DNA

35
detection, the use of fluorescence FOBS have been widespread because amplification is
often necessary to detect low levels of biomolecules at UV and visible wavelengths.
Perhaps due to the small size of these molecules, intensity or absorption based sensors are
not sensitive enough. More recently, SPR has evolved to become one of the most
commonly used methods for protein characterization. SPR was originally used with a
chip but there have been a few successful studies combining SPR and optical fibers. The
ng/mL sensitivity provided by SPR suits many clinical applications. While fluorescence
provides an attractive platform in terms of selectivity, its sensitivity is surpassed by SPR.
In addition, fluorescence methods require multiple steps for the preparation of the sensor
or the sample with fluorescent labels. Little research has been done in combining tapered
fibers with SPR and that is currently one area of focus in our group.
As for applications, one possible area of growth is the use of SPR or intensitybased FOBS for the detection of protein and DNA hybridization. The hybridization of
DNA is increasingly being used for pathogen detection, and most methods used to date
involve fluorescence. Another area of growth is drug screening using FOBS by making
chemical modification on the fiber surface. Because of recent concerns of security, there
will likely be a lot of research activity in the detection of bio-threat agents such as
anthrax and smallpox. Detection of pathogens also remains important in maintaining a
safe environment and food supply. In addition, clinical applications of FOBS will
continue to flourish along the advancement of modern medicine. After surveying the
large number of studies on FOBS, it appears that detection of cells using antibodies as
recognition molecules is lacking across all platforms, and this is one important area that
is likely to grow in the future.

36
There are many challenges in the development of FOBS. One challenge is the
high LOD faced by intensity-based TFOBS. Thus far the most popular method to
overcome this challenge is to utilize labels to amplify the signal associated with the
presence of the target. However, the disadvantage of fluorescence is the limited lifetime
of fluorescence molecules and the need to prepare a second labeled antibody for
detection. SPR is an attractive method because it does not require any labeling. As a
result, there has been a shift in protein and increasingly DNA detection to SPR; however,
some disadvantages of SPR include its large size and high cost. The development of fiber
optic use with SPR should alleviate some of these problems. One area of TFOBS which
has been investigated to a limited extent is the use of longer wavelengths. The use of
longer wavelengths has been used with some success in chemical sensing, but has not
caught on in biosensing because of the challenges associated with water absorption at
long wavelengths. If long wavelengths are to be used extensively in TFOBS, it is
necessary to functionalize the surface of the TFOBS so that water is excluded, or to find a
wavelength where the absorption of water is minimal.
As TFOBS evolves, new efforts will be focused on enhancing the sensitivity and
selectivity. It appears that methods based on SPR and fluorescence have reached a
plateau in terms of detection limit, and one possible future direction is to combine the two
methods so that the SPR signal is enhanced. Improved surface chemical modification and
stability of the recognition molecule can also increase the sensitivity and robustness of
TFOBS, especially for intensity-based TFOBS because it is the most sensitive when
molecules are bound to its surface. As was shown in this review, there is a solid
foundation of work to support the use of TFOBS and a wide variety of applications.

37
Given its promising advantages, it is likely that TFOBS will remain a popular choice for
detection in the future.

38

Table 2-1: TFOBS for pathogen and toxin measurements

Target Analyte

LOD

Matrix

Taper Geometry

Fiber Type

Bacillus anthracis

3.2E5
spores/mL
8 x 10(4)
spores/mL
pM

buffer

BT

Polystyrene MM

buffer

NA (chip)

NA (chip

Leaky wave (SPR)

73

buffer

Uniform

MM

71

1 ng of protein
A per milliliter
0.016 dB/h/No,
Initial number
(No): 10-800 *

ND

ND

MM plastic

PBS

BT

MM

Fluorescent
intercalating agents
Fluorescence
sandwich assay
Absorption

E. coli O157:H7

1 CFU/ml

MM polystyrene

Fluorescence
sandwich assay

33, 34

Salmonella

50 CFU/g

Waveguide

fluorescence

36

Salmonella

10(4) cfu/ml

ground
beef Uniform
samples
spent irrigation RAPTOR uniform
water used in the
sprouting
of
sprout seeds
Hotdog samples RAPTOR uniform

Waveguide

Fluorescence

74

Salmonella

10(4) cfu/mL

Nutrient broth

TT

MM

Fluorescence
sandwich assay

37

Staphylococcal Enterotoxin
B Concentration

Min: 0.5 ng/ml

(buffer)

CTT

ND

Fluorescence

47

C. Botulinum toxin A,
Pseudexin Toxin

Min: 30 pM (C.
Botulinum toxin
A),
60 pM
(Pseudexin)

CTT

MM

Fluorescence

75

Bacillus subtilis var. niger

LacZ DNA in Escherichia
coli
Staphylococcus aureus
Escherichia coli O157:H7
Concentration

Buffer, human
serum, urine, and
aqueous extract of
ham
ND

Detection Principle

References
41

49

31

38

39

buffer

Clostridium-Botulinum
Toxin-a

5 ng/mL

E. coli lipopolysaccharide
endotoxin

Min: 10 ng/ml
Min: 100 pg/ml
(buffer)
Max: 1 ng/mL
(river water)

Buffer, river water

Ricin Concentration

Listeria monocytogenes

5 x 10(5) cfu/ml

Listeria monocytogenes

5.4 x 10(7)
cfu/ml
4.3x10(3)
CFU/ml

Listeria monocytogenes

TT

MM

Fluorescence sandwich
assay

CTT

ND

Fluorescence

52

CTT

MM (plastic clad
silica)

Fluorescence

51

frankfurter sample

RAPTOR uniform

Waveguide

Fluorescence

40

Hotdog samples

RAPTOR uniform

Waveguide

fluorescence

39

Buffer

Uniform

MM polystyrene

Fluorescence
sandwich assay

38

Buffer and plama

50

Abbreviations: BT = Biconical Taper, TT = Tapered Tip, CTT = Combination Taper Tip, SM = Single Mode, MM = Multimode, ND
= Not Described; * = the change in dB per hour per number of cells at inoculation.

39

40

Table 2-2: TFOBS for biochemical measurements

Target Analyte

LOD

Chinese Hamster Ovary

Cell Concentration

Min: 0.2 M
(NADH),
0.5
M (NADPH)
Min:
105
cells/ml

Paraoxon

Sub ppm

STAT3

ND

NADH, NADPH
Concentration

Matrix

Taper Geometry

Fiber Type

Detection Principle

References

buffer

BT

SM

Absorption

42

buffer

BT

SM

Absorption

42

TT

MM

Chemiluminescence

44

Uniform decladded tip

MM

Fluorescence sandwich
assay

buffer
Buffer

46

Abbreviations: BT = Biconical Taper, TT = Tapered Tip, CTT = Combination Taper Tip, SM = Single Mode, MM = Multimode, ND
= Not Described.

40

41

Table 2-3: TFOBS for clinical measurements

Target Analyte

LOD

Protein A

1 g/mL

BSA

2.5 g/ml

Ovalbumin

2.5 g/ml

Hemoglobin

2.5 g/ml

IgG

20 fM

IgG

75 pg/mL

Protein C

0.1 g/mL

Protein C

0.5 g/mL

Protein C

0.5 g/mL

Protein S

0.5 g/mL

Antithrombin III (ATIII)

30 g/mL

Plasminogen (PLG)

30 g/mL

B-type natriuretic peptide (BNP)

0.1 ng/mL

Matrix
ND
Buffer
buffer
Buffer
buffer
Serum and
jejunal fluids
diluted with
buffer
buffer
plasma
plasma
Plasma
Plasma
Plasma
Plasma

Taper Geometry

Fiber Type

Detection Principle

References

NA (chip)

NA

Leaky wave (SPR)

76

BT

ND (plastic
clad silica)

Absorption

55

BT

ND (plastic
clad silica)

Absorption

55

BT

ND (plastic
clad silica)

Absorption

55

TT

MM

Fluorescence

57

BBT

SM

Fluorescence

12

TT

ND

Fluorescence

58

TT

MM

Fluorescence
sandwich assay

Uniform

MM

Fluorescence
sandwich assay

Uniform

MM

Fluorescence
sandwich assay

Uniform

MM

Fluorescence
sandwich assay

Uniform

MM

Fluorescence
sandwich assay

Uniform

MM

Fluorescence
sandwich assay

59

18

18

18

18

18

41

42

cardiac troponin I (cTnI)

1 ng/mL

C-reactive protein (CRP)

1 g/mL

Myoglobin (MG)

75 ng/mL

L. donovani Antibody
Concentration

Min: 0.244
ng/ml

Bovine Serum Albumin (BSA)

7.4 ng/mL

Progesterone

Ng/mL

Adriamycin

0.01 g/mL

Cytochrome c

ND

Cytochrome c

2.5 g/ml

Yersinia pestis fraction 1

50 400 ng/mL
(R)

cTnI

31 pM

BNP

26 pM

Intracellular Benzopyrene Tetrol

6.4 pM 1.6
nM 19 (R)

Benzo\c\phenanthridinium
alkaoids

ND

Plasma
Plasma
plasma
serum
buffer
Buffer
blood
Cell
buffer
Buffer, serum,
plasma, and
whole blood
plasma

Uniform

MM

Fluorescence
sandwich assay

Uniform

MM

Fluorescence
sandwich assay

Uniform

MM

Fluorescence
sandwich assay

CTT

MM (plastic
clad silica)

Fluorescence

17

chip

chip

SPR

77

ND

ND

Fluorescence

64

Straight core tip

MM

Fluorescence

16

TT

MM

Fluorescence

65

BT

ND (plastic
clad silica)

Absorption

55

BT

MM

Fluorescence

66

TT

MM quartz

TT

MM quartz

TT 19, 79

ND 19, 79

Fluorescence

19, 79

Chip

NA

SPR

80

Fluorescence

53

SPR
Fluorescence Energy
Transfer

60

plasma

Myoglobin

101000 ng/ml
(R)
2.9 ng/mL

Myoglobin

5 nmol/L

Fumonisin B1 Concentration

cell
buffer
methanol/waterextracted corn
buffer
buffer

tip

MM (plastic
clad silica)
MM

Uniform probe

MM

TT

Nano gold particle

enhanced
fluorescence
Nano gold particle
enhanced
fluorescence

18

18

18

78

78

61

42

43

Thrombin

Min: 1 nM

Thrombin

0.01 NIH ml1

RNA

pM

DNA

70 fM

interleukin-1 (IL-1), interleukin-6

(IL-6), and tumor necrosis factora. (TNF-alpha)

1 ng/mL

DNA

5 nM

Buffer
Buffer

Buffer
Buffer
Buffer and
spiked cell
culture medium
(CCM)
buffer

Fluorescence
Coagulation of
fluorescently labelled
fibrinogen to
unlabelled fibrinogen
bound to the surface
of the fibre optic

63

SM

Fluorescence

72

Decladded fiber core

MM

Fluorescence

68

ND

MM

Fiber-optic surface
plasmon resonance
(SPR)

ND

MM

Fluorescence

NA (spheres)

NA

Uniform

MM

TT

62

67

69

Abbreviations: BT = Biconical Taper, TT = Tapered Tip, CTT = Combination Taper Tip, SM = Single Mode, MM = Multimode, ND
= Not Described, NA = Not Appicable

43

44

3. EFFECTS OF GEOMETRY ON TRANSMISSION AND SENSING

POTENTIAL OF TAPERED FIBER SENSORS

3.1 Introduction

The sensitivity of TFOBS depends strongly on the geometry 23, 42, 81. In the past, a
few reports have appeared on the effects of geometry on sensing ability

1, 8, 9, 20

. Review

of the current literature indicates that a systematic study of direct effects of taper
geometry, namely the lengths of convergent and divergent sections, waist diameter and
its length on sensor performance have not been a central focus in the context of
biochemical applications. Our experience has been that the tapered fibers sensing
capability is strongly dependent on its geometry, and in this chapter we provide insights
into this fairly complex process.
We investigated tapered fibers with waist diameters from 6 - 12 m drawn from 8
m core/125 m cladding with a total taper length of 600 m to 1.2 mm. We showed
that: (1) No change in transmission was observed due to the presence of E. coli with
tapers that showed no relative change in transmission in water compared to air. (2)
Tapers that exhibited a significant difference in transmission in water compared to air
gave weak response to the presence of the E. coli. Of these, tapers with low waist
diameters (6 m) showed sensitivity to E. coli at 7,000 cells/mL and higher
concentration. (3) Tapers that showed modest difference in water transmission compared
to air, and those that had small waist diameters gave excellent response to E. coli at 100
to 7,000 cells/mL.

In addition, mathematical modeling showed that:

(1) at low

wavelength (470 nm) and small waist diameter (6 m), transmission with water in the

45
waist region is higher than in air. (2) Small changes in waist diameter (~0.05 m) can
cause larger changes in transmission at 470 nm than at 550 nm at waist diameter of 6 m.
(3) For the same overall geometry, a 5.5 m diameter taper showed larger refractive
index sensitivity compared to a 6.25 m taper at 470 nm. These results and our
experimental observations indicate that the taper geometry not only affected the
magnitude of sensor response, but also its direction. The purpose of this chapter is to
examine more closely the influence of tapered fiber geometry on transmission
characteristics, and its effects on sensing potential. We are particularly interested in
identifying those characteristics of the taper that are suitable for efficient biosensing.

3.2 Physics of Sensing

3.2.1 Wave propagation in absorption sensors

The types of waves which propagate in optical fibers are determined by the
number of modes present. The number of modes that can be supported in the fiber is
determined by the V-number,
V=

2 a0

nco 2 ncl 2

(3 1)

where a0 is the radius of the core. The index of the core is nco and that of the cladding is
ncl.When V<2.405, only the lowest order mode is supported, and as V increases, number
of modes increase. Although in a single-mode (SM) fiber only the lowest order is
supported, in the tapered region higher order modes can potentially be supported because
the difference in refractive index between the core and the sample is ten times larger

46
(~0.12) compared to a regular fiber (~0.01). Higher order modes have a larger fraction of
the power in the evanescent domain. The evanescent light can be absorbed or scattered
by the analyte sample outside the fiber, affecting light transmitted and detected at the
output end of the fiber. The percentage of the evanescent field is about 15-18 % in a
normal SM fiber, and can be increased to near 100% by tapering the fiber 82. Reducing
the core radius of a fiber increases the strength of the evanescent field, and increases the
interaction of the transmitted light with the analyte. However, if the fiber diameter
changes abruptly, V-number mismatch occurs because the V-number of the tapered fiber
immersed in aqueous sample is much larger than the V-number of the fiber with silica
cladding, resulting in considerable signal loss

20

. Hence, a fiber that has a gradually

decreasing radius is desirable for biosensing applications.

3.2.2 Wave Propagation in continuous bi-conical tapered fibers

Continuous bi-conical tapered fibers are made by heat-pulling an optical fiber

without removing the cladding. When V-number of the taper becomes less than unity, the
core is too small to contain the light and light guidance is determined by the original
cladding which acts as the core and the external medium of index next which serves as the
cladding. The new V-number is called Vclad where the parameter, a0 in Eqn. (3-1) is
replaced by the radius of the overall fiber, b(z), and is given by
Vclad ( z ) =

2 b( z )

ncl 2 next 2

(3 2)

Note that in tapered fibers, V-value in Eq. (3-2) is generally referred to as Vclad to
distinguish it from Vcore, given in Eq.(3-1).

47

3.3 Materials and Methods

3.3.1 Fiber Tapering

A silica, single mode (at 1300 nm), Corguide optical fiber (Corning Glass Works,
NY, attenuation at 1300 and 1500 nm of 0.36 and 0.26 dB/m, respectively) with a core
diameter of 8 m and an overall diameter of 125 m was used in all experiments. The
polyacrylic sheathing was removed by a heated fiber stripper (AMHERST FiberOptics).
The fiber was cleaned with isopropanol, and the ends were cut clean using a fiber
cleaver (Ericsson EFC11). To fabricate the fusion-spliced tapers, the fiber was inserted
into a programmable fusion splicer (Ericsson FSU 975) and held together by clamps on
both sides. Electric current was applied via a pair of electrodes for up to 60 s while the
taper was pulled by a pre-set program. Electric current levels and time can be altered in
program parameters pre-defined by the splicer hardware. If an asymmetric taper was
desired, one of the clamps was used as a free weight attached to one end of the fiber.
Heat-drawn tapers, on the other hand, were fabricated using a torch and manual pulling
apparatus. This method is described in detail elsewhere 22, 42.

3.3.2 Taper Characterization

Pictures of the taper were taken via a video camera installed inside the fusion
splicer. The dimensions of the taper were measured using the Scion Image software
(Scion Corp., MD) using a calibration standard. The input end of the fiber was connected
to a LS-450 blue or white LED light source (Ocean Optics) and the output end was

48
connected to a CCD detector (USB2000, Ocean Optics). The light output was monitored
in a 60-min period, and the variations were less than 0.5%. Spectra were obtained for
each tapered fiber in air, de-ionized water (referred to as water henceforth) and in cell
suspensions. Spectral characteristics of the light source were obtained by connecting an
un-tapered naked fiber from the light source to the CCD detector. The signal detected by
the CCD detector was recorded onto a computer through the USB port and using the
OOIBase32 software (Ocean Optics). The cell suspensions containing Escherichia coli,
strain JM101 (ECJ) at concentrations of 100, 1,000, 7,000 and 7 million cells/mL served
as test analyte samples. No special sensor surface preparation such as a recognition
molecule or any other chemical treatment was made other than rinsing the taper with
water.
Escherichia coli, strain JM101 (ECJ) was grown in Luria broth overnight,
centrifuged, and washed three times in 100 mM phosphate buffered saline (PBS), and
then deactivated with 1% chlorox solution. A stock cell sample containing 7 billion
cells/mL was made in water. Samples needed for the present study were prepared by
dilution of this mother mixture. The bacterial concentration of the samples containing
7,000 and 7 million cells/mL were determined by trypan blue staining (for ease of
visualization) and then counting them using a haemocytometer. The lower concentration
samples of 102 and 103 were obtained by dilution and no direct enumeration was done.

3.3.3 Numerical Simulation of Light Transmission in a Tapered Fiber

The numerical simulation of light transmission through a tapered fiber can

provide useful insight in interpreting experimental responses.

We used the simplified

49
mode theory based on linearly polarized modes (LP) to determine the behavior of light in
the tapered region. The LP mode theory allows the use of field descriptions in terms of
the X and Y components of the fields instead of the complex field analysis based on the
radial and azimuthal coordinates

82

. When light is launched normal to the interface, the

only modes that are excited are the LP0m modes. The transverse components of the
electrical field associated with the light inside the fiber are:
J 0 (U 0 m R),

E X (r ) = A J 0 (U 0 )
K (W ) K 0 (W0 m R ),
0 0

R 1

R > 1

(3 3)

where r is the radial axis U and W are constants. The parameter R is the normalized radial
coordinate, r/a0. J0(.) and K0(.) are the Bessel and modified Bessel functions of zeroth
order, respectively, representing the field in the core and the cladding. A is a constant
which can be determined from orthogonality principle. The subscript m represents the
various LP0m modes that can be present in the fiber, each of which has circular symmetry.
The constants U and W depend on the wavelength and refractive index of the core:
U 0m

2 2 2

2
n

= a 0

0m
co

2
2
W0 m 2 = a 02 0 m 2
ncl

(3 4a )
(3 4b)

where c is the speed of light and is the propagation constant. When m=1, we have only
the fundamental mode.

50
As the radius decreases, Vcore decreases, and when core radius is very small, the
core is far too small for light to be confined it, so it spreads into the cladding. As the core
radius approaches zero, the cladding behaves as the core and the medium that surrounds
the taper behaves as the cladding. This phenomenon and the basis of the following model
are described in detail elsewhere 83.
In the taper region where Vcore<1 and Vclad>2.405, many modes can be supported
since the refractive index difference between the cladding and the external medium is
large. Since the tapering is a perturbation, coupling among modes will occur. Because of
the radial symmetry of the perturbation, coupling will only occur among circularly
symmetric modes LP01, LP02, and others

15, 83

. The contracting and expanding regions of

the taper can be approximated using stepwise linear approximation. For a converging
portion of the taper, the radius on the right is smaller than on the left. At the ith step, the
parameters U0m and W0m and are analogous to the constants U and W in a constant radius
core. They can be expressed using the local radius i as
i 2

U 0m

2 2 2

i 2
n

cl
0m

(3 5a )

i2

2
2
W0im 2 = i 2 0i m 2
next

(3 5b)

The index of the medium surrounding the fiber is next. Note also that the index of the
cladding replaces the index of the core (Eq. (3-4)). The V-number for each step is given
by:
Vi =

1/ 2

i ncl 2 next 2

(3 6)

51
The values of U, W and can be calculated using the following approximation for LP
modes,
2.405e 1/ V for LP01

i
U i = 5.52e 1/V for LP02

1/ V i
for LP03
8.86e
i

(3 7)

The values of U from Eq. (3-7) can be used to calculate the corresponding values of W
and for the modes using Eq. (3-5b). The relationship between the modal amplitudes of
the LP0m modes of the ith step and LP0q modes of the (i+1)th step is:

A mi E mi ( r ) e j m z =
i

m =1

q =1

B qi + 1 E qi + 1 ( r ) e

j qi + 1 z i + 1

(3 8 )

E(r) in Eq. (3-8) is the electric field, m is the propagation constant on the left and q is
the propagation constant on the right. It has been assumed that E(r) are orthonormal. That
is,

E (r ) rdrd = 1

(3 9)

In Eq. (3-8), Am is the amplitude of the modes on the left and Bq is the amplitude of the
modes on the right. The amplitude on the right is obtained by applying the orthogonality
principle:
B qi + 1 e

j qi + 1 z i + 1

A mi e j m z C n m ; p q
i

(3 1 0 )

C nm ; pq = 2 E qi +1 ( r ) E mi ( r ) rdr
0

(3 11)

52
In the tapered region, light is coupled among the various LP0m modes. When Vcore=1,
power in the LP01 cladding mode is transferred to LP01 core mode and appears at the
output end of the fiber. The light remaining in other modes stays in the cladding and is
lost. A MATLAB program was used to determine the amplitude and output power.
Parameters such as taper geometry, wavelength and number of steps were systematically
varied, and the resulting changes in power transmitted to the output end were calculated.

3.4 Results and Discussion

3.4.1 Characteristics of Tapers

Several tapers were fabricated using the method described in section 3.1.
Micrograph of a typical fabricated taper is shown in Figure 3-1, along with a schematic
representation of the taper. The convergent, divergent and waist lengths are labeled as a,
b, and c respectively. In the schematic, the core in the waist region is shown to be very
thin. This is because, when the original fiber of 125 m is drawn down ten-fold, the core
diameter reduces to 0.8 m and therefore is almost non-existent. The physical dimensions
and optical characteristics of tapers that were used in sensing experiments are
summarized in Table. 3-1. According to the table, the fusion splicer made fibers (labeled
as FS) were short, ranging from 0.96 to 2.48 mm. Since the arc in the splicer is stationary
and the pulling-tension is applied uniformly on either side of the arc, the tapers tended to
be symmetric. By adjusting the tension applied to one side of the fiber holder,
asymmetric tapers were fabricated successfully. As summarized in Table 3-1, the ratio of
divergent length to convergent length, a parameter that characterizes the degree of

53
asymmetry was varied from 0.99 to 3.74. The heat drawn (HD) fibers were significantly
longer and were in the range of 3.82 to 7.56 mm. The ratio of divergent to convergent
length was 0.51 to 2.55 with most of them at a value less than unity. The waist length
was short in fusion spliced fabricated tapers (0.04 to 1 mm) compared to heat drawn taper
(0.2 to 2.58 mm). The waist diameter of the taper was reproducible within 0.5 m using
the fusion splicer but not easily reproducible with the heat drawn apparatus.

d
a

Figure 3-1 Top: Micrograph of a typical taper. Bottom: Schematic of a tapered fiber. Lengths a, b, c, are
along the z axis, while d is the diameter of the waist region.

54

3.4.2 Air-Water Transmission Ratios

The efficiency of the tapers to operate as sensors was explored by exciting them
with a white LED whose spectral output was in the range of 400 to 800nm. The light
transmission was measured with the tapered fiber waist exposed to air and submerged in
water. The reason for the choice of these two media, namely air and water, was that they
provide the most difference in refractive index that is expected to be present in the waist
region for biological samples which are normally in aqueous solutions and have indices
close to that of water (1.33). For example, whole cell of E.coli B/r H266 growing in
minimal medium have a refractive index of 1.3870.002

84

. The tapers that exhibited

weak change in transmission when the medium surrounding the waist was changed from
air to water, also showed weak sensitivity to ECJ suspensions. As summarized in Table
3-1, the tapers FS-51, HD-1, HD-2, HD-5 and HD-9 exhibited water to air transmission
ratio of nearly unity at 470 nm. Two examples, HD-1 and HD-2, are shown from the
various tapers that were characterized in Figure 3-2. The responses were normalized with
respect to source transmission of a plain fiber, and then again normalized so that the
value is unity at 400 nm. Thus normalized transmission represents relative changes when
an analyte is added to the taper, and allows us to compare results obtained from various
tapered fiber sensor experiments. As shown in Figure 3-2, both HD-1 and HD-2 show
weak responses to cell suspensions. The magnitude of change in transmission for ECJ
concentration was less than 5%.

55

1.8

1.6

1.15
1. 5

100

1.5

1.1

1. 45

1.6

1. 4

1.4

Air

1.3

7,000

1.2

Water

1. 25
1. 2
1. 15
1. 1
1. 05
1
400

410

420

430

440

450

1,000

1.1
1
0.9
0.8

0.95

1.4

0.9
0.85
500

7,000

520

540

560

580

600

100

1.2

1,000
Water

Air
0.8

0.7
0.6
400

1.05

1. 3

Normalized Transmission

N orm aliz ed Transm ission

1. 35

450

500

550

600

650

700

750

Wavelength,nm

800

0.6
400

450

500

550

600

650

700

750

800

Wavelength, nm

Figure 3-2 Transmission characteristics of two tapers fabricated using heat drawn (Left: HD-1. Right: HD2) whose air to water transmission ratio is near unity. Sensitivity to cell concentrations as high as 7,000
cells/mL showed almost no response over the entire wavelength investigated. Small changes are observed
for higher EC concentration.

3.4.3 Response of Fusion Spliced Tapers to Low Concentrations of E. coli

Tapers that showed no relative change in transmission in water compared to air,

also showed no or low response to the presence of ECJ suspensions. Both symmetric and
asymmetric tapers of small and large waist diameters show this behavior, pointing to the
lack of sensing ability of these tapers.

56

Several tapers exhibited a significant difference in transmission in water

compared to air. For example, FS-1, FS-2 showed nearly 50% reduction in water. These
tapers also showed little or no ability to detect the presence of the ECJ cells (spectra not
shown). In these cases, small changes in indices due to the presence of ECJ suspension
were not sufficient to produce the changes in transmission, resulting in poor sensitivity.
Any impact on the light through the fiber was already saturated from change to water
itself, such that the presence of ECJ suspension had little further impact. Other tapers that
had this characteristic property, but were of smaller waist diameter showed weak
sensitivity (FS2, FS4; spectra not shown). Most of such tapers that exhibited a significant
difference in transmission were symmetric tapers. The asymmetric tapers, on the other
hand, showed lower relative transmission through water. Such tapers allowed further
modulation in transmission due to the presence of ECJ and subsequent absorption when
the concentration of the bacteria was high. The sensitivity was also determined by the
operating wavelength as will be shown through simulation. To illustrate the above
general observation, we examine specific cases in detail.
Responses of three fusion spliced tapers to air, water and low concentration of the
bacteria ECJ are shown in Figure 3-3.

Several other tapers with almost identical

geometric parameters exhibited similar behavior, and are not shown. For example, FS162 showed response similar to FS-158. The tapers FS-158 and FS-164 have nearly the
same geometry except for the waist length and waist diameter. The waist length of FS
158 is about 30% longer, and waist diameter about twice as large compared to FS-164.
The taper, FS-2 has nearly the same convergent length as the others, but its waist length

57
is almost ten times that of FS-164, and its divergent length is twice as long as that of FS158 and FS-164.

Its diameter is midway between the FS-158 and FS-164.

The

transmission response given in Figure 3-3 shows that the symmetric taper, FS-158 with a
waist diameter of 9.5 m was responsive to ECJ concentration as low as 100 cells/mL.
The responses to 1,000, 7,000, and 7 million cells/mL are clearly discernable, and the
transmission increases progressively with ECJ concentration. On the other hand, the
taper, FS-164 which has a shorter waist length, and a smaller waist diameter is sensitive
only at cell concentrations greater than 1,000 cells/mL. The longer taper, FS-2 with an
intermediate waist diameter of 6.2 m was responsive at 7,000 cells/mL and higher. Note
also that transmitted power is not a direct function of cell concentration. It is less for
7,000 cells/mL than for 1,000 cells/mL, while transmission for 7x106 cells/mL is higher
than that for 7,000 cells/mL. It is also to be noted that all three tapers are responsive in
the 450 nm to 650 nm wavelength range. In this wavelength range, bacterial suspensions
are known to absorb and thus a larger fraction of evanescent light in the above tapers
were probably be absorbed resulting in reduction in transmission. Note also that water/air
transmission ratio of FS 158 is higher than unity (1.23 at 470 nm) while that of FS-164
and FS-2 are less than unity (0.49 and 0.48).

58

N orm aliz ed Transm ission

1.1
1

7 million
7,000

0.9
0.8
0.7

100
1,000
Water

0.6
0.5

Air

0.4
0.3
0.2
400

500

600

700

800

Wavelength, nm

N orm alized T ransm ission

1.1
Air

1
0.9
0.8
0.7

Water
1,000

0.6
7,000

0.5
0.4

100
7 million

0.3
0.2
400

500

600

700

800

Wavelength, nm
1.1
N orm alized T ransmission

1
0.9

Air

0.8
100

0.7

Water

1,000

0.6
0.5
7,000

0.4
0.3

7 million

0.2
400

500

600
Wavelength, nm

700

800

Figure 3-3 Transmission characteristics of three short tapers: FS-158, FS-164 and FS-2. FS-158
and FS-164 have nearly the same values of a, c and d, while the waist length (b) is longer in FS

59
158 compared to FS-164. The taper FS-2 has same value of a, and twice the length c, and a very
long b with an intermediate waist diameter, 6.3 microns.

In Figure 3-4, we compare relative transmission at 470 nm for a number of tapers

that were fabricated using the fusion splicer. In general, we see two types of responses.
In the first type, the transmission either increases or decreases monotonically, as ECJ
concentration increases. The tapers, FS-158 and FS-162 are two good examples of this
behavior. In the second type, an initial increase for low cell concentration (for example,
less than 1,000 cells/mL) is seen followed by a decrease at higher cell concentration. The
tapers, FS-163, FS-164, and FS-4 are examples of this type of behavior. We also found
that tapers such as FS-1, FS-2 and FS-3, that showed an increase in transmission at low
ECJ concentrations, followed by decrease at intermediate concentrations and then an
increase at 7 million cells/mL. Although this type of response was uncommon among the
tapers we tested, they are included here for completeness.

60

Transmission at 470 nm

1.1

0.9

0.8

0.7

0.6
FS-158

FS-162

FS-163

FS-164

FS-1

FS-2

FS-3

FS-4

Taper No
Water

100 cells/mL

1,000 cells/mL

7,000 cells/mL

7 million cells/mL

Figure 3-4 Transmission characteristics of Fusion Splicer fabricated tapers at 470 nm. Data were
normalized with respect to PBS, and ordinate value is relative transmission. Most of the fibers show
modest response for 100 to 1,000 cells/mL, but most show good response for 7,000 and 7 million cells/mL.
See for example, FS-2.

3.4.4 Response of Heat Drawn Tapers to Low Concentrations of E. coli

Heat drawn tapers (see Table 3-1) have typically a much longer convergent, waist
and divergent sections, each in the order of millimeters compared to FS-tapers. One notes
from Figure 3-5 that transmission of HD-14 taper is responsive to the entire ECJ
concentration tested, 100 to 7 million cells/mL. On the other hand, HD-7 was responsive
at low cell concentration, but was not responsive at high cell concentration.

The change

from water to 100 cells/mL at 470 nm was nearly a 7% change in transmission. Further
increase in concentration caused further decrease in transmission, but the change was less
pronounced. The decrease in transmission is presumably due to absorption of evanescent
light by the cells. On the other hand, for HD-14, at low cell concentration 100 to 1,000

61
cells/mL transmission increased, and upon further increase in cell concentration the
transmission decreased. That is, at high cell concentration lower transmission is seen due
to increases cellular absorption of evanescent light. In general, the absorbance is
proportional to the length of a uniform fiber, and we believe that this phenomenon is
responsible for the monotonic relationship between transmission and concentration in the
case of HD-7 for all cell concentration tested and in HD-14 at cell concentration higher
than 1,000 cells/mL.
With regard to wavelength response with HD-7 lower transmission was observed
in 400 - 470 nm region, followed by an increase to over 150% relative transmission at
610 nm. HD-14, on the other hand, showed an initial increase to over 140 % followed by
a sharp decrease to below 100%.

The reversal of the responses is likely to be due to

refractive index effects, and is further discussed under simulation results.

62

Norm aliz ed Transm ission

1.6

Water

Air

1.4

100
1.2

1,000
1

7,000
0.8
0.6
0.4
400

450

500

550

600

650

700

750

800

750

800

Wavelength, nm

Norm aliz ed Transm ission

1.6
1.4
1.2

Air

1,000

100

0.8
0.6

7,000

Water
0.4
400

450

500

550

600

7 million
650

700

Wavelength, nm

Figure 3-5 Normalized transmission characteristics of heat-drawn tapers. HD-7 and HD-14 have an
overall length and waist diameter of 3.82 and 3.99 mm, and 14.2 and 20.6 m. HD-14 (bottom panel) is
responsive at both low and high cell concentrations while HD-7 (top panel) shows strong response at low
cell concentration, and is relatively insensitive at higher cell concentration.

Of the fourteen heat-drawn fibers, we present the transmission response

(normalized at 470 nm with respect to water) for a representative set in Figure 3-6.
Analogous to the behavior of fusion spliced tapers, HD tapers showed two basic
characteristics. In one case, tapers such as HD-4 and HD-7 showed a monotonic decrease
in transmission; their sensitivity was found to be strong or weak at low cell concentration
and appeared to depend strongly on the waist diameter. In this particular case, HD-4 has
a waist of 6.3 m while HD-7 is 14.2 m. HD-4 showed a decrease from 0.98 at 1,000

63
cells/mL to 0.47 at 7,000 cells/mL. In the other case, HD-11, HD-12 and HD-14 showed
a slight increase from water to 100 - 1,000 cells/mL and then a decrease in transmission
for higher concentrations. As stated earlier, in these tapers at low concentration the
refractive index of cellular suspension influence transmission response which causes the
increase in transmission. At higher concentrations, the absorption of the evanescent light
by the cells dominates the response. The response suggests that tapers fabricated by heatdrawing have excellent potential as biosensors. In the next section we attempt to provide
quantitative discussion on light transmission through tapers to provide insights into the
taper responses discussed above.

1.1

Normalized Transmission

1
0.9
0.8

0.7
0.6
0.5
0.4
HD-4

HD-7

HD-11

HD-12

HD-14

Heat Drawn Taper

Water

100 cells/mL

1,000 cells/mL

7,000 cells/mL

7 million cells/mL

Figure 3-6 Relative transmission response of heat-drawn tapers at 470 nm when exposed to dilute ECJ cell
suspensions. Transmission was normalized with respect to values obtained with water at 470 nm for each
taper. Tapers such as HD-7 show good overall sensitivity for low ECJ concentration while tapers such as
HD-14 show sensitivity at higher cell concentration.

64

3.4.5 Transmission in tapered fibers: Simulation Results

In general, we expect that when a taper waist is in air, a greater fraction of the
light is transmitted through the fiber compared to the case when the waist is surrounded
by water. This is because Vclad value is higher when surrounded by air. In general, the
larger value of the Vclad, greater and tighter is the confinement of light and higher is the
transmission through the core. The tapers fabricated in this study exhibited this behavior
with a few exceptions, and is summarized in Table 3-1. This property alone does not
point to the utility of the tapers as sensors. Certainly, the light transmission is a function
of the lengths of the contracting, waist and expanding regions of the taper along with
waist radius and the operating wavelength. It is suggested that the transmission in water
may be higher than that in air when one considers all geometric aspects of the taper. The
light transmission characteristics of the tapers were simulated by calculating the
transmission through tapers of various lengths and waist radii. Three modes, LP01, LP02
and LP03 were used in the simulation 82, 83. Since the light will be launched normally, the
only modes to be excited will be the LP0m modes, with a majority of the light staying in
the LP01 mode. Sample simulation results, illustrated in Figure 3-7, show changes in
transmission for two geometries, a short symmetric taper and a long asymmetric taper, as
a function of waist diameter.

In Panel A, the taper geometry simulates that of a

symmetric taper, while in Panel B, the simulation is for a long taper similar to a heatdrawn taper. The transmission is normalized with respect to air, so that a value of 1.2
indicates a transmission increase of 20% in water compared to air. The results in Figure
3-7 show that as the waist radius increases, the difference in transmission between water

65
and air decreases. However, at intermediate values the ratio may be higher or lower than
unity, particularly at smaller waist diameters. For specific values of the radius, one notes
that the transmission through water is higher than in air. At a longer wavelength (550
nm, for example) and for the same waist diameter values, the difference in transmission
between air and water differ by less than 10%.

To explore further, simulation was

undertaken by varying the diameter of the waist in much smaller steps of 0.001 m.
Computed transmission results are shown in Figure 3-8 for two starting diameters, 5 m
and 6.25 m. The transmission characteristics change rather significantly for small
changes in diameter.

For example, a 5.54 m diameter taper exhibits 30% higher

transmission in water while a 5.58 m waist diameter taper transmits 20% less
transmission. The differences, however, become smaller for larger waist diameters. The
example of 6.25 m in Figure 8 shows that the changes in transmission were less than 10
%.

66

Normalized Transmission

1.5
1.4

A. 470 nm

1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
4

12

16

20

Taper Waist Diameter (microns)

1.5

Norm alized Transm ission

1.4

B. 550 nm

1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
4

12

16

20

Taper Waist Diameter (microns)

Figure 3-7 Transmission in water normalized with respect to air as waist diameter is altered. Panel A. A
short symmetric taper: a= 0.425, b=0.325, c=0.500 mm. Operating wavelength = 470 nm. Panel B. A
longer asymmetric taper: a=2.25, b=0.245, c=4.5 mm. Operating wavelength = 550 nm. Note that larger
undulations in transmission occur at lower wavelength. V-number is higher at 470 nm.

Norm alized Transm ission

67

1.4
1.3

5.5 m

1.2

6.25 m

1.1
1
0.9
0.8
0.7
0.6
0

0.02

0.04

0.06

0.08

0.1

Change in waist diameter (m)

Figure 3-8 Transmission characteristics of a taper (a= 2.25, b=0.25, c=4.5 mm) in water at 470 nm as a
function of change in waist diameter. Transmission is normalized with respect to transmission in air at the
corresponding geometric values. Smaller starting diameter tapers show large changes in transmission for
small (0.01 m) changes is waist diameter.

The simulation results merely serve as a guide to the analyses of the experimental
data on the tapered fibers. It is important to recognize that in the simulation we have
considered only the effect of refractive index (air and water) in the waist region, while in
the actual sensing experiment, the cells absorb at the operating wavelength, and the
resulting sensor response is a complex interplay of these two phenomena. In addition,
because the cells constitute particulate matter, there is an additional complexity of
inhomogeniety in the sample surrounding the taper waist.

Furthermore, the cell

attachment or adsorption onto the taper surface is often not uniform as we showed in our
earlier report 22, thus compounding the sensor response further.

68

3.5 Conclusions

Of the experiments that were conducted, several conclusions can be made on the
geometry of the taper and its effect on transmission. The results showed that the
transmission characteristics of the taper with water and air surrounding the waist are good
indicators of the performance of the tapers as sensors. The hypothesis that no differential
transmission between air and water clearly pointed to tapers that had no sensitivity. The
sensitivity of the tapers is clearly determined by the profile parameters, namely the
lengths of the contracting, expanding and waist regions and their relationship to the waist
diameter and the operating wavelength. A taper that showed moderate change in
transmission, also showed good sensitivity to cell suspensions.

69
Table 3-1 Characteristics of Sample Fabricated Tapers by the two method: Fusion Splicer (FS) and Heat
Drawn (HD)
Taper
Designatio
n

a
m

b
m

c
m

d
m

Transmission
Ratio at 470
nm (water/air)

Asymmetry
Ratio c/a

Overall
length
m

FS-1
FS-2
FS-3
FS-4
FS-41
FS-42
FS-50
FS-51
FS-52
FS-54
FS-55
FS-56
FS-158
FS-162
FS-163
FS-164

434
448
344
394
390
391
440
391
460
390
368
396
461
459
454
463

187
455
198
505
275
549
940
996
41
172
620
335
82
65
90
57

929
905
1285
1426
668
665
1096
879
460
954
856
821
470
483
434
457

7.9
6.3
9.5
3.2
14.2
12.7
5.0
3.2
3.2
6.0
3.0
11.0
9.5
9.5
7.9
4.8

0.49
0.48
0.70
0.52
0.74
0.69
1.40
1.06
0.41
0.47
1.10
0.61
1.23
0.74
0.49
0.49

2.14
2.02
3.74
3.62
1.71
1.70
2.49
2.25
1.00
2.45
2.33
2.07
1.02
1.05
0.96
0.99

1550
1807
1826
2324
1333
1605
2476
2266
961
1516
1844
1552
1013
1007
978
978

HD-1
HD-2
HD-3
HD-4
HD-5
HD-7
HD-8
HD-9
HD-10
HD-11
HD-12
HD-13
HD-14

2218
2273
3784
3023
2938
2218
1109
1129
1074
2218
2218
2218
1109

230
763
1559
2514
2580
470
2110
1477
1032
202
245
1406
1109

1910
1800
2218
1509
1649
1129
1523
1481
2742
1625
1354
1296
1769

24.0
27.0
11.0
6.3
7.9
14.2
4.8
7.9
6.3
11.1
11.1
9.5
20.6

1.02
1.06
0.97
1.30
1.03
1.28
1.33
1.04
1.25
0.56
0.87
0.85
0.86

0.86
0.79
0.59
0.50
0.56
0.51
1.37
1.31
2.55
0.73
0.61
0.58
1.60

4358
4836
7561
7046
7167
3817
4742
4087
4848
4045
3817
4920
3987

70

4. DETECTION OF E.COLI O157:H7 USING ANTIBODY-IMMOBLIZED

TFOBS

4.1 Introduction

Escherichia coli is a common microorganism found in the intestinal tract and it is

usually harmless.

It becomes pathogenic in debilitated or immunosuppressed host.

Escherichia coli O157:H7 (EC) is a pathogenic variant that causes food borne illness,
hemorrhagic colitis, hemolytic-uremic syndrome, and diarrhea. This pathogen is
abundant in undercooked or raw ground beef, milk, fruits and vegetables

85

. It can be

transmitted through food and the use of common facilities. At least 20,000 cases of food
poisoning by EC occur in the US alone each year

85

. Since a dose as low as 10

microorganisms of this pathogen is enough to cause infection, diagnosis requires rapid

and sensitive detection

31

. There is a need for rapidly detecting the presence of the

pathogen EC in aqueous samples. Current approach of culturing on selective media and

then counting colonies formed does not give contemporaneous information on the
presence of EC in process systems.
Fiber-optic biosensors (FOBS) have been widely investigated as sensors for
chemicals

28, 86, 87

, biological entities

31, 42, 65, 71

, and for physical properties

typically make use of sensing mechanisms such as fluorescence

absorption

31, 42

20

88, 89

. FOBS

and evanescent field

. Fluorescence-based sensors have used fluorescent-labeled secondary

antibodies. In these sensors, the fluorogenic molecules must be within a few microns of
the fiber surface for effective sensing. Sensor geometries that have been explored include
fiber tips 37, 65, U-shape 15, 90, and tapered fibers 1, 42.

71

In the area of pathogen detection, FOBS have been used for Streptococcus
Salmonella

37

, and L donovani antibodies

17

45

. In most of the studies done to date, the

cladding on the fiber was removed such that the evanescent field propagating outside the
core can interact with the analyte. In the study done by Ferreira

31

, the interaction

between whole cell EC and the evanescent light wave was carried out in a clad-stripped
FOBS. The FOBS measurements showed different response for each phase of bacterial
growth in the medium. While this approach was good to characterize growth, it is hardly
a detector since it had no antigen-recognizing chemistry on the surface. In a study
performed by Kishen 45 the fiber was etched to remove the cladding. Then, the surface of
the core was treated and coated with a thin film of porous glass using the sol-gel
technique. A photosensitive indicator was immobilized within the coating to monitor the
sucrose-mediated growth of Streptococci 45. In another study 17 a clad-stripped fiber was
tapered to a core size about half of its original size. The tapered fiber was immobilized
with purified cell surface protein of L. donovani, which has high affinity for the antibody.
It was used to detect antibodies to L. donovani present in twelve patients without any
false negative results. Such an approach showed good selectivity as it did not give false
positives from the sera of leprosy, tuberculosis, typhoid, and malaria patients.
From the foregoing discussion it is clear that methods to increase evanescent field
can significantly enhance the sensor capabilities. Of specific interest is the property
making the sensor selective to the target antigen, EC. In this study, we achieve that by
covalently linking an antibody to EC on the tapered fiber surface.

72
4.2 Physics of Tapered Fiber Sensing

Optical fibers are cylindrical waveguides with an inner core typically made of Gedoped silica surrounded by a cladding of pure silica. Light propagating through optical
fibers has two components: the guided component inside the core, and an exponentially
decaying or evanescent component propagating through the lower index cladding. Fibers
are normally designed to be insensitive to the outside environment by having a cladding
that is much thicker than the core.

Thus, even though the evanescent component

propagates outside the core, its magnitude decays rapidly to almost zero due to the thick
cladding, and any remaining field that interacts with the medium outside the cladding is
so small that it has negligible effect on the overall transmission. However, if the cladding
is partially or totally removed and replaced by a medium to be sensed, the evanescent
field interacts with the medium to a far greater extent. Thus, large and significant
changes in the transmitted power occur, and that forms the basis for sensing.
The basic sensing mechanism of evanescent field absorption sensors is based on
the total internal reflection (TIR) and evanescent field propagation. TIR takes place at
the core/cladding boundary.

Beyond the core/cladding interface, there is an

exponentially decaying field in the cladding (rarer medium) whose evanescent field
penetration depth is given by 91:

dp =

2 nco 2 sin 2 ncl 2

(4 1)

where is the wavelength of transmitted light, is the incident angle at the core/cladding
interface, and nco, ncl are, respectively, the refractive indices (RI) of the core and
cladding. Analytes located on the surface within the depth of evanescent field (Eq.(4-1))

73
are sensing targets.

In the biosensing applications, the refractive index of aqueous

sample is typically 1.33, and that of the original cladding and core are 1.458 and 1.46
respectively.

Thus, the evanescent field depth useful for sensing at 470 nm is

approximately 0.13 m. Given that antibodies and other recognition molecules are about
10-12 nm, the evanescent field depth is reasonable to interact with target antigens in
sample solutions.
Another consideration in sensing is the group of modes propagating in a fiber is
determined by the V number of the fiber 83:
V=

2 a

nco2 ncl2

(4 2)

where, a is the radius of the core. When V= 2.405, only the lowest order mode is
supported, and if V is greater than 2.405, other modes also propagate. When a fiber is
tapered, V decreases and when V is less than unity, the core is too small to guide the light
and guidance is provided by the original cladding, which acts as the core with the
external aqueous medium acting as the cladding. The light guidance is determined by
Vclad, and can be determined from Eq (4-2), by re-writing it as:

Vclad =

ncl 2 next 2

(4 3)

where is the radius of the fiber and next is the index of the aqueous surrounding the
fiber. Note that in tapered fibers, V value in Eq. (4-2) is generally referred to as Vcore to
distinguish it from Vclad of Eqn. (4-3). The strength of the evanescent field is typically
very low in a normal single mode fiber, and can be significantly enhanced by tapering.

74
The evanescent light can be absorbed or scattered by the analyte outside the fiber,
and some of it couples back into the fiber and is detected at the distal end of the taper.
Since the propagated light is directly related to the magnitude of the evanescent field,
changes in transmitted light reflects changes in the evanescent light.

4.3 Materials and Methods

4.3.1 Fiber tapering

A silica fiber (nominally single mode at 1300 nm, corguide optical fiber, Corning
Glass Works, NY, attenuation at 1300 and 1500 nm of 0.36 and 0.26 dB/m, respectively)
with a core diameter of 8 m and total a diameter of 125 m was used for all the
experiments.

The polymeric sheathing was removed completely by a heated fiber

stripper. The fiber was then cleaned with isopropanol and the ends were cut clean using a
fiber cleaver (Ericsson EFC 11-4). Tapered regions were fabricated by two different
methods. In the first method, the fiber was inserted into the programmable fusion splicer
(Ericsson FSU975). Electric current was applied via a pair of electrodes for up to 60
seconds while the taper was pulled automatically. Various current levels (3-13 mA) and
pull times (2-30 s) were used to produce fibers of varying taper diameters and lengths.
The second method of obtaining tapered fibers was by heat pulling using micropropane torch, as shown by a schematic in Figure 4-1. A 30-35 cm long fiber without the
polymeric sheathing was mounted on the apparatus with two paper clips of identical
weights (2.8 g) on either end to provide tension to the fiber. A micro-flame (Model 6000,
Microflame, Inc., MN) connected to propane and oxygen tanks was used to taper the

75
fiber. The flame was positioned such that the fiber was approximately one-third distance
from the top end of the visible end of the flame. The tapering took a few seconds, and
the flame was removed as soon as the paper clip touched the stop. After the formation of
the taper, both ends of the fiber were cleaved using a fiber-optic cleaver (NO-NIK) to
give clean-cut ends. The fiber was placed in an optical fiber holder to be used in the
experiments. Tapers prepared by these techniques retain their optical characteristics by
virtue of relative arrangement of core and cladding, the presence of both core and the
cladding makes them ideal for sensing. Optical micrograph of a typical fabricated taper
is shown in Figure 4-2. The taper geometry is characterized by three parameters: the
convergent length (Lc), waist length (Lw), divergent length (Ld) and the waist diameter
(Dw). By the approach used in fabricating tapers, it was not always possible to get the
same values for Lc,Lw, Ld and Dw. Thus, each tapered fiber fabricated was characterized
for its transmission in air and then in water.

76

2 mm

4 mm

M icroflame torch

Magnification

Fiber

Weight
Fiber Tapering stand

Heat

Figure 4-1 Some of the fibers discussed in this paper were fabricated using a simple fiber tapering device.
Fibers without sheathing were mounted on a stand and two pieces of weights were attached to the ends to
provide tension required for tapering. The fibers were then heated with a flame while carefully monitoring
the diameter of the tapers. When the desired diameters were reached, the two ends of the fibers were
clipped, their diameters were measured using a calibrated image processing software in an optical
microscope.

77

Figure 4-2 100X magnification of a tapered fiber. The lighter region in the center of the fiber is the core
and the dark region surrounding the core is the cladding.

Micrographs of the taper were taken using an IMT-2 optical microscope

(Olympus, Japan) equipped with a video camera (Cohu Corp., Japan) linked to a
computer. Its dimensions were then measured using the Scion Image software (Scion
Corp., MD) after a calibration was performed according to the microscope objective
used.

4.3.2 Experimental arrangement

Using an experimental setup similar to Figure 4-3, light transmission through the
fiber was measured. The light sources used were one of the following: LS-450 Blue
LED (Ocean Optics) and a 75W Xenon arc lamp (Ushio Inc., Japan). The selected light
source was coupled to the fiber via an SMA or an FC connectors. For the 470 nm source,
a CCD detector (USB2000, Ocean Optics) was used for detecting light transmission and

78
spectra were characterized using the OOIBase32 software. In the case of 75W Xenon arc
lamp, the transmitted light (470 nm, with slit width of 5 nm at excitation and 10 nm at
emission monochromator, respectively) was detected using a photomultiplier tube (PTI
701) connected to a computer running Felix software.

Reaction
LED
470

CCD

details

PC

Fiber

Fiber Holder
LED

To CCD Spectrometer
Analyte Chamber

Figure 4-3 Typical experimental arrangement for detecting pathogens. Light from a LED is launched from
one end of the fiber. The tapered section of the fiber is held in a plexiglass fiber holder. Light transmitted is
detected by a CCD spectrometer at the distal end, which is read by a computer.

4.3.3 Antibody immobilization

The immobilization of antibody on the tapered surface consisted of three steps

adapted from

92

. These were: cleaning, amination of glass and covalent linking of the

antibody. Cleaning of the surface was done sequentially with methanol-hydrochloric acid
solution (1:1 v/v), concentrated sulfuric acid, hot sodium hydroxide, and finally boiling
water. The surface was rinsed between each washing steps with deionized water. The
cleaning procedure produces reactive hydroxyl groups on tapered surface.

After

79
cleaning, the tapered surface was silanylated with 1% 3-aminopropyl-triethoxysilane
(APTES; Sigma-Aldrich) in deionized water at pH 3.0 (adjusted by hydrochloric acid, 10
N) and 75oC for 2 hours. APTES reacts with glass leaving a free amine terminal for
further reaction. The carboxyl group present in the monoclonal antibody to EC, IgG2
(Kirkegaard and Perry Lab (KPL), Gaithersburg, MD), was activated using the zero
length cross linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC; SigmaAldrich) and promoted by sulfo-N-hydroxysuccinimide (Sigma-Aldrich). EDC converts
carboxylic groups into reactive unstable intermediates susceptible to hydrolysis. SulfoNHS replaces the EDC producing a more stable reactive intermediate that is susceptible
to reaction with amines. Covalent coupling of the stable intermediate with the silanylated
tapered fiber surface was carried out at room temperature for 2 hours. The taper surface
with the immobilized antibody was used to detect the pathogen EC.

4.3.4 Pathogen Detection experiments

An EC stock solution (7x109 cells/mL) was prepared as per the vendors (KPL)
rehydration protocol in 10 mM phosphate buffered saline (PBS) solution pH 7.4. Lower
concentrations (7x107 cells/mL, 7x105 cells/mL, 7x103 cells/mL, and 70 cells/mL) of the
antigen were prepared in PBS (pH 7.4) by serial dilution. 150 L of EC samples of
different concentrations were loaded into the analyte chamber ensuring complete
immersion of the taper. After recording the transmission during the attachment of EC to
the fiber, the sample was pipetted out and the chamber was loaded with a buffer, either
HCl/PBS buffer at pH of 2.3 or Glycine-HCl/ethylene glycol (1:1 v/v) buffer at pH 1.7 to
characterize the release of antigen from the fiber taper.

80

In order to evaluate the selectivity of the antibody immobilized taper to the

pathogenic bacteria exclusively, the taper response to non-pathogenic E. coli in solution
was measured.

Test samples containing both a wild strain and the pathogen were

prepared. Stock solution containing EC was mixed with a wild strain of E. coli (JM101)
in volumetric proportions of 0%, 50% and 70%. The total bacterial count was kept
constant at 7x107 cells/mL. The detection experiments with these samples were carried
out in the same manner as with pure EC.

4.3.5 Sample preparation for Electron Microscope

To prepare this sample, a tapered fiber was immobilized with anti-EC as

described in section 4.3.3. The antibody-immobilized taper was immersed in an EC
solution for 1h. Subsequently, the taper surface was rinsed 5 times with PBS, pH 7.4 and
air dried, and cut to fit onto a SEM sample holder. The taper was attached to the sample
holder using double-sided SEM tape and the holder was then dried in an oven overnight
at 70oC. Once the sample was dried, it was gold-sputtered. Pictures of the sample were
obtained using an Environmental Field Emission Scanning Electron Microscope (ESEM),
Model XL30 ESE-FEG (FEI, OR).

81

4.4 Results and Discussions

4.4.1 Tapered Fiber characterization

Optical characteristics of fabricated tapers were obtained by comparing

transmission in air and in water. A sample response is shown in Figure 4-4 for a fusion
drawn taper. The refractive index of the medium surrounding the taper is 1.0 for air and
1.33 for water. As shown in Figure 4-4A, the transmission decreased by 56% when water
was added. The diameter of the waist used was 7 m and the total length (Lc+Lw+Ld) was
approximately 700m. Interestingly, the transmission did not always decrease when
water was added. In Figure 4-4B, the taper with a slightly larger at the waist, 7.9
microns, and longer exhibited exactly the opposite behavior. Both of these fibers were
fabricated in the fusion splicer. Table 4-1 shows the dimensions of several similarly
fabricated tapers and their change in transmission when water was injected. These results
show that when water was added, the transmission may increase, decrease or remain
nearly constant.

82

2500

1,600
Air

Water

Transmission [counts/s]

Transmission [Counts/S]

2000
1,200

800

Wate r

400

0
420

1500

Air

1000

500

0
440

460

480

500

520

Wave le ngth [nm ]

420

440

460

480

500

520

Wavelength [nm]

Figure 4-4 Comparison of transmission spectra in air and in water. Fabricated tapers were mounted on a
fiber holder and transmission through them were measured using a 470 nm light source. DI water was
added to the sample chamber and the resulting spectrum was compared to the spectrum in air. Panel A:
Diameter of the taper was 7.0 m. Overall length of taper was 700 m. Panel B: Diameter of the taper was
7.0 m. Overall length of taper was 880 m. In many tapers upon addition of water transmission decreased
(example: A) while in others transmission increased (example: B). This behavior is very strongly dependent
on taper geometry.

4.4.2 EC Detection in aqueous samples

An antibody-immobilized tapered fiber (diameter of 6 m) was mounted in a fiber

holder and EC (concentration 7x107 cells/ml) was injected into the analyte chamber. The
light transmission (470nm) was recorded during pathogen attachment.

Figure 5-5A

83
shows the response when EC was first injected. Immediately upon addition of the EC
sample, there was a rapid increase in transmission due to the change in refractive index of
the medium. Subsequently, a gradual and exponential decrease in transmission occurred
due to EC attachment to the sensor surface.

Cells that attached to the antibody

immobilized on the taper surface absorb light from the evanescent field of the fiber, thus
reducing transmitted light. When the attachment had reached equilibrium, no further light
can be absorbed and the magnitude of transmission reached a constant value.
In order to visually verify that EC attachment to sensor surface, the tapered fiber
was examined in an electron microscope. A micrograph showing EC attachment can be
seen in Figure 4-6.

The micrograph shown in Fig. 4-6A was obtained at 60 min.

exposure with EC at 7 107 cells/mL. One notes that the surface coverage of sensor
surface is nearly complete, and that many cell fragments (less than 1 microns) can be
seen. At the same time, two whole bacteria are seen that appear to be well attached to
surface in Fig. 4-6B. The antigen from the vendor contained both whole cells and cell
fragments. The micrograph in Fig. 4-6A shows these cell fragments.

4.4.3

E. coli Release

The antigen attached to the sensor may be released by altering the pH as the
antibody-antigen binding is pH-dependent. In the present application, the sensor with EC
attached was subjected to a glycine-HCl/ethylene glycol buffer (pH 1.7). The response
(see Figure 4-5B) showed an increase in transmission, which is opposite to the
attachment response. The increase occurred because cells released into the bulk no longer
absorbed the evanescent light, therefore an increasing amount of light was transmitted.

84
When cells released reached an equilibrium, the amount of light transmitted did not
increase any further, causing the transmission to reach a constant value. Comparison of
the two panels in Figure 4-5 indicates that the transmission change due to attachment and
release are equal in magnitude, but opposite in direction. The change is approximately

Change in Transmission [A U]

5% of the original intensity.

A
200

100

Change in Transmission [A U]

10

15

B
200

100

0
0

10

15

Time [min]

Figure 4-5 Detection and release of EC on antibody immobilized tapered fiber. EC detection and release
experiment was carried out using a 8.8 m diameter tapered fiber. The antibody immobilized taper was
soaked in PBS before antigen containing solution was injected into the analyte chamber for attachment.
After complete attachment, release buffer (glycine-HCl/ethylene glycol buffer, pH 1.7) was injected into
the chamber to release attached antigen. Transmitted light was measured using a CCD detector. AU is
arbitrary unit of light.

85

Figure 4-6 SEM of EC attached to the antibody immobilized tapered fiber. Panel A: Image of a tapered
fiber with EC cells and their fragments attached to the surface at time= 60 min. Panel B: At a slightly
higher magnification, one can see EC cells attached to the surface in a different section of the fiber. The
fiber was sparsely populated with EC.

86

4.4.4 Effect of EC concentration on sensor response

The tapered fiber response for various EC concentrations is shown in Figure 4-7
for two different tapered fibers with waist diameters of 10.2 and 12.3 m. The trend of
the response is not much different from that of Figure 4-5, for the 10.2 m fiber (Panel
A), while for the 12.3 m fiber (Panel B), the response is exactly the opposite, but
somewhat similar in magnitude. As was mentioned earlier, the two different responses
are due to the geometry of the taper. These two fibers showed opposite responses when
one compared their transmission characteristic in air in relation to that in water. More
significantly, the effect of concentration on the magnitude of change in transmission has
an inverse behavior. That is, total steady state change at 1 h was higher for lower
concentration (70 cells/mL) compared to a more concentrated one (7,000 or 70,000
cells/mL).
The results show a sharp increase in transmission before it starts to decrease
gradually as the concentration increases. Normally, one would have expected to see a
steady decrease in transmission with increase in concentration. Such a behavior occurs
with homogeneous analytes (with no specific attachment to the surface of the taper)
around the taper. With particulate matter, such as the pathogenic bacteria, that attach to
the taper surface in our experiments, the behavior appears to show a dichotomous trend.
This can be explained as follows. When the concentration is very low, a very small
number of pathogen attach to the surface. Such small concentrations are insufficient to
produce significant absorption of the evanescent light. At the same time, these few
pathogens on the surface of the waist with a refractive index (~1.33) much less than that

87
of the cladding, act as a quasi periodic grating on the surface. Such a grating can induce
coupling of modes

93-95

. This process results in the transfer of power in the higher order

modes to the lowest order mode, leading to an increase in its power. This is the cause of a
sharp increase in transmission at very low concentrations. The requirement for such
coupling is that the spatial periodicity of the attached bacteria should match the
difference in propagation constants of the modes.

This will be easily met at very low

concentrations. As more and more bacteria attach, the grating structure vanishes, the
bacteria become predominantly absorbent leading to a steady decrease in transmission.
As the taper surface becomes fully covered with the pathogen, a saturation point is
reached and the transmission reaches a constant value. In Figure 4-7, one notes that the
extent of coverage on the surface depends on the concentration in the liquid sample.
Higher the concentration, lower is the transmission. The attachment of cells, as noted in
Fig 4-7, causes decrease or increase in transmission through the taper. The direction of
change is always the same for a particular taper, as is established in Figure 4-7 with
repeat experiments.

Change in Transmission [Counts/S]

88

4,000

3,000

70 pathogen/mL

2,000
7,000 pathogen/mL

1,000

PBS

0
0

10

15

20

25

30

Time [min]

Figure 4-7 Effect of pathogen concentration of sensor response. Panel A: Change in transmission for the
attachment of various concentration of EC for antibody immobilized taper fiber with waist diameter of 10.2
m. Panel B: Change in transmission for the attachment of various concentration of EC for antibody
immobilized taper fiber with waist diameter of 12.3 m. The change in transmission obtained were in the
opposite direction compared to panel A. Such a reversal in transmission pattern is probably due to the
difference in actual shape of the taper. Detector was a PMT (PTI 701).

4.4.5 Loss of sensitivity with use

A 9.3 m fusion splicer drawn fiber was used for EC detection repeatedly by
adding pathogen and release buffer in an alternate fashion, with PBS rinses in between to
clean and maintain the antibody at a pH of 7.4. The response of the fiber is given as
changes in transmission, and is plotted in Figure 4-8 as a function of use. Note that the
magnitude of total change decreased with each successive use. The change of
transmission decreased from 118 AU in the first use to less than 30 AU in the fourth use.
This suggests that either antibody was lost from the surface or the binding affinity was
reduced. One possible explanation for this behavior is that acidic conditions of the buffer
altered the conformation of the antibody and caused it to lose activity. The ethylene
glycol in the buffer was selected for its ability to neutralize the electrostatic attractions
between the antigen and the antibody, thereby weakening the non-covalent bonds

89
between them. It is of interest to determine the number of use-cycles one can obtain.
The data in Figure 4-8 shows that multiple uses may be possible, and that the release

Change in Transmission [A U]

buffers may need to be optimized to maximize re-use.

Use #4

-20
-40
Use #3

-60
-80

Use #2

-100
-120

Use #1

-140

10

Time [min]
Figure 4-8 Change in Transmission vs. Time for four cycles of sensor use.

4.4.6 Selectivity of antibody to immobilized fiber

A flame drawn fiber with 9.3 m waist diameter and 2 mm length taper was used
to check the selectivity of antibody immobilized taper. Pathogen samples were prepared
from a solution of EC (7x107 cells/mL) and a wild strain (JM101). Figure 4-9 shows the
responses of three samples: 0%, 50% and 70% by volume of EC mixed in with JM101.
The taper was soaked in release buffer (HCl, pH 2.3) for 45 minutes after each sample
was added and then soaked in PBS for 10 minutes to bring the pH of attached antibody
back to 7.4. For the wild strain (0% pathogen), the results indicate a small change(-700
AU) in transmission. The change in transmission is likely just due to changes in

90
refractive index as the solution was slightly cloudy to the naked eye due to the presence
of the non-pathogenic variant. Although these cells did not attach to the antibody on the
surface, they still would have caused a change in the refractive index. However, as the
concentration of EC increased, they attached to the antibody, absorbed light, and results
in decrease in transmission as seen in Figure 4-9. Intermediate concentrations (10%,
20%, 80% and 90%) were used, but are not included in the figure for clarity of
presentation. In Figure 4-9, the decrease in transmission is proportional to the increase in
concentration of EC in the sample solution. Also, the addition of the 100% pathogen
resulted in a transmission that followed close to the 70%, and reached a constant value in
one hour. The results in Figure 4-9 show that by immobilizing antibody to the antigen,
one is able to distinguish the presence of target species. The noisy response is probably
due to the presence of the non-pathogenic variant that does not bind, but does seem to
affect the evanescent field, albeit transiently.

91

Change in Transmission [A U]

1,000
0

0%

-1,000
-2,000

50%

-3,000
-4,000

70%

-5,000
0

10

20

30

40

50

60

70

Time [min]

Figure 4-9 Effect of pathogenic and non-pathogenic EC mixture. Percent above refers to % of cells that are
pathogenic. Experiment performed on a 9.5 m diameter tapered fiber to check selectivity of immobilized
antibody to its complementary antigen. When 0% pathogen (100% wild strain JM101 ) was injected
around the taper, there was no significant change in transmission through the taper over time. When a
solution containing an EC and the wild strain is added to the solution, the cells bind to the antibody thus
resulting in a decrease in transmission through the fiber. As the concentration of EC is increased to 50%
and 70%, there is a greater binding of pathogen to the antibody on the surface and thus greater change in
transmission occurs. AU is arbitrary unit of light.

4.0 KINETICS OF PATHOGEN ATTACHMENT FOR VIRGIN SENSORS

The rate of attachment of the antigen EC to the tapered fiber surface depends
directly on the bulk concentration in sample, and the number of available attachment sites
on the surface. Let N be the maximum number of attachment sites and n be the number
of sites that are occupied at any time. The bulk concentration of pathogen changes during
a detection experiment. A balance on the antigen gives:
V C0 n
Cb = s b

Vs

(4 4)

92
where Cb0 is the initial pathogen concentration and Vs the sample volume.

When

Vs C b0 >> N , the above equation can be approximated as C b = C b0 . Thus:

dn
= kC b ( N n )
dt

( 4 5)

which results in:

n
= 1 e kCbt
N

(4 6)

where, k is the attachment or binding rate constant and t is time. If I is the intensity of
light detected at the distal end of the fiber, and if we assume as an approximation that
changes ( I ) in transmitted light is proportional to the number of attached pathogen,
I = n

(4 7)

where is a sensitivity factor that depends on the fiber geometry and fiber surface
preparation. When all the N sites are occupied, the total change in transmitted light is:
I * = N . Thus, Eq. (4-6) can be re-written as:

I = I * (1 e kC b t )

(4 8)

*
Since ( I ) was measured as a function of time, a plot of ln ( I * I ) vs. Cbt

should result in a straight line, and the value of binding rate constant (k) can be
determined from the slope. All detection experiments were analyzed by the method
described above, and a sample set of results are shown in Figure 4-10, and a summary of
kinetics obtained with the same tapered fiber is given in Table 4-2. These results are
typical of several detection experiments done in our laboratory. In Figure 4-10, a sample
analysis is given for a 8.8 m fiber. Note that the response exhibits two distinct rates, an

93
initial rate that is more rapid than the rate that sets in after the initial 10-15 minutes. As
shown in Table 4-2, the initial rate constant varied in the range of 1.2x10-8 min-1
(cells/mL) -1 to 1.1 x10-9 min-1 (cells/mL) -1, while the final rate constant ranged from 2.5
x10-9 min-1 (cells/mL)

-1

to 6.1 x10-9 min-1 (cells/mL)

diameter 8.1 and 12.3 m.

-1

for the two tapers of waist

It is important to point out that each of the tapered fiber

sensors (fibers A, B, C, ... F) was immobilized separately and the experiments were
carried out over a six-month period. While the range of initial rates varied by almost an
order of magnitude, the cell attachment rate showed a much smaller variation, within a
factor of 2. It is suggested that this variation is in part due to the taper geometry (the
parameters: La, Lw, Ld and Dw), and to a lesser extent due to immobilization variability.
The two distinct rates observable in all experiments appear to be due to the nature
of the antigen used. Upon closer examination of the antigen provided by the vendor
(KPL, Inc.), it became clear that antigen sample is a mixture of cell fragments and whole
cells.

The electron micrographs given in Figure 4-6 provide a visual of the cell

fragments. We believe that the more rapid initial rate is due to the cell fragments. These
fragments are significantly smaller in dimension, and thus have a higher transport rate to
the sensor surface compared to the larger whole cell.

The slower or final rate is

characteristic of whole cell attachment. Larger particulate matter has a slower transport
rate.
In order to investigate the attachment kinetics further, the data of repeat-use
experiments (Figure 4-8) was analyzed for attachment kinetics, and the results are
summarized in Table 4-3. It was suggested earlier that there was a loss of sensitivity of
the fiber may be due to loss of antibody activity. The results in Table 4-3 indicate that

94
the initial rate falls much closer together (1.4 x 10-8 to 0.8 x 10-8 min-1 (cells/mL) -1 )
while the final rate variability remained approximately the same. While a detailed study
of antibody activity on sensor surface is needed to obtain a more accurate picture, it
suffices to note that cell attachment rate (final rate) is in the range of 2 to 5 x 10-9 min-1
(cells/mL) 1. In a parallel investigation in our laboratory with cantilever sensors, we
immobilized the same antibody and antigen on glass borosilicate glass, and the kinetics of
attachment obtained ranged from 3x10-9 min-1 (cells/mL)-1 to 5x10-9 min-1 (cells/mL)-1 96.
The closeness of the results obtained by the two independent experimental methodologies
provides stronger support for the validity of the two sensing platforms.

ln[(I* I)/ I*)]

0
Final slope =3.8E-08 min

-1

-2
-1

-1

Initial slope =1.2E-08 min (pathogen/mL)

-3
-4
0E+0

1E+8

2E+8

3E+8

4E+8

5E+8

Cb t [min (pathogen/mL)]
*
Figure 4-10 Rate analysis of ln ( I I ) vs Cb*t.
I *

6E+8

7E+8

95
6. CONCLUSIONS

The tapered fibers with waist diameters in the range of 4 to 12 microns, and with
antibody immobilized on its surface provided excellent response to the antigen, E. coli
0157:H7. Of special mention is that such sensors show responses even when antigen
concentration is as low as 70 cells/mL. Tapered fiber sensors showed high selectivity to
the complementary antigen when contaminating wild strain was present in the same
sample. A first order kinetic model of binding kinetics was proposed, and showed good
agreement with experimental observations. The specific binding rate constant for whole
cells was found to vary in the range of 2.5 x10-9 to 6.1 x10-9 min-1 (cells/mL) 1.

96
Table 4-1 Transmission of light through six different tapered fibers fabricated using a fusion splicer. The
percentage change in transmission in water compared to that in air depends on the geometry of the taper.

Fiber

Lc, m

Ld, m

1
2
3
4
5
6
7
8

540
560
700
430
640
440
340
430

470
530
440
580
590
550
340
430

Appx. Lt,
m
20
20
20
20
20
20
20
20

Dw, m
9.5
9.2
7.6
11.2
9.4
9.7
7.0
7.9

Transmission in Water /
Transmission in Air
2.63
0.99
1.56
0.98
0.98
0.97
0.56
1.22

Table 4-2 The rates of attachment (k) of EC (7x107 cells/mL ) calculated from attachment on six different
flame-drawn fiber tapers.

Fiber

L c,
m

Ld , m

Lw, m

Diameter
m

[min-1(pathogen/mL)-1]

kinitial

A
B
C
D
E
F

620
440
630
540
620
660

540
590
580
490
590
430

20
20
20
20
20
20

8.1
11.1
9.3
9.1
8.8
12.3

1.1X10-9
1.4X10-8
7.7X10-9
7.5X10-9
1.2X10-8
1.4X10-8

kfinal

[min-1
(pathogen/mL)-1]

3.6X10-9
5.2X10-9
2.5X10-9
3.2X10-9
3.8X10-9
6.1X10-9

Table 4-3 Rates of attachment (k) obtained from four use-cycles of EC (7x107 cells/mL) attachment shown
in Figure 4-6.

Use Cycle
1st
2nd
3rd
4th

kinitial
[min-1(pathogen/mL)-1]
8.0X10-9
1.0X10-8
1.4X10-8
1.4X10-8

kfinal
[min-1(pathogen/mL)-1]
2.4X10-9
2.0X10-9
3.8X10-9
5.2X10-9

97

5.0 REAL-TIME MONITORING OF BOVINE SERUM ALBUMIN AT

FEMTOGRAM/ML LEVELS ON ANTIBODY-IMMOBILIZED TAPERED
FIBERS

5.1 Introduction

One of the challenges in the field of protein science is the real-time observation of
protein-protein interactions at sub-picogram/mL concentrations without extensive sample
preparation or labeling. Real-time observation provides specific information such as
binding specificity, concentration effects, binding kinetics, and affinity 97. Determination
of protein characteristics, particularly at low concentrations, has significant applications
in biotechnology, medical diagnostics, drug discovery, and bioterrorism monitoring.
Previous work in this field used the Surface Plasmon Resonance (SPR) method to
observe and characterize the protein behavior 97. SPR based methods rely on the changes
in refractive index at the substrate surface where the protein binding occurs. The lower
limit of sensing in some of the typical devices based on SPR is in the ng/mL range

97

Like SPR based devices, tapered fiber optic based devices respond to changes in the
evanescent field arising from the alterations in refractive index due to protein binding
close to the taper surface 83, 98. Since the evanescent field dimensions are on the order of a
few nanometers to a few hundred nanometers, extremely small refractive index
perturbations on the sensor surface due to protein binding can lead to significant changes
in the optical throughput

4, 99

. When the surface of the tapered fiber is prepared with

specific proteins or functional groups, target molecules bind specifically to the modified
surface. This causes modulation of refractive index at the surface leading to changes in
optical throughput 100. If the interaction region is made small, such as in a biconical taper,

98
opportunity exists to observe binding at an extremely dilute concentrations of the analyte
50, 66, 101, 102

. Thus, tapered fibers have great potential to monitor protein interactions at

extremely low concentrations.

In this work, we immobilized an antibody to BSA and used biconical tapers at
1550 nm to observe binding of BSA and the antibody. Our results show sensitivities
down to a concentration of 10 fg/mL.

5.2 Physics of Sensing in tapered fibers

It is known that tapered fibers exhibit excellent sensing capabilities due to the
existence of a strong evanescent field in the tapered region. By having a protein layer
bound on the surface of the tapered region, the characteristics of the light propagating
through the tapered region and consequently, the optical throughput can be altered.
Regardless of whether the protein is absorbing or non-absorbing at the operating
wavelength, the changes in refractive index surrounding the tapered fiber region leads to
changes in optical throughput

4, 83, 98-100

. The interaction of light with the protein is

dependent on the strength and penetration of the evanescent field in the tapered region.
Typical protein molecules are 3 10 nm, and thus, when attached to the surface would be
in the evanescent field

4, 99

. Since the evanescent field decays exponentially from the

surface, the optical throughput is more sensitive to the protein bound to the surface than
the protein in bulk solutions.
Due to the inherent sensitivity of the tapered fiber towards changes in refractive
index, there have been past attempts at looking at protein interactions using tapered
fibers. Most if not all of these methods use ultraviolet (UV) to visible wavelengths, and,

99
most depend on labels attached to the fiber or the protein. For example, BSA was
previously measured using fiber-optic evanescent wave and dye based sensor and the
detection limit was 20 g/mL

55

. We postulate that improved label-free sensing can be

achieved with 1550 nm for the following reasons: 1) At 1550 nm, waist diameters of the
fiber in the range of 8 - 12 m produce sufficiently strong evanescent field for detection,
2) such taper diameters are easily fabricated compared to the smaller waist diameters (~1
m) required for optimal operation at 470 nm

4,

99

, 3) use of a standard

telecommunication single mode fiber at 1500 nm eliminates mode excitation problems

observed at shorter wavelengths 8. An additional practical benefit of using longer
wavelengths is the availability of stable, low cost lasers and detectors, particularly in the
1300-1500 nm range, commonly used in telecommunications.
Thus far, the authors are unaware of any reports of using longer wavelengths in
the context of tapered fiber geometry for label-free monitoring of protein interactions.
However, other optical methods have been used to detect proteins with limited success in
term of concentration levels. In a study on the resonance of whispering gallery modes, the
adsorption of BSA on amine-modified silica glass microparticles was measured for
concentrations of 1.5 M

103

. Protein studies have also been conducted using optical

interferometer geometries and have had success at the detection of Protein G on IgG with
a limit of 750 fg/mm2 . Such low concentrations of analytes correspond to refractive
index changes of on the order of 10-4

104

. With the well known optical properties of

tapers, namely, enhanced interaction at longer wavelengths

4, 83, 99

, and ability to detect

small changes in index using optical techniques, the possibility exists that TFOBS
possesses the ability to detect very low concentration levels of proteins from the protein

100
adsorption and the consequent changes in index from the presence of analytes in the
tapered region.

5.3 Materials and Methods

5.3.1 Fiber tapering

Silica single mode optical fibers (Corning Glass Works, NY, attenuation at 1300
and 1500 nm of 0.36 and 0.26 dB/m, respectively) with a core diameter of 8 m and an
overall diameter of 125 m were used for all the experiments. The polymeric sheathing
was removed completely over a distance of 5 cm by a wire stripper following a 10minute immersion in acetone.

The tapers were fabricated using the programmable

Ericsson FSU 975 fusion splicer, by applying electric current via a pair of electrodes for
up to 60 seconds while the fiber was pulled automatically. Current levels of 3 to 13 mA
and pulling times of 2 to 11 s were used to produce tapers of varying taper diameters and
lengths.

5.3.2 Permanent attachment of the tapered fiber to a fiber holder

The fiber was then fixed using epoxy (Cotronics, NY) between two pieces of
plexiglass (Figure 5-1a). The thicker piece of plexiglass has a 200 L sample chamber
which is exposed to the taper. Analytes were introduced into the chamber either by
pipeting or pumped through a tube by a peristaltic pump (Fisher Scientific).

101
5.3.3 Permanent connectorization of tapered fibers

Approximately 10 cm of protective tubing was inserted onto the fiber on both

sides of the fiber holder. The tubing was then held in place using epoxy. The ends of the
fiber were connectorized and polished using the method described by the manufacturer
(Thorlabs, NJ).

5.3.4 Experimental arrangement

Using the experimental setup shown in Figure 5-1b, light transmission through the
fiber was measured. The source used was a 1550 nm DFB laser (Anritsu GB5A016) with
maximum rated output power of 20 mW and peak wavelength of 1546.4 nm. In our
experiments, the laser was typically operated at 5 mW, well below its peak power. Since
the fibers were already connectorized, a FC-FC mating sleeve (Thorlabs, NJ) was used to
connect the fiber to the light source, and the other end of the fiber was connected to an
optical spectrum analyzer (ANDO AQ-6310B).

102

(a)
Tubing

Sample addition/removal port

Epoxy

FC-Connector
Tapered region

Sample holder

(b)
Thorlabs TED200
Temperature controller
u
er
rits 16
An 5A0 Las
GB 0nm

5
15

Thorlabs LDC202 Laser

diode controller

sensor

Ando
AQ-6310B
Spectrum
Analyzer

FC-FC adapter
Figure 5-1 (a) Experimental arrangement. The fabricated taper was fixed to the base with epoxy. The two
ends were connectorized with FC adapter. (b) Cross-sectional view of the fiber holder. The sample chamber
has a 200 L volume. The two ports on the topside are used for sample addition and removal using a
standard 200 L pipetter.

5.3.5 Antibody immobilization

The antibody immobilization method used in this study was adapted from
Bioconjugate Techniques with modification for the fiber surface and geometry

92

. The

tapered region of the fiber was prepared prior to every sensing experiment using the
following method: It was cleaned by 0.5 M sulfuric acid, for 30 minutes, Piranha solution

103
(hydrogen peroxide and 6 M sulfuric acid in a volume ratio of 3:7) for 10 minutes, and
hot 0.1 M sodium hydroxide for 10 minutes. Following each cleaning step, the sample
holder and fiber were rinsed several times with de-ionized water. The cleaning procedure
produced reactive hydroxyl groups on tapered surface. The tapered surface was then
silanylated with 0.4 % 3-aminopropyl-triethoxysilane (APTES; Sigma-Aldrich) in deionized water at pH 3.0 (adjusted by sulfuric acid) and 75 oC for 2 hours. The fiber was
then dried overnight in an oven at 50 oC. The APTES reaction produces a free amine
group at the glass surface, which becomes available for further reaction with carboxylic
groups in the antibody to form a peptide bond. The polyclonal antibody to BSA (antiBSA; Sigma Catalog # B1520) contains carboxyl groups which were activated using 1ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC; Sigma-Aldrich) and stabilized by
sulfo-N-hydroxysuccinimide (Sigma-Aldrich).

EDC converts carboxylic groups into

reactive unstable intermediates susceptible to hydrolysis. However, Sulfo-NHS replaces

the EDC producing a more stable reactive intermediate which catalyzes reaction with
amine groups. Covalent bonding of this intermediate with the silanylated taper fiber
surface was carried out at room temperature for 2 hours. During this time period,
transmission through the fiber was monitored, which provides useful data on the extent of
antibody immobilization.

5.3.6 Absorption Characteristics of BSA and antibody to BSA

A BSA stock solution (1 mg/mL) was prepared in 10 mM phosphate buffered

saline, pH 7.4 (PBS). Lower concentrations (1 g/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL,
100 pg/mL, 10 pg/mL, 1 pg/mL, 100 fg/mL) were prepared in PBS (pH 7.4) by serial

104
dilution. The absorption of BSA was measured in a cuvette using the same 1550 nm laser
that was used in the taper detection experiments. A single mode FC fiber was used to
connect the laser diode to the input of the cuvette holder (Ocean Optics, FL). Another FC
fiber was connected from the output end of the cuvette holder to the same spectrum
analyzer as the one used in sensor experiments. Transmission characteristics of various
BSA solutions, made by serial dilution in the concentration range of 10 pg/mL to 1
ng/mL, were measured. In the same way absorption of antibody to BSA at 500 g/mL
was also determined.

5.3.7 Protein attachment and release experiments

Solutions of BSA from 10 fg/mL to 1mg/mL were prepared. Only one

concentration was used in each experiment of attachment and release. After the taper
surface was immobilized with the antibody, it was rinsed with PBS, and 200 L of BSA
was injected into the sample compartment and the transmission of light through the fiber
was recorded. After 30 minutes, the BSA solution was removed and the sensor was
rinsed with PBS. Then, PBS adjusted to a pH of 2 by H2SO4 was added to release the
attached BSA. The acidic PBS weakens the binding of BSA to the antibody because it
changes the conformation of the protein, thus the protein is released from the surface.
After this the fiber surface was regenerated from step 1, which is the cleaning sequence
with acid, base, and modification by APTES. Once the fiber surface was silanized the
cycle of antibody immobilization, BSA attachment, and BSA release was repeated.

105

5.3.8 Multiple step attachment experiment

The multiple step attachment experiment was performed on three fibers with up to
five different solutions of BSA, whose concentration range from 100 fg/mL to 10 ng/mL.
The experiments were done initially starting with initial concentrations of 100 fg/mL. In
the last experiment, the initial concentration was set at 10 fg/mL. The BSA solutions
were added in order from the lowest to the highest concentration, with removal of each
sample after collection of data for up to 40 minutes, and then addition of the next
concentration.

5.4 Results and Discussions

5.4.1 Tapered Fiber characterization

Several tapers were fabricated during the course of the current study. In Figure
5-2, typical optical micrograph of a taper obtained is shown. The sensors typically have a
convergent length of 200 500 m, waist of 50 200 m, and divergent length of 500
1200 m. The waist diameters ranged between 6 and 12 m. The tapers were tested for
continuity and sensitivity to the presence of liquids by monitoring the optical throughputs
when the waist was dry and when it was wetted with PBS.

106

100 m

Figure 5-2 Micrograph of a typical tapered fiber taken using a light microscope camera.

5.4.2 Immobilization of Antibody to BSA

Reaction of anti-BSA with the amine-modified taper surface was monitored over
the course of two hours by measuring the transmission through the fiber. This was
performed with most of the tapers that were subsequently used in BSA detection
experiments. Two typical transmission responses during anti-BSA immobilization are
shown in Figure 5-3. The temperature was controlled by an incubator and was maintained
at 30 +/- 0.5 0C. It is seen that the transmission gradually increased and reached saturated
levels. Saturation usually occurred within two hours, and in Figure 5-3 the responses are
shown for up to 80 minutes because of the attainment of steady transmission. During the
immobilization, bonding of antibody to the fiber surface resulted in a small increase in
fiber diameter as well as change in refractive index at the taper surface. This type of
surface reaction is analogous to adsorption of a protein onto a surface and can be
modeled with the Langmuir model of adsorption. The antibody is about 13 x 6 nm, thus
the taper diameter is likely to change non-uniformly by about 12 to 26 nm, and such a
change is expected to alter the transmission characteristics. The measurements are shown
as change in transmission following sample addition. From Figure 5-3 one can see that
the total change was 5 dB for one experiment, and 1.5 dB for the other. The difference in
magnitude between the two responses resulted because the fiber surface was cleaned

107
thoroughly between experiments, and antibody immobilization does not result in the
same spatial coverage of antibody on the fiber surface every time. However, Figure 5-3
shows that the antibody immobilization follows the Langmuir adsorption model closely
in both experiments, as discussed later in section 5.5.

40

4.5

35

Experiment 1

30

Temperature

3.5
3

25

2.5

20

15

1.5

10

1
Experiment 2

Antibody

0.5

Temperature (C)

Change in Transmission (dB)

0
0

20

40

60

80

time (min)

Figure 5-3 Transmission change in dB vs. time for antibody immobilization in two separate experiments is
plotted. Temperature is held constant at 30 oC 0.5 oC as indicated. The experiments were performed in
an incubator to keep the temperature stable.

5.4.3 Absorption characteristics of BSA and antibody to BSA

While the absorption of BSA in the UV wavelength region is well known

105

, its

absorption behavior in the near-IR region has not been characterized. The absorption of
BSA and anti-BSA at 1550 nm was measured in a cuvette of 1 cm optical path using the
same laser source that was used in the sensing experiments. The concentration of BSA
used ranged from 10 pg/mL to 1 ng/mL. The concentration of anti-BSA used was 0.5
mg/mL. Upon introduction of the sample, the change in transmission stayed within noise

108
levels of the instrument, 0.1 dB, compared to transmission in PBS, pointing to negligible
or no absorption. This confirmed our belief that BSA and anti-BSA do not absorb at 1550
nm, in the concentration range tested with an optical length of 1 cm. Since optical path in
the taper is much shorter than 1 cm, it is reasonable to conclude that changes in
transmission observed in TFOB experiments are due to refractive index effects, and that
absorption effects are absent.

5.4.4 Detection of BSA Attachment and Release

Both the attachment and release responses are shown in Figures 5-4 for
concentrations of 10 pg/mL. When BSA was injected into the sample holder,
transmission decreases due to change in surface refractive index caused by the presence
of BSA. A low pH PBS was then added to change the conformation of the proteins so as
to loosen the binding between the antibody to BSA and the BSA. When BSA is loosened
and released from the antibody, transmission increases back almost to the starting value.
Similar experiments were performed with other tapers using different initial
concentrations, and we conclude that experimental results are reproducible with the
different fibers. Similar to the antibody attachment to the fiber surface, the binding of
BSA and anti-BSA appears to follow the Langmuir adsorption model as discussed in
section 5.5. However, here the time scale for saturation is about 10 minutes whereas it
was about 1 hour for the antibody.

109

Change in Transmission (dB)

0.5
0.4

Release

0.3
0.2
0.1
0
-0.1

10

15

20

-0.2

25

30

35

Attachment

-0.3
-0.4
Time (minutes)

Figure 5-4 BSA attachment and release of 10 pg/mL sample. The attachment response was obtained when
the tapered region was first exposed to 10 pg/mL of BSA. Data was collected for 30 minutes, rinsed with
PBS, and then the tapered region was exposed to pH2 PBS. The low pH denatures the antibody and
changes its conformation, resulting in the weakening of the bond between the antibody and BSA, and
causes BSA to be released. Consequently, the transmission changes due to attachment and release are in
opposite direction and have approximately the same magnitude.

5.4.5 Multiple step attachment experiment

Having established that both antibody immobilization, and subsequent attachment

and release of the antigen BSA can be measured, a series of experiments was conducted
to examine the lower limit of detection.
In three separate experiments, the taper was exposed to 100 fg/mL of BSA for 30
to 40 minutes. The transmission was recorded every five minutes and the raw data is
presented in Figures 5-5. One can see that although the experiments were performed
using different fibers after thorough cleaning, the direction of transmission change is the
same, and the transmission changes are in the same order of magnitude. Based on the

110
largest change of 5 dB that resulted from 100 fg/mL, it is reasonable to assume that even
lower concentrations can be sensed.

Change in Transmission (dB)

0
-1

Experiment 1

-2
Experiment 2

-3
-4

Experiment 3

-5
-6
0

10

15

20

25

30

35

40

Time (minutes)

Figure 5-5 Attachment of 100 fg/mL of BSA to antibody-immobilized tapered fibers for three separate
experiments. In each experiment, the BSA was exposed to the antibody-immobilized taper for 40 minutes.

In fact, the ability to sense 10 fg/mL BSA was observed in one of the experiments
shown in Figure 5-5. In that experiment, the taper was first exposed to 10 fg/mL of BSA
for 30 minutes, and then exposed to higher concentrations up to 10 pg/mL, and the data
was plotted in Figure 5-6. Data was collected automatically every minute. Like the
responses shown in Figure 5-5, the transmission decreased as a function of time as BSA
attached to the antibody. The steady state value of transmission reached at each
concentration also decreased. The relationship between concentration-dependent steady
state transmission and time was indicated by connecting the steady state values reached
subsequent to each addition by a dotted line on the same graph.

111

-32.7

31
Temperature

10 fg/mL

-33.1

100 fg/mL

-33.3

1 pg/mL

29
27

10 pg/mL
25

-33.5
-33.7

23

-33.9

Transmission

21

-34.1
-34.3
50

100

Temperature (oC)

Transmission (dB)

-32.9

150

200

250

19
300

Time (minutes)

Figure 5-6 Semi-batch staircase experiment showing attachment of BSA from10 fg/mL to 10 pg/mL.
Temperature was maintained at 30 oC 0.5 oC using an incubator . Temperature variation is also shown.
The BSA solutions were added sequentially from lowest to highest upon removal of the previous solution.
The dark line with peaks represents transmission through the fiber. The peaks correspond to time instants
when the samples were introduced. The dotted line at the bottom represents the trend exhibited by the
steady state value of transmission with respect to time.

From these results, it is seen that the change in transmission is not linearly
proportional to concentration. We believe that the reason for this is that at small
concentrations, the surface of the fiber is not saturated with the antigen BSA. Estimate of
antibody/antigen surface coverage of the fiber can be made with a few simplifying
assumptions. Using a convergent length of 400 m, waist length of 100 m, divergent
length of 1000 m, and waist diameter of 12 m, the surface area is estimated as 2.8 x
o

10-7 m2. Given the diameter of gyration of each BSA molecule is 35.9 and BSA has a
molecular weight of 67 kD, it is estimated that it would require 6.9 x 109 molecules to

112
completely cover the taper surface if uniform coverage is assumed. A 0.2 mL sample
containing 10 pg/mL of BSA has enough BSA molecules to cover less than 0.26% of the
taper surface. In the ideal case it would require about 4 ng/mL of 0.2 mL BSA sample to
saturate the surface of the fiber. Due to the likelihood that antibody coverage of surface is
likely to be less than 100%, it is suggested that the concentration required for saturation is
less than 4 ng/mL.
Transmission changes observed are caused by the evanescent field interaction
with the surface layer of antigen. Once the concentration approaches ng/mL levels, the
surface of the fiber would become saturated with a single layer of BSA. Additional BSA
molecules would need to attach on top of the layer closest to the fiber surface. However,
the evanescent field magnitude decays away from the surface. Therefore the effect of any
BSA on top of the first layer result in much less change. In addition, the condition for
immobilization varies from one experiment to another. It is possible that nonlinearity was
observed in terms of transmission change based on concentration because at the lowest
concentration, the bulk refractive index is approximately the same as that of water, and
the small number of BSA moledule on the fiber surface acts as isolated points of high
refractive index perturbation. When the concentration increases to saturation point, the
fiber surface is covered with a monolayer of BSA which has a higher refractive index
than water. Thus the transmission changes that can occur at either high or low
concentration depend on the condition of the fiber surface. With improvements in
immobilization repeatability and mathematical modeling, it should be possible to obtain a
more predictable relationship between the transmission changes and analyte
concentration.

113
Another feature of the staircase experiments given in Figure 5-6 is that the
existence of variation in the rate of attachment at different concentrations. It appears that
the rate is more rapid initially, when the sensor surface is virgin, and becomes slower as
binding sites become less available, as in the case of higher concentrations of analyte.
The dotted line shown in Figure 5-6 represents the steady state values of transmission
achieved after the addition of each concentration. Based on the changing slope of this
dotted line, it appears that there is a decrease in rate of attachment as concentration
increases.

5.5 Kinetics of BSA attachment to antibody-immobilized sensors

It is useful to obtain an estimate of the kinetics of antibody immobilization and

protein attachment on antibody-immobilized surfaces, as it is useful in the design of
TFOB. The immobilization and detection responses show exponential behavior, similar
to the adsorption process often referred to as Langmuir kinetics. The Langmuir kinetics
model can be expressed as 106:

= 1 e

obs

(5 1)

where (0 1) is the fractional coverage of the reactive sites, the NH2 groups on the
fiber surface or immobilized antibody at time, t. The parameter, k obs , is the observed
binding rate constant, which depends on the bulk concentration of the reactant. We
hypothesize that the measured transmission is indicative of the antibody or BSA
attachment, and therefore express the Langmuir model as follows:

( I ) = ( I ) (1

e kobst

(5 2 )

114
where (I ) is the change in transmission at time, t , and

(I )

is the steady state

transmission change. Taking the natural log on both sides of Eq. (5) we obtain:
( I ) ( I )
ln
= k obs t

( I )

(5 3)

The above suggests that the characteristic rate constant k obs during initial time can
be determined from a plot of the left hand side versus t in Eq. (5-3) where we include
only data obtained during the first few minutes, when bulk concentration is nearly
constant. We expect the observed rate constant, kobs, to differ at different concentrations
of BSA and the antibody. Based on our data, the rate constant was found to be in the
range of 0.02-0.08 min-1 for the 10 pg/mL BSA, and 0.02-0.05 min-1 for the anti-BSA.
These values are comparable to 0.036 and 0.018 min-1, which was obtained for antiBacillus anthracis rabbit and goat antibody respectively, in our laboratory for flat glass
surfaces, at room temperature 96. In general, antibody immobilization took a longer time
(about 40 to 60 minutes) while BSA attachment was of a shorter duration, typically 30
minutes. This time frame is again comparable to reported results from our laboratories 96.

5.6 Effect of concentration of refractive index

The effect of refractive index on transmission was reported by Lukosz using

integrated optical sensors

107

. It has been suggested that there are many reasons for the

change in index caused by the presence of the analyte in the tapered fiber. The index
changes can occur from the changes from the bulk index, i.e., simply from the
concentration and volume of the analyte. Index changes also arise from the adsorption of
the analyte onto the surface. Furthermore, the index change from adsorption may change

115
the real part of the index of the cladding surface, the imaginary part of the cladding index,
or both. It has also been suggested that index changes may arise from the mechanical
stress induced by the adsorption of the molecules to the surface. We are in the process of
determining the precise mechanism for the index change and the magnitude of such index
changes.
Based on previous studies on tapered fibers it is reasonable to assume adsorption
is the primary cause of the changes in optical throughput. Our results in terms of the
saturation of the response with time and concentration clearly point to adsorption since
the responses observed by us mirror those observed by others

108-110

. Thus we conclude

that the changes in transmission is primarily arising from the change in refractive index
from surface adsorption

108-113

. It must also be kept in mind that the protein binding in

this study is highly dependent on the conditions under which antibody was first
immobilized. As such, there is a high probability of locally high concentrations of BSA
bound to parts of the taper 110.

5.7 Conclusions

Tapered fiber optic biosensors exhibited strong transmission changes at 1550 nm

when antibody binds to the surface and when the antigen (BSA) binds to the antibody.
The limit of detection of BSA was found to be 100 fg/mL. We observed the sensing
ability at 10 fg/mL as well as shown in Figure 5-6. We are currently in the process of
exploring experiments at levels lower than 10 fg/mL. The response of TFOB was found
to be about one hour for antibody immobilization, and about 30 minutes for the antigen
binding. The immobilization of antibody and binding of BSA to antibody can be modeled

116
after Langmuir adsorption kinetics, and the observed rate constants are in agreement with
other studies in literature

106

. Based on studies done on other types of sensors we

postulate that the transmission change we observed is dominated by the refractive index
change caused by adsorption on the surface of the fiber. While this study only marks the
beginning of the use of TFOBS to investigate protein-protein interaction, its response to
low concentrations leads us to believe that with improved fabrication controls it will
become a very promising platform to study protein-protein interactions. We are also
attempting to improve our handling approaches so that these sensors can be used
repeatedly.

117

6.0 MODEL PROTEIN DETECTION USING ANTIBODY-IMMOBILIZED

TAPERED FIBER OPTIC BIOSENSORS (TFOBS) IN A FLOW CELL AT 1310
NM AND 1550 NM

6.1 Introduction

Tapered fiber optic biosensors (TFOBS) have been widely investigated for the
monitoring of physical properties
17, 22, 35, 42, 58, 66

3, 88, 114-116

, chemicals

5, 30, 117-120

, biological molecules

. The use of TFOBS for detection has many applications in environmental

monitoring, drug screening, clinical diagnostics, and defense. Some of the advantages of
TFOBS include flexibility, ease of use, affordability, and ability to perform sensing using
a small amount of samples.
Previously, we showed that as low as 10 fg/mL of BSA can be detected by an
intensity-based TFOBS at 1550 nm in stagnant conditions 56, in spite of BSA being nonabsorbing at this wavelength. While the stagnant configuration provides valuable insight
on detection and sensing, the development of sensors using continuous flow is important
in enhancing sensor performance, for the following reasons: 1) it results in a more
convenient process, since sample injection can be automated, 2) flow reduces nonspecific surface adsorption, 3) intensity-based TFOBS are sensitive to the mechanical
movements caused by batch sample injection. We hypothesize that the transmission
changes of the TFOBS due to mechanical movement will be eliminated in continuous
flow mode. To date, most of the work done in flow configuration are based on SPR
sensors

121-123

or fluorescence fiber optic sensors

124, 125

because these two methods are

already well-developed for stagnant detection. There has been one recent study on the
detection of hydrocarbons in water using IR absorption spectroscopy in a flow

118
configuration

119

. Thus far, there have been no reports on the label-free detection of

proteins in flow configuration using intensity-based sensors at IR wavelengths. Based on

our success with the detection of BSA at 1550 nm in stagnant conditions, we became
interested in exploring the application of the sensor in a flow cell device. In this report,
we demonstrate the continuous detection of various concentrations of BSA starting from
1 pg/mL, and the detection of the target BSA in the presence of a contaminating protein,
Ovalbumin (OVA). All the experiments were performed using both 1310 nm and 1550
nm distributed feedback (DFB) lasers simultaneously, and in a flow configuration. To
better understand the effects of bulk refractive index changes on transmission, we first
obtained the non-specific response of the TFOBS in flowing glucose solutions.

6.2 Physics of Sensing in tapered fibers

Optical fibers are cylindrical waveguides made with silica, and consist of a core
surrounded by a cladding. The core is doped with Ge to make its refractive index (RI)
slightly higher than that of the cladding. Light propagates through an optical fiber by total
internal reflection (TIR), which occurs when light hits the core-cladding interface and is
reflected. Even though most of the light is reflected, there is a small component of light,
known as the evanescent field, which propagates in the cladding. In a standard fiber
where the cladding is much thicker than the core, the evanescent field decays to near zero
value at the fiber surface. This standard arrangement does not allow the evanescent field
to interact with the environment surrounding the cladding. However, if the fiber is
tapered down to a diameter less than the core diameter, the light is no longer guided by
the core but is guided by the cladding, and the evanescent field not only interacts with the

119
surrounding environment, but its magnitude is also enhanced in that tapered region.
When a liquid medium is placed at the tapered region, the evanescent field interacts with
the medium through absorption and scattering.
The amount of light that can be absorbed and scattered depends on the penetration
depth, which is defined as the position away from the core/cladding interface when the
light decays to 1/e of its magnitude at the surface. As discussed previously, the use of
long wavelengths results in higher penetration depth and is therefore beneficial to sensing
56

.
While the penetration depth depends only on the wavelength in a uniform fiber,

the evanescent field strength is determined by the diameter and the taper geometry in a
tapered-fiber. In a tapered geometry, mode coupling occurs at the tapered region because
of changes in the cladding index and the analyte index. The coupling causes the
magnitude of the evanescent field and transmission of the fiber to change. When a sample
is introduced to the sensor, changes in the RI surrounding the cladding of the tapered
region occur, and this change is reflected in the evanescent strength and output power. As
confirmed in a previous study, the transmission changes depend the lengths, diameters
and slopes of the contracting and expanding regions of the taper

126

. Previously, it was

established that long wavelength laser at 1550 nm is suitable for the detection of BSA 56
even though BSA does not absorb at this wavelength. It was the first reported use of such
long wavelength for protein detection, since most if not all the previous studies were
based on the use of visible or UV sources.

Here, we extend our studies to the

simultaneous use of both 1550 nm and 1310 nm, in a flow arrangement. In addition, we

120
show the detection of BSA in the presence of OVA, and determined the non-specific
response of the sensor using glucose solutions.

6.3 Materials and Methods

6.3.1 Flow Cell Apparatus

The flow cell apparatus is outlined in Figure 6-1. The chamber is a cylindrical
well 4 mm in diameter and 3 mm in depth, drilled into a 6 mm thick plexiglass (~3 cm X
5cm). The taper was positioned such that it was within the sample chamber, which was
approximately 40 L in volume. The sample chamber is sealed by attaching a 2 mm thick
plexiglass on top. Four reagent reservoirs (5 25 mL), each connected to an on/off valve,
were fed into a 4-port manifold. The output of the manifold was connected to the inlet of
the flow cell fiber holder (FCFH) using a 1.14 mm diameter polyethylene tube (Warner
Instruments Inc.) The output of the flow cell was directed to a peristaltic pump (Fisher
Scientific, Variable Flow Mini-Pump). The output of the pump was re-circulated back to
the reagent reservoirs or to waste by manual transfer. The pump was operated at a flow
rate of 0.5 mL/min in all experiments reported. The reservoirs contained antibody,
antigen, PBS, or release buffer. The reservoir containing the desired reagent was turned
on via the on/off valve while other reagents were in the off mode. All experiments were
performed in a temperature-controlled water bath maintained at 28.80.1 oC and the
temperature of the water bath was recorded throughout each experiment.

121
Re-circulation Loop

0.5 mL/min

PBS

Antigen

Release buffer

Antibody

Reagent Reservoirs

TED200

LDC202

V1

V2

V3

LDC202

1310 nm

1550 nm
On/Off
valves

TED200

V4

Y-coupler
4-port
manifold

FC Connector

Sensor Flow Cell (SFC)

outlet

Water
Bath

Peristaltic Pump
0.5 mL/min

Spectrum
Analyzer
inlet

Computer

Figure 6-1 Experimental arrangement. The two sources are connected to the input of the TFOBS by a Ycoupler. The output of the sensor was connected to the spectrum analyzer, whose input was fed into the
computer for storage and analysis. The sample chamber has a volume of 50 L. The samples are pumped
into the chamber at 0.5 mL/min from the reservoir. Temperature was maintained at 29 0.5 oC by a water
bath.

6.3.2 Optical Arrangement

Using the experimental setup shown in 6-1, light transmission through the fiber
was measured. The light sources used were a 1550 nm DFB laser (Anritsu GB5A016)
and a 1310 nm DFB laser (QPhotonics). The two sources were connected to the input of
the sensor via a 2X2 optical coupler. A FC-FC mating sleeve (Thorlabs, NJ) was used to

122
connect the sensor to the output of the coupler. The output end of the sensor was
connected to a spectrum analyzer (ANDO AQ-6310B). A LabView application on a PC
acquired the measured spectrum peak transmission at a sampling rate of one
measurement per minute.

6.3.3 TFOBS Fabrication

Single mode silica optical fibers (Corning Glass Works, NY, attenuation at 1300
and 1500 nm of 0.36 and 0.26 dB/m, respectively) with 8 m core and total diameter of
125 m were used in all experiments. The polymeric sheathing was removed completely
over a distance of 5 cm by a wire stripper following a 10-minute immersion in acetone.
The fibers were tapered using the programmable Ericsson FSU 975 fusion splicer. The
fiber was held together by clamps on both sides. Electric current was applied via a pair
of electrodes for up to 60 seconds while the taper was pulled automatically. Current
levels of 3 to 13 mA and pulling times of 2 to 11 s were used to produce fibers of various
taper diameters and lengths. Figure 6-2 is a schematic of a tapered fiber, where a is the
convergent length, b is the waist length, c is the divergent length, and d is the
diameter of the waist region. After tapering, the fiber was fixed and enclosed using epoxy
(Hardman) between the fiber holder components described in section 6.3.1.
Approximately 10 cm of protective tubing was inserted onto the fiber on both sides of the
fiber holder. The tubing was then held in place using epoxy. The ends of the fiber were
connectorized and polished using the method described by the manufacturer (Thorlabs,
NJ).

123

Figure 6-2 Schematic representation of a tapered fiber. Lengths a, b, c, are along the z-axis, while d is the
diameter of the waist region. Length of the waist region is denoted by b, convergent length is denoted by a,
and divergent length is denoted by c.

6.3.4 Response of TFOBS to liquid refractive index changes

In order to characterize optical properties of the fabricated, we measured

transmission properties under various refractive index fluids placed in the tapered region.
The transmissions at 1310 and 1550 nm were monitored and recorded simultaneously
using the spectrum analyzer and LabView program. Once the transmission stabilized in
air, de-ionized (DI) water was flowed in at 0.5 mL/min. Various concentrations of
glucose solutions were then flowed through the chamber holder containing the taper, with
de-ionize (DI) water flowed in to rinse out the taper in between glucose solutions.

6.3.5 Antibody immobilization procedure

The antibody immobilization method used was adapted from Hermanson with
modification for the fiber surface and geometry. Prior to each experiment, the taper was
cleaned with 1 M hydrochloric acid for 30 minutes, sulfuric acid for 10 minutes, and 1 M
sodium hydroxide for 10 minutes. Following each cleaning step, the sample holder and

124
taper were rinsed several times with de-ionized water. The cleaning procedure produced
reactive hydroxyl groups on tapered surface. The tapered surface was then silanylated
with 3-aminopropyl-triethoxysilane (APTES; Sigma-Aldrich) in de-ionized water for 224 hours. The fiber was then dried overnight in a vacuum oven at 40 oC. The APTES
reaction produces a free amine group at the glass surface, which becomes available for
further reaction with carboxylic groups in the antibody to form a peptide bond. The
polyclonal antibody to BSA (anti-BSA; Sigma Catalog # B1520) contains carboxyl
groups which were activated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC; Sigma-Aldrich) and stabilized by sulfo-N-hydroxysuccinimide (Sigma-Aldrich).
EDC converts carboxylic groups into reactive unstable intermediates susceptible to
hydrolysis. However, Sulfo-NHS replaces the EDC producing a more stable reactive
intermediate which catalyzes reaction with amine groups. To prepare the antibody, 0.4
mg of EDC and 1.1 mg of sulfo-NHS was added to each mL of antibody solution and
reacted for 30 minutes at room temperature. Then, 1.4 L of 2-mercaptoethanol was
added to quench the EDC. This intermediate was added to the silanylated tapered fiber
surface and covalent bonding was carried out at room temperature for 2 hours, at stagnant
conditions. At the end of antibody immobilization, Hydroxylamine was flowed in to
regenerate the carboxylic groups of the antibody. Then, 10 g/mL of OVA was flowed in
to block the amine sites not already bonded to the antibody. After the transmission
reached steady state upon adding OVA, PBS was flowed in to cleanse the chamber of any
residual OVA until transmission reached steady state.

125
6.3.6 Multi-step detection and release experimental procedure

The multi-step attachment experiment for pure BSA was performed on three
fibers with up to five different BSA concentrations, from 1 pg/mL to 10 ng/mL. The BSA
solutions were flowed into the sample chamber continuously at 5 mL/min from the
lowest to the highest concentration. Each sample solution (4mL) was re-circulated for
~30 minutes until steady state in transmission was reached. After steady state was
obtained with one concentration, the next higher concentration of BSA was pumped in, in
a re-circulation mode. This process was repeated until the highest concentration was
reached. After the attachment of the highest concentration of BSA was completed, pH 2.4
PBS was flowed in to release the proteins.
A similar multi-step experiment was performed like the one previously described,
but with an equal mixture of BSA and OVA at 1 pg/mL each. 1 pg/mL BSA was first
flowed in for 30 minutes, and then pH 2.4 PBS was flowed in to release it. After this
sequence, PBS was flowed in to rinse the flow cell and the sensor, and 10 g/mL of OVA
was flowed in to block NH2 groups so that only specific binding between the antibody
and antigen will be recorded. Then, PBS was flowed in to stabilize the transmission.
Once the transmission became stable, the 1:1 mixture of BSA and OVA was flowed in
and released.

126
6.4 Results and Discussions

6.4.1 Optical characterization of TFOBS

Prior to examining TFOBS detection response, it is useful to obtain optical

characteristics and sensitivity of TFOBS to the presence of liquid of known refractive
indices. In this study, glucose solutions of various concentrations were chosen to provide
refractive index changes. Glucose solutions varied from 1 g/mL to 0.1 g/mL and were
maintained at 28.80.1 oC by the water bath during the experiment. The refractive indices
(RI) of glucose solutions are linear to their concentrations, and are given by n = n0 + C ,
where n is the glucose solution RI, n0 is the RI of pure water, and C is the concentration
of glucose in g/100mL

127

. At 850 nm, was determined to be 1.43 103

127

. At 1310

nm, the RI of water is 1.32; and at 1550 nm, the RI of water is 1.315 128. Because glucose
is weakly absorbing at the concentration range used (with absorbance of the maximum
concentration being 0.0376 129) and has not been reported for near-IR wavelengths, we
assume that the relationship applicable is

n = 1.32 + 1.43 103 C at 1310 nm, and

n = 1.315 + 1.43 103 C at 1550 nm. Therefore, the range of concentration used here
corresponds to the RI range of 1.315 to 1.334. Based on the transmission observed with
respect to glucose concentration, we can determine whether the TFOBS is sensitive to
small RI changes.
Figure 6-3a depicts the TFOBS response to liquid refractive index changes,
determined by first flowing in DI water, followed by glucose solutions from low to high
concentrations. Transmission increased by 6.83 dB at 1310 nm and 4.68 dB at 1550 nm
when DI water was first flowed into the sample chamber and came in contact with the

127
initially dry TFOBS. Since the flow rate was 0.5 mL/min and the sample holder had a
hold-up volume of 40 L, it was expected that only 4.8 seconds were necessary for the
sample holder to fill with water. It was observed during the initial wetting of the fiber that
the signal increased to steady state within one minute, which is the minimum sample rate
of the spectrum analyzer, and consistent with the 4.8 seconds sample chamber fill time.
The sample holder was filled within 4.8 seconds, giving slightly less than one minute for
the signal to respond, resulting in immediate change from one data point to the next upon
liquid flow into the chamber. After steady state was obtained, 1 g/mL to 0.1 g/mL of
glucose was flowed sequentially, allowing sufficient time to reach steady state between
changes. No change in transmission was noted until the concentration reached 0.01 g/mL
of glucose. Having established that 0.01 g/mL is the minimum glucose concentration
required for a measurable response, the range of concentration was narrowed down to
0.01 to 0.1 g/mL, with increments of 0.01 g/mL in between each concentration. Figure 3b
shows the transmission response at 1310 nm for 0.01 to 0.1 g/mL of glucose, which
corresponds to a RI range of 1.321 to 1.334. Figure 6-3c shows the response at 1510 nm
for the same concentration range, corresponding to a RI range of 1.316 to 1.329. Figure
3d was derived from Figure 6-3b and 6-3c, and shows the change in transmission through
the TFOBS plotted against RI. From Figure 6-3d, it can be seen that the change in
transmission is significant and TFOBS behavior depends on the wavelength. At 1310 nm,
as refractive index increased from 1.321 to 1.334, the change in transmission increased
monotonically, and the sensitivity as defined by the slope of change in transmission vs.
change in RI is 2.42 dB/ 0.01 RIU. However, the change in transmission can increase or
decrease depending on the wavelength and the range of RI used, as shown by the

128
response of the TFOBS at 1550 nm, where the transmission decreased with a sensitivity
of -3.37 dB/ 0.01 RIU in the RI range of 1.316 to 1.322. Then, the transmission increased
with a sensitivity of 2.56 dB/ 0.01 RIU from the RI range of 1.322 to 1.329. It is
interesting to see that the transmission behavior between 1310 nm and 1550 nm are
similar in terms of direction and sensitivity in the RI range of 1.322 to 1.329. The
behavior of 1310 nm in the range of 1.316 to 1.322, and the behavior of 1550 nm in the
range of 1.329 to 1.334 are unknown and would be an interesting topic of study in the
future.
The previous experiments using glucose involved gradual switching of
concentrations in increments of 0.01 g/mL. It would be interesting to determine how
effective the flow cell is in switching the concentration of glucose abruptly from 0 to 0.09
g/mL. Therefore, PBS, DI water, and 0.09 g/mL of glucose were flowed through a
TFOBS in alternating fashion and in repeated cycles, and the response is shown in Figure
6-4. From this result, it can be seen that the response was reversible when one switched
from glucose to water and vice versa. Since the transmission returned back to the original
value within only 3 minutes once 0.09 g/mL of glucose was replaced with DI water, we
conclude that the flow cell did not retain residual glucose, and the flow system cleaned
out the sample chamber very effectively.

129
D

D C

D
-22

-19

Transmission (dB) at 1550 nm

-19.2

-22.5

-19.4
-23

1550 nm

-19.6

-23.5

-19.8
-20

-24

-20.2

1310 nm
-24.5

-20.4
-20.6

-25
50

b)

100

150
200
Time (minutes)
HD

-27

250
I D

300
D

G D

-27.5
Transmission (dB, 1550 nm)

Transmission (dB) at 1310 nm

a)

D
-28

E
-28.5

-29

-29.5

-30
-30.5
1310 nm
-31
0

100

200
300
Time (minutes)

400

500

Figure 6-3 a) Non-specific response of a TFOBS. A = 1 g/mL to 1 mg/mL of glucose, B = 0.01 g/mL of
glucose, C = 0.1 g/mL of glucose, D = DI water. There was no response from 1 g/mL to 1 mg/mL of
glucose, but there was a slight response starting at 0.01 g/mL of glucose. There was a large response at 0.1
g/mL of glucose. The dimension of this TFOBS in m is a = 360, b = 134, c = 500, d = 8.
b) Non-specific response of a TFOBS at 1310 nm for the concentration range of 0.01 to 0.1 g/mL glucose.
A = 0.01 g/mL, B = 0.02 g/mL, C = 0.03 g/mL, D = DI water, E = 0.04 g/mL, F = 0.05 g/mL, G = 0.06
g/mL, H = 0.07 g/mL, I = 0.08 g/mL, J = 0.09 g/mL, and K = 0.1 g/mL; all concentrations are for glucose
in DI water. The dimension of this TFOBS in m is a = 412, b = 223, c = 522, d = 8.
c) Non-specific response of a TFOBS at 1550 nm, for the same experiment as the one illustrated in b).

130
Figure 6-3 (continued) d) Change in transmission of the TFOBS in b) and c), compared to the transmission
response of DI water, vs. refractive index, based on the concentration of glucose in the 0.01 0.1 g/mL
range. The slopes of the straight trend lines m1, m2, and m3 correspond to sensitivities in dB/RI for the RI
range through which the straight lines were drawn.
D

c)-26.5

D
C

D
G

D
I

D
K

-27
Transmission (dB, 1550 nm)

-27.5

1550
-28

-28.5

-29

-29.5
0

100

200
300
Time (minutes)

400

500

4.00

Change in Transmission (dB)

d)

m3 = 242

3.00

2.00

1310 nm

1.00

0.00

-1.00

-2.00
1.315

1550 nm
m2 = 256
m1 = -337

1.32

1.325
1.33
Refractive Index (RI)

1.335

1.34

131

-18

D
-28.1
-28.3

-19
1550 nm

-19.5

-28.5
-28.7

-20

-28.9

-20.5
-21

1310 nm

-29.1

Transmission (dB) at 1310 nm

Transmission (dB) at 1550 nm

-18.5

-29.3

-21.5
-22
150

170

190
210
230
Time (minutes)

250

-29.5
270

Figure 6-4 Transmission of a non-antibody modified TFOBS. There was no difference in transmission
caused by PBS and DI water, but there was a large response caused by 0.09 g/mL glucose. After flowing in
0.09 g/mL glucose, the transmission was restored to the original value by the introduction of DI water.
Further flow of 0.09 g/mL glucose yielded the same response as previous additions. D = DI water, G = 0.09
g/mL glucose. The dimension of this TFOBS in m is a = 360, b = 134, c = 500, d = 8.

6.4.2 Response of TFOBS to antibody immobilization

In the previous section, the sensitivity of TFOBS to homogeneous bulk liquid

refractive indices was established along with fluid renewal characteristics of the flow
cell. However, the response of TFOBS to antibody and antigen on the surface may be
quite different from the response to bulk liquid RI. One reason for this is that the
magnitude of the evanescent field is the largest at the surface and decays exponentially
away from the surface. Another issue is that protein mixtures are not homogeneous near
the surface of the TFOBS due to the presence of antibodies, whereas the glucose
solutions are homogeneous everywhere. Although the response of TFOBS in bulk liquid

132
is an interesting topic, it is of little value in sensing because analytes in the bulk cannot be
attached specifically to a recognition molecule. Therefore, it is useful to determine the
response of amine-modified TFOBS to antibody immobilization.
Activated antibody to BSA was flowed into the flow cell, and the reaction of antiBSA with the amine-modified taper surface was monitored under stagnant condition over
the course of 2 hours by measuring the transmission through the fiber. Typical
transmission response obtained during the surface-immobilization step is shown in Figure
6-5a and 6-5b, for 1310 nm and 1550 nm respectively. One can see that transmission
gradually changes over the first hour of reaction. The bonding of antibody to the fiber
surface resulted in a very small increase in fiber diameter (~ 10 nm) and a change in
refractive index at the taper surface. This type of surface reaction is analogous to
adsorption of a protein onto a surface and can be described by the Langmuir model of
adsorption. From Figure 6-5a, one can see that the total change in transmission was 1.3
dB for 1310 nm and from Figure 6-5b, the change is 4.3 dB for 1550 nm, which are very
significant. The magnitude and direction of transmission change depends on the fiber
geometry and wavelength. In this case, the direction of transmission change at 1310 nm
was the opposite to that at 1550 nm. When hydroxylamine was added, a 0.3 dB change in
the transmission was observed at 1310 nm, and a 2.2 dB change was observed at 1550
nm. Since hydroxylamine caused only a modification of a carboxyl group on the antibody
that is already surface-immobilized, it is suggested that the response was indicative of
conformational change of the antibody.
It is useful to obtain an estimate of the kinetics of antibody immobilization and
protein attachment on antibody-immobilized surfaces, and compare the rates of

133
attachment to previously reported values. The immobilization and detection responses
show exponential behavior, similar to the adsorption process often referred to as
Langmuir kinetics. The Langmuir kinetics model can be expressed as 106:

= 1 ek
where

(0 1)

obs

( 6 1)

is the fractional coverage of the reactive sites at time t. The

parameter, k obs , is the observed binding rate constant, which depends on the bulk
concentration of the reactant. We hypothesize that the measured transmission is
indicative of the antibody or BSA attachment, and therefore express the Langmuir model
as follows:

( I ) = ( I ) (1

e k obs t

where (I ) is the change in transmission at time, t , and

(6 2 )

(I )

is the steady state

transmission change. Taking the natural log on both sides of Eq. (6-2) we obtain:
( I ) ( I )
ln
= k obs t

( I )

(6 3)

The above suggests that the characteristic rate constant k obs during initial time can be
determined from a plot of the left hand side versus t in Eq. (6-3). In the case of the
antibody immobilization, since the reaction occurred under stagnant condition, which
causes the bulk concentration to change, we included only data obtained during the first
few minutes, when bulk concentration was nearly constant. However, the kinetic analysis
of BSA detection was done throughout the entire period of attachment because detection
was done in flow mode, resulting in constant bulk concentration. The plot described by

134
Eq. (6-3) is shown in Figure 6-5c for antibody immobilization. Based on this plot and
those obtained from the responses of other fibers, the rate constant was in the range of
0.004-0.09 min-1 for anti-BSA, and are comparable to 0.036 and 0.018 min-1, obtained for
anti-Bacillus anthracis in our laboratory on flat glass surfaces, at room temperature

130

The large range of rates seen in anti-BSA attachment may be due to the changes in
silanization procedures, such as changing the time of immobilization from 2 hours to 24
hours in room temperature in some cases in this study.

135

a)

-27.6

HA
PBS

-27.8
AB
Transmission (dB, 1310 nm)

-28
-28.2
-28.4
-28.6
-28.8
-29
-29.2
-29.4
30

80

130

180

230

Time (minutes)

b)

AB

-23

Transmission (dB, 1550 nm)

-24
-25
HA

-26

PBS

-27
-28
-29
-30
30

80

130

180

230

Time (minutes)

Figure 6-5 Response of a TFOBS to antibody immobilization. Dimension of the TFOBS in m is a = 465,
b = 296, c = 340, d = 5. 100 g/mL of antibody to BSA was injected and the transmission was recorded.
After 2 hours of antibody immobilization, hydroxylamine was flowed in for 40 minutes, followed by PBS,
until the transmission reaches steady state. AB = antibody, HA = hydroxylamine. a) The response from
antibody at 1310 nm. b) The response from antibody at 1550 nm. c) ln (1-theta) vs. time to determine the
rate of immobilization of the antibody.

136

c)

0
-0.5

ln (1-theta)

-1
-1.5
-2
-2.5
-3

y = -0.0652x

-3.5
-4
0

10

20

30
Time (minutes)

40

50

60

Figure 6-5 (continued)

6.4.3 Multi-step detection and release of BSA

Based on the strong response of the TFOBS to antibody immobilization, the

sensors were tested for detecting the target antigen, BSA. Typical multi-step detection
and release responses observed are shown in Figure 6-6a and 6-6b, for 1310 nm and 1550
nm respectively, for concentrations of 1 pg/mL to 100 pg/mL of BSA. Similar to the
antibody attachment to the fiber surface, the binding of BSA and anti-BSA appear to
follow the Langmuir adsorption model. However, here the time required to reach steady
state was about 30 minutes whereas it was about 1 hour for the antibody immobilization.
At 1310 nm, the transmission decreased 0.12 dB for 1 pg/mL and 10 pg/mL, and
decreased 0.29 dB for 100 pg/mL. At 1550 nm, the transmission increased 0.12 dB for 1
pg/mL, increased 0.27 dB for 10 pg/mL, and increased 0.26 dB for 100 pg/mL. When the
release solution (pH 2.4 PBS) was circulated through the sample chamber, the resulting
response was in the opposite direction to the attachment response, and reached levels

137
similar to the transmission value prior to BSA attachment, indicating that all the attached
BSA were released. Aside from the result shown in Figure 6-6a and 6-6b, a similar result
was obtained for the attachment of 1 pg/mL to 10 ng/mL of BSA, with 0.46 dB decrease
for 1 pg/mL of BSA, ~ 0.41 dB decrease for 10 pg/mL of BSA, and about 0.27 dB
decrease for 10 ng/mL of BSA, at both 1310 nm and 1550 nm. Attempts were made to
detect lower concentrations of pure BSA in the flow configuration. However, unlike in
the stagnant condition where 10 fg/mL of BSA were routinely detected, the lowest limit
of detection for the flow cell was 1 pg/mL of BSA. One possible reason for this
difference is the tapers used in this study were shorter and more symmetric than those in
the previous report

126

. In this study, the dimensions of the taper used for the result in

Figure 6-6a in m were a = 480, b = 213, c = 500, d = 5. In the earlier study, the tapers
were of similar geometry to the one used in Figure 6-6. However, all of them had
diameters greater than 6 m, and a majority was longer in length than the ones used in
the present paper. Furthermore, many of the tapers used in the earlier study were much
more asymmetric. We determined

126

in the wavelength of 400 to 800 nm that slight

changes in dimensions result in drastic differences in the non-specific TFOBS response to

solutions of E.coli JM101. Experimentally it was found that asymmetric tapers of 4 mm
in length were superior in detecting low concentrations (100 and 1000 cells/mL)
compared to the symmetric tapers. Furthermore, mathematical modeling showed that for
the same overall geometry, a 5.5 m diameter taper showed larger refractive index
sensitivity compared to a 6.25 m taper at 470 nm. Therefore, it is likely that the
geometric parameters influenced the detection limit in this study as well.

138
Kinetics analysis similar to that of antibody immobilization was performed on
results presented in Figure 6-6a. The analysis is shown in Figure 6-6c for the 1 pg/mL
and 100 pg/mL of BSA, while that of 10 pg/mL was omitted for clarity but the rate was
still determined. The rates were 0.089 min-1 for 1 pg/mL, 0.075 min-1 for 10 pg/mL, and
0.028 min-1 for 100 pg/mL. In general, antibody immobilization took a longer time (about
40 to 60 minutes) while BSA attachment was shorter, typically 30 minutes. A similar
study in our laboratory using cantilevers to detect BSA in flow had obtained a rate of
0.103 to 0.111 min-1 96. The rate constants obtained in this study are lower in comparison,
perhaps because in the previous study the flow rate was 1 mL/min. Also, the
concentration of BSA used in the previous study was 1 mg/mL, which is at seven orders
of magnitude higher than those used here.

139
A

a)

Change in Transmission (dB)

-0.1
-0.2
-0.3
1310 nm

-0.4
-0.5
-0.6
0

b)

50
A

0.7

100
Time (minutes)
C

150

200

0.6

Change in Transmission (dB)

0.5
1550 nm

0.4
0.3
0.2
0.1
0
0

50

100
Time (minutes)

150

200

Figure 6-6 The change in transmission of two antibody-immobilized TFOBS. a) Response at 1310 nm.
BSA of 1 pg/mL to 100 pg/mL were attached sequentially to this fiber, and then pH 2.4 PBS was used to
release the protein. A = 1 pg/mL BSA, B = 10 pg/mL BSA, C = 100 pg/mL BSA, D = pH 2.4 PBS. The
dimension of this TFOBS in m is a = 480, b = 213, c = 500, d = 5. pH 2.4 PBS causes the opposite
change in transmission, with the almost same value as the value prior to any BSA addition, thus confirming
the attachment of BSA in the first place. b) Repsonse at 1550 nm for the same experiment as that done in
a). c) ln (1-theta) vs. time plot used to determine the rate of attachment for 1 pg/mL and 100 pg/mL of
BSA.

140

c)

0
-0.2

y = -0.0246x

-0.4

ln (1-theta)

-0.6

100 pg/mL

-0.8
-1
-1.2
y = -0.1106x

-1.4

1 pg/mL

-1.6
-1.8
-2
0

10
Time (minutes)

15

20

Figure 6-6 (continued)

6.4.4 Multi-step detection of BSA and BSA in the presence of OVA

Having determined that pure BSA at 1 pg/mL can be detected, we investigated the
selective sensing characteristics of the sensor. We used the protein OVA as a
contaminating protein for the detection of BSA. A multi-step experiment similar to the
one in section 6.4.3 was performed to determine if there is a difference in the rate and
change in transmission between pure BSA and BSA in the presence of OVA. The results
of this experiment are shown in Figure 6-7a as total transmission vs. time at 1310 nm,
and in Figure 6-7b as changes in transmission vs. time at both wavelengths. It was found
that at 1310 nm, both the rate and the change in transmission were similar for pure BSA
at 1 pg/mL and a 1:1/v:v mixture of BSA and OVA, both at a concentration of 1 pg/mL.
However, there was no detectable transmission change for 1 pg/mL of BSA at 1550 nm,
and there was only a 0.4 dB change for the 1:1/v:v mixture of BSA and OVA at 1550 nm.
The changes in transmission at 1310 nm were between 0.4 to 0.7 dB, slightly higher than

141
the response of the multi-step attachment experiments in section 4.3. Kinetic analysis
shows that the rate for 1 pg/mL of pure BSA was 0.03 min-1 whereas the rate for the
mixture of BSA and OVA was 0.058 min-1.

142

Transmission (dB, 1310 nm)

a)

-21.5

-22

-22.5

-23

-23.5

1310 nm

-24
120

b)

160

200
Time (minutes)

240

0.6
0.4

Change in Transmission (dB)

280

1550 nm - 1:1

0.2

1550 nm - 1 pg/mL BSA

0
0

10

20

30

40

50

-0.2
-0.4

1310 nm - 1:1

-0.6
-0.8

1310 nm - 1 pg/mL BSA

Time (minutes)

Figure 6-7 a) The response at 1310 nm of an antibody-immobilized TFOBS. The response of 1 pg/mL of
BSA in the presence of 1 pg/mL of OVA is shown here. O = 1 g/mL OVA, P = PBS, A = pH 2.4 PBS, M
= 1:1/ v:v mixture of 1 pg/mL BSA and 1 pg/mL OVA. The dimension of this TFOBS in m is a = 400, b
= 245, c = 574, d = 5. Response at 1550 nm is similar but not shown because the magnitudes of the
transmission changes are much smaller. b) The same data as a), plotted in terms of change in transmission
compared to the transmission due to PBS prior to protein addition. 1:1 represents the mixture of 1 pg/mL
OVA and BSA. Time scale was adjusted such that the sample addition for each solution is at time zero. c)
ln (1-theta) vs. time, plotted to determine the rate of attachment.

143

6.5 CONCLUSIONS

In this study, the response of TFOBS to liquid refractive index was determined by
various concentrations of glucose and TFOBS were found to be sensitive to small
changes in refractive index. TFOBS exhibited significant changes in optical transmission
when exposed to BSA and antibody to BSA at 1310 nm and 1550 nm. The response to
antibody immobilization and BSA binding was modeled after Langmuir adsorption
kinetics. The detection of 1 pg/mL of BSA and BSA in the presence of OVA was found
to be feasible in flow configuration. Finally, it was shown that the non-specific response
of TFOBS is reversible when one switches from glucose to water repeatedly.
Reversibility of the sensor is a powerful feature of TFOBS which has not been previously
shown in the stagnant condition. The use of flow cell with TFOBS is an important step in
the development of intensity-based TFOBS because it reduces the possibility of
transmission changes due to external factors that are not associated with the presence of
the samples.

144

7.0 LABEL-FREE DETECTION OF DNA HYBRIDIZATION

7.1 Introduction

Detection of DNA interactions at femtomolar concentrations has many

applications, such as biotechnology

131

, genetics 68, medical diagnostics

132, 133

, pathogen

detection 71, 134, and drug screening 80. To date the most popular methods of detection use
single strand DNA probes as recognition molecules

68, 71, 124, 133, 135

. Most of these

methods depend on a fluorescent label in the target strand to provide and amplify a signal
68, 70, 71, 136

. Although the use of fluorescent labels improves sensitivity and selectivity,

they require trained personnel, ultra-clean facilities, and highly time-consuming sample
preparation. Furthermore, nucleic acids are often present in such small amounts that they
need to be amplified using polymerase chain reactions (PCR) such that enough DNA is
present to cause a change in sensor signal. As a result, it is highly desirable to reduce or
eliminate the need for labeling and amplification, in order to develop a rapid, reliable,
and user-friendly sensor.
For the past few years, Tapered Fiber Optic Biosensors (TFOBS) have been
investigated in our laboratory as an alternative detection device for biomolecules. TFOBS
have been widely investigated for the monitoring of physical properties
chemicals

5, 30, 117-120

, and biological molecules

17, 22, 35, 42, 58, 66

3, 88, 114-116

. TFOBS has several

applications in environmental monitoring, drug screening, clinical diagnostics, and

national security. From a detection standpoint, some advantages of TFOBS include the
exposure of evanescent field beyond the surface of the sensing region, fast and real-time
response, and the ability to respond without labels. Based on the femtogram to pictogram

145
per mL sensitivity we have obtained for protein Bovine Serum Albumin (BSA) at near-IR
wavelengths and flow conditions

56

, we postulate that DNA can be detected using

modification to our current system, resulting in label-free and continuous detection in the
flow setting, using near-IR wavelengths.
In this study, TFOBS are coated with gold and housed in a flow cell. Thiolated
15-mer single stranded (ssDNA) probes were immobilized on the TFOBS gold surface.
Complementary 10-mer ssDNA target strands were then detected while they hybridized
with the immobilized probes at concentrations as low as 750 fM. The sensor also showed
selectivity against a single nucleotide mismatch.

7.2 Physics of Sensing

Optical fibers are light transmission waveguides that are made out of silica. They
consist of a cylindrical core surrounded by a cladding. The core is doped with Ge such
that its refractive index (RI) becomes slightly higher than that of the cladding. Since the
core has a higher RI than the cladding, light is reflected along the fiber axis when it
reaches the core-cladding interface, and therefore propagates through the optical fiber by
total internal reflection (TIR). Aside from the large portion of guided light in the core,
there is a small component of light, known as the evanescent field, which decays
exponentially away from the core surface and propagates in the cladding. In the majority
of uniform-sized fibers, the cladding is much thicker than the core, making the
evanescent field to decay to near zero value at the fiber surface. As a result, these
standard fibers do not predispose the interaction of the evanescent field with the
environment outside the cladding. However, should the fiber be reduced down to a

146
diameter less than the core diameter, light would no longer be guided in the core, but
would instead spread out to be guided by the cladding. With tapered fibers, the
evanescent field not only extends beyond the cladding, but its magnitude is also enhanced
in that tapered region. When a liquid medium is placed at the tapered region, the
evanescent field interacts with the medium through absorption and scattering.
The amount of evanescent light that is available for sensing depends on the
penetration depth, which is defined as the position away from the core/cladding interface
when the light decays to 1/e of its magnitude at the surface. As discussed in our previous
publication, penetration depth is wavelength dependent, and the use of long wavelengths
results in higher penetration depth 137. The evanescent field strength is also determined by
the diameter and the taper geometry. This is due to the mode coupling that occurs at the
tapered region from the changes in the cladding and analyte RI 83. Mode coupling causes
the magnitude of the evanescent field and transmission of the fiber to change. Due to
mode coupling, changes in RI at the tapered region are reflected in the sensor output
when a sample is introduced. As confirmed in a previous study, some factors which affect
a tapers sensitivity include lengths, diameters and slopes of the contracting and
expanding regions of the taper 126.

147

7.3 Materials and Methods

7.3.1 Flow Cell Apparatus

The flow cell apparatus was described in our previous work (currently under
review) and the diagram was previously shown in Figure 6-1. The chamber consists of a
cylindrical well 4 mm in diameter and 3 mm in depth, in a 6 mm thick plexiglass (~3 cm
X 5cm). The tapered region was positioned within the sample chamber, which has a
volume of approximately 40 L. Epoxy was used to seal the sample chamber to a 2 mm
thick plexiglass. Four reagent reservoirs (5 25 mL), each connected to an on/off valve,
were fed into a 4-port manifold, which was in turn fed to the inlet of the flow cell fiber
holder (FCFH) using a 1.14 mm diameter polyethylene tube (Warner Instruments Inc.)
The output of the flow cell was fed to a waste container or re-circulated by a peristaltic
pump (Fisher Scientific, Variable Flow Mini-Pump). The flow rate used in all
experiments reported was 0.5 mL/min. The reservoirs contained four separate reagents:
ssDNA probe, target ssDNA, TE buffer, and 1-mercapto-1-hexanol (MCH). The reservoir
containing the desired reagent was turned on via the on/off valve while other reagents
were in the off mode. All experiments were performed in a temperature-controlled water
bath maintained at 28.8 0.1 oC and the temperature of the water bath was recorded
throughout each experiment.

148
7.3.2 Optical Arrangement

The light sources used for all experiments reported were a 1550 nm DFB laser
(Anritsu GB5A016) and a 1310 nm DFB laser (QPhotonics). As shown in Figure 6-1, the
two sources were connected to the input of the sensor via a 2X2 optical coupler. A FCFC mating sleeve (Thorlabs, NJ) was used to connect the sensor to the coupler. The
output end of the sensor was connected to a spectrum analyzer (ANDO AQ-6310B) using
FC connectors. A LabView program measured spectrum peak transmission at each
wavelength and recorded the data at a sampling rate of one measurement per minute.

7.3.3 TFOBS Fabrication

TFOBS were fabricated using a programmable Ericsson FSU 975 fusion splicer.
The fabrication of TFOBS was described in detailed in our previous work

56, 126

. Single

mode silica optical fibers (Corning Glass Works, NY, attenuation at 1300 and 1500 nm of
0.36 and 0.26 dB/m, respectively) with 8 m core and total diameter of 125 m were
used for all TFOBS fabrication.
Immobilization on a gold surface is convenient for the attachment of DNA probes
because sensors can be sputtered with gold enabling the formation of self-assembled
monolayer (SAM) of thiolated ssDNA probes. After coating the taper with a thin film of
polyurethane, a 50 nm gold layer was sputtered the taper in a Denton Desk II System
(Denton Vacuum, New Jersey) at 1 mTorr.
After the gold surface was formed, the fibers were fixed and enclosed using epoxy
(Hardman) between the FCFH components described in section 7.3.1. Approximately
10 cm of protective tubing was inserted onto the fiber on both sides of the fiber holder.

149
The tubing was then held in place using epoxy. The ends of the fiber were connectorized
and polished using the method described by the manufacturer (Thorlabs, NJ).

7.3.4 Reagents

All reagents were purchased from Sigma, unless otherwise noted. Single-stranded
thiolated 15-mer oligonucleotide probe from Bacillus 16S-rRNA sequence HS-C6H12-5GGAAGAAGCTTGCTT-3, its complementary 10-mer target 5-AAGCAAGCTT-3,
and 10-mer target with a single mismatch to the probe sequence were purchased from
Integrated DNA Technologies (Coralville, IA). The 15-mer probe was used as a model
system for immobilization, while the 10-mers were used as target analytes. Lyophilized
DNA samples were reconstituted in TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0)
with 1M NaCl. It was diluted in TE buffer to desired concentration prior to use. 1 M
NaCl was used to bring the hybridization temperature of probe-target DNA strands to
desired temperature of operation (26 C),
temperature 30C

138, 139

which is 4C

lower than the melting

. 1-mercapto-6-hexanol (MCH) at1M was freshly prepared in

TE buffer for each experiment.

7.3.5 Probe Preparation

Thiolated single stranded DNA (ssDNA) probes were supplied in disulfide form
and were reduced prior to use. After re-constitution, each aliquot of ssDNA contained
250 L at a concentration of 65.8 M. The disulfide linkages of the thiolated DNA was
reduced using 100 mM DTT at room temperature and pH 8. 3.9 mg of dithiothreitol (DTT)

150
was added to each aliquot and allowed to react at room temperature for 30
minutes. Excess DTT was removed with G-25 dry columns to ensure that no DTT
remains to compete with the probe molecules on the sensor surface. Excess DTT was
removed with Sephadex G-25 columns (Pure Biotech LLC, NJ) following the vendors
protocol. The purified ssDNA was diluted in TE buffer to a desired concentration
(approximately 80 nM) and used within 1 hour. No attempts were made to optimize
probe surface concentration.

7.3.6 Experimental apparatus, probe immobilization and hybridization detection

All experiments were conducted in the specially constructed flow apparatus

described in Section 7.3.1. The TFOBS flow cell was firmly secured in a temperature
controlled water bath maintained at 28 0.1 0C. The TFOBS was connected to an optical
spectrum analyzer (Ando AQ-6410B) interfaced to a PC running a custom written
LabVIEW data acquisition program. The transmission at 1310 and 1550 nm was
collected at 1 minute intervals. The intensity of the TFOBS was monitored during ssDNA
immobilization. All measurements were performed at 1310 and 1550 nm simultaneously
in the flow setting. Flow rate was kept at a constant value of 0.5 mL/min in all
experiments. First, TE buffer was flowed through the sensor in recirculation mode until
the transmission has been stable for 30 minutes. Then, a 1 M ssDNA probe stock
solution was flowed in recirculation mode until the transmission reaches a new steady
state. Freshly prepared 1M solution of 1-mercapto-1-hexanol (MCH) in TE buffer was
then flowed in for 40 minutes to remove any ssDNA that was non-specifically attached to
the sensor. MCH is used to reduce non-specific adsorption because it competes with

151
probe bases which are weakly adhered to the sensor surface while keeping covalently
bonded thiolated probe molecules intact. MCH also helps extend DNA and increase the
probability of contact since it scatters the DNA to enhance the accessibility. After the
addition of MCH, TE buffer was flowed in for 40 minutes followed by the target ssDNA.
The test sample solutions were circulated for 30-45 minutes until the transmission
reached a constant value.

7.4 Results and Discussions

7.4.1 Probe immobilization

ssDNA probe immobilization was monitored by recording the transmission at

1310 and 1550 nm. A sample result is shown in Figure 7-1. The change in transmission
was obtained by subtracting the transmission when sample was first introduced from the
transmission at each instant. One notes from this figure that the transmission response is
similar to antibody immobilization response we obtained in an earlier publication

56

There was a change of 0.7 dB at 1310 nm, and it was 1.6 dB at 1550 nm, which points to
the dependence of sensitivity and optical behavior on wavelength.

152

Change in Transmission (dB)

2
1550 nm

1.5

1
1310 nm

0.5

0
0

10

20

30

40

50

60

-0.5
Time (minutes)

Figure 7-1: The response of the TFOBS during thiolated ssDNA probe immobilization.

7.4.2 Hybridization of complementary ssDNA

Several experiments were performed with the 10-mer that was complementary to
the ssDNA probe. They were conducted to determine response of immobilized probe to
sequential addition of increasing concentrations of complementary target DNA. A typical
hybridization response of complementary strands at four concentrations of 750 fM, 7.5
pM, 750 pM, and 750 nM is shown in Figure 7-2. After the probe immobilization and a
TE buffer flush, 1 M MCH was flowed in until steady state transmission was reached.
Then sequential injection of 750 fM, 7.5 pM, 750 pM, and 750 nM complementary single
stranded DNA containing samples were flowed in. For each of step, 4 mL of sample was
loaded into a clean reservoir, and was pumped into the flow circuit in a once through

153
mode. As the content in the reservoir reached close to empty the outlet of the flow cell
was returned to the reservoir so that the sample was in a recirculation mode. Since the
flow loop volume is 4 mL, the circulating sample concentration was not diluted. The
result was displayed in terms of changes in transmission vs. time, where each data point
was the difference between the transmission at a certain time and when sample was first
introduced. The response to 750 fM, 7.5 pM, 750 pM, and 750 nM were 0.15 dB, 0.2 dB,
0.35 dB, and 0.35 dB respectively. This shows that DNA hybridization can be detected at
as low as 750 fM. The TE buffer response prior to sample addition was used as control.

DNA attachment 1310 nm

0.15

Change in Transm ission (dB)

0.1
0.05
0
-0.05 0

10

20

30

40

50

Control - 1310
0.75 pM - 1310

-0.1

7.5 pM - 1310

-0.15

0.75 nM - 1310

-0.2

0.75 uM - 1310

-0.25
-0.3
-0.35
-0.4
Time (min)

Figure 7-2: Response of the TFOBS at 1310 nm for 750 fM to 750 M of complementary 10-mer ssDNA
probe.

154
7.4.3 Hybridization of single mismatch ssDNA

It is desirable to test the selectivity of the TFOBS by using a mismatch 10-mer

sequence to determine the effect of a single nucleotide mismatch. The procedure for this
experiment is similar to the one with pure 10-mer. However, mismatch probes of all
concentrations were introduced before introducing complementary strands. After ssDNA
probe immobilization, TE buffer was flowed in for 40 minutes, followed by a target with
single mismatch 5-AAGCGAGCTT-3, introduced from the lowest to the highest
concentration. After the transmission reached steady state, the complementary target was
flowed in from low to the high concentration. A smaller response or different rate of
attachment is expected with the single mismatch compared to the complementary target.
A typical hybridization response of single-mismatch strands at concentrations of 750 fM
and 7.5 pM, as well as complementary strands at concentrations of 750 fM and 7.5 pM, is
shown in Figure 7-3. As can be seen in this figure, the responses due to the mismatch
sequence were significantly smaller for 750 fM and 7.5 pM.

155

Change in Transmission (dB)

-0.05

10

15

20

25

30

-0.1
-0.15
1310 nm - 0.75 pM mismatch

-0.2

1310 nm - 7.5 pM mismatch

-0.25

1310 nm - 0.75 pM target

-0.3

1310 nm - 7.5 pM target

-0.35
-0.4
-0.45
-0.5
Time (minutes)

Figure 7-3: Response of TFOBS due to a mismatch sequence and complementary sequence.

7.5 Conclusions

Through the use of the model system, we have demonstrated that DNA
hybridization can be detected using the TFOBS. The notable aspects of our detection
system are intensity-based sensing, the use of near-IR wavelengths, and the ability to
detect label-free DNA. The detection limit is comparable to many detection systems, and
there should be some effort dedicated to lowering the limit. After the optimization of
detection limit, the next step would be the detection of DNA from live cells.

156

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VITA

Angela Shing Yan Leung was born in Hong Kong and immigrated to Canada in
1990. She attended the University of Toronto from 1999-2003, where she obtained a
Bachelors in Applied Science in Biomedical Engineering.
In 2003, Angela began her graduate studies at Drexel University, and her studies
were supported by teaching and research assistantships. Her publications include:
Leung A.; Rijal, K.S.; Shankar, P.M.; Mutharasan, R., Biosensors and Bioelectronics,

2006, 21(12): 2202-2209.

Rijal, K.S.; Leung A.; Shankar, P.M.; Mutharasan, R., Biosensors and Bioelectronics,

2005, 21(6): 871-880.

Leung A.; Shankar, P.M.; Mutharasan, R., Sensors and Actuators B, 2007, 123(2):

888-895.
Leung A.; Shankar, P.M.; Mutharasan, R., Sensors and Actuators B, in press,

doi:10.1016/j.snb.2007.03.010.
Leung A.; Shankar, P.M.; Mutharasan, R., Sensors and Actuators B, submitted

December 20, 2006.

Leung A.; Shankar, P.M.; Mutharasan, R., Evanescent Field Fiber Optic Biosensors:

Fabrication, Antibody Immobilization, and Detection, Optical Fibers Research

Advances, Nova Science Publishers, accepted for publication.

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