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OPTICAL METROLOGY AND INSTRUMENTATION Assignment

Fiber Optic Bio sensors - An analysis

Submitted By Name: Sankar Ganesh Matric No : G1301028D

Introduction Evolution of Fiber optic bio sensors Knowledge is the manifestation of perfection already in man said Vivekananda, a famous Indian Philanthropist long time back. This holds good to every action what we do in our day to day life. A recent survey says every man spends half of his time in finding new and simpler ways to do his day to day activities. This may sound a little weird because this makes us to think that a person is lazy, but we fail to realize that only such thinking paves way for new inventions in Science. Today the whole world is looking upon the upcoming technology of Rapid prototyping which is also known as 3D printing, Direct digital manufacturing or additive manufacturing to create to create a certain leap in the field innovative production. The evolution of this technology is also considered to be Third industrial revolution. It gives the opportunity to print an object of any shape with complex features in limited period of time. The manifestations of this technology lead to the invention of Tissue engineering. Now people are in the verge of trying to print all the possible organs of human body. We also know that hip and knee replacement has already become a hug hit in the medical field. Today bio engineers are thinking to fabricate artificial kidney, heart, lungs and other organs. For this dream to come true a lot measurement instruments in the medical field becomes inevitable. The basic measuring instrument which can interact with tissue and cells is Fiber Optic Bio Sensor. Bio sensors are analytical instruments which comprises of optical fibers and biological recognition molecules. An optical fiber is a thin, strong, flexible and transparent wire through which light can be transmitted with minimum loss over long distances by the principle of Total internal reflection. Sensing layer composed of bio recognition molecules generates light which is coupled to the fiber end. The light transmitted through the optical fiber measures the different optical phenomenon like absorption, luminescence, evanescence when the sensing layer and analyte interaction takes place. Fiber-optic biosensors (FOBS) are optical fiber-derived devices which use optical field to measure biological species such as cells, proteins, and DNA. In traditional immunological methods, it measures the presence or concentration of an analyte which may be a protein in a solution through the use of an antibody or immunoglobulin. Because of their efficiency, accuracy, low cost, and convenience, FOBS, mainly Tapered ber-optic biosensors (TFOBS), which are a type of FOBS are promising alternatives to traditional immunological methods for bimolecular measurements. In order to amplify sensitivity and selectivity, TFOBS are often used with various optical transduction mechanisms such as changes in refractive index, absorption, uorescence, and Surface Plasmon Resonance.

Principle of Operation The combination of optical and electrode lead to the formation of the word optrode. It is a fiber optic based measuring device which can measure the concentration of a specific chemical or a group of chemicals in the test sample. Basic components of an optrode are 1. Light Source 2. Optical fiber to transmit light 3. Sensing material which is immobilized to the surface end of the fiber 4. Detector to measure output light. Latest advancements in bio-optrode make it feasible for the development of nanoscale bio optrodes, which enables measurement inside single living cells, and the development of multi analyte and reagent less bio-optrodes. The sensing element is the main component of optrode. it undergoes physiochemical transformation that changes its optical properties during its interaction with the analyte. This change in the optical properties can be interpreted to analyte concentration. The optical signals can be measured by emitting light from the light source into the optical fiber to the end of the fiber, where sensing element is immobilized. The same fiber or a set of other fibers are used to guide the output light to the detector where the reflected, emitted or absorbed light is measured. Materials of biological nature such as enzymes, anti bodies and cells are trapped on optical fibers and used for specific recognition of different analytes. Due to the fact that most of the biological sensing elements and analytes do not have significant spectral properties, the bio recognition events are transduced to optical signals like fluorescence and absorbance by uniting optically responsive reagents to the sensing elements. In fluorescence based optical bio sensors, the incident fluorescent light is measured and then transmitted through the optical fiber and the excited light after transmission is also measured. The difference in the amount of fluorescence intensity is directly proportional to the analyte concentration. Biosensors have the capability to detect biological species selectively. They may be of the type electrical, optical, mechanical or chemical. Examples of biological recognition molecules include enzymes, antibodies, and oligonucleotides. A biosensor is said to be ideal only if it responds to low concentration of analytes and discriminates among various species. Some of the applications of the biosensors are biomarker detection for medical diagnostics and pathogen and toxin detection in food and water. Fiber optic biosensors use optical fibers as the transduction elements and use optical methods like evanescent sensing for detection. Most of the evanescent FOBS are Tapered ber-optic biosensors (TFOBS)

Optical bers consist of a cylindrical core and a surrounding cladding, both made of silica. The core is generally doped with Germanium to get a difference in refractive index between the core and cladding, which results in light propagation by total internal reection (TIR). As in a uniform diameter fiber, the evanescent eld decays to almost zero within the cladding. Light propagating in uniform-diameter cladded bers cannot interact with the bers surroundings.

Optical fibers had applications in sensing which requires light to interact with the fibers surroundings. This could be achieved in many ways like, if the cladding of a ber is reduced or removed, the evanescent eld can interact with the surroundings. The distance to which the evanescent eld extends beyond the core-cladding interface is described by the penetration depth, which is the distance where the evanescent eld decreases to 1/e of its value at the core-cladding interface and is mathematically expressed as

where x is fiber core distance starting from centre, x= 0 at the core-cladding interface, magnitude of the eld at the interface, and dp is the penetration depth. The penetration depth is given by

is the

Where is the wavelength of the light source, the incident angle of light at the core/cladding interface is , nco and ncl are the refractive indices (RI) of the core and cladding, respectively. The earliest designs of FOBS involved a simple design where the cladding was removed uniformly over a distance and replaced with an analyte. The penetration depth is too small for sensing. Considering a case where refractive index of the analytes and core are approximately 1.33 and 1.45 respectively, then the penetration depth was calculated to be very small. For the purpose of sensing, the penetration depth needs to be increased depending on the size of the target analyte.

Apart from the penetration depth, light propagation through an optical ber can be described in more detail by the wave theory. The properties of light in the ber core are determined by the number of modes N. In a multimode ber, there is a one to one relationship between the modes and angle of incidence. The number of modes is directly related to a dimensionless parameter known as V number, given as

where is the radius of the core, is the wavelength of the light, ncore and n clad are the refractive index correspondingly of the core and cladding. The number of modes in a multimode ber,N, is proportional to

The increase in evanescent eld forms the basis for increased sensitivity. Several attempts have been made to increase the penetration depth by bending, tapering, altering the light launching angle, and increasing the wavelength. The Effects of tapering on the evanescent eld Tapering exposes the evanescent field to the surroundings and increases the evanescent field magnitude and penetration depth. Tapering can be performed by removing the cladding and then tapering the core, or keeping both the core and cladding in place and tapering the entire ber. In a study by Mignani et al., the optical characteristics of a de-cladded uniform-core ber were compared to that of a de-cladded tapered core ber in the context of evanescent absorption. Fractional power () is dened as the fraction of power in the evanescent eld compared to the total power in the ber. While absorption characteristics of both bers followed the LambertBeers Law, the fraction of power () differed signicantly. After analysis it was indicated that tapered core bers are 10 times more sensitive. The Effects of bending on evanescent eld Bending of optical ber results in loss in light and increase in evanescent eld and is performed on a uniformly de-cladded fiber. Fiber bending creates higher order modes which results in the evanescent eld having greater penetration depth. Khijwania et al. absorbed that U probes had superior sensing ability than straight probes and U probes had high evanescent field. In straight probes, angle of ray propagation is constant and thus the number of ray reections as well as the evanescent absorption coefcient is inversely proportional to the core radius. As the angle of ray propagation is constant in straight probes, the penetration depth is comparatively less. But in U probes, the angle of propagation decreases and the penetration effect increases and thus the sensitivity increases. Sensitivity increases when bending radius decreases, but only up to a certain value, after which the sensitivity decreases. This

decrease in sensitivity at small bending radius is due to the evanescent eld overlap resulting in power transfer between the two arms without propagating through the bent sensing region. The different authors observed signal enhancements of up to 9 in bent bers over straight bers.

Effect of launch angle on the evanescent eld In a single mode ber, light is launched over a very small angle because only the lowest order mode is supported. However, in multimode bers light can be launched at different angles since these angles correspond to modes, and several modes are supported simultaneously. The evanescent wave penetration was shown by Ahmad et al. to be enhanced by as much as 300% in certain designs of tapered bers.

The Effect of wavelength on evanescent eld Penetration depth increases with the wavelength of the transmitted light. Research has been done on the use of bers at near-IR or IR wavelengths, but based on the analysis, the data and reports available for biosensing after this research is limited. The application of IR wavelengths with intensity based tapered fiber optic biosensors (TFOBS) was investigated and was shown to result in high sensitivity at low concentrations.

Tapered ber geometries Tapered FOBS (TFOBS) is the most common method to increase the evanescent field of FOBS. There are two types of TFOBS currently used in biosensing - tapered tips and continuous tapered bers. A tapered tip consists of an optical ber which gradually decreases in diameter until it becomes a tiny tip, and the tip is the sensing element. Tapered tips can be further divided into the most commonly used geometries: step-etched, conical, and combination tapers. A continuous tapered ber consist of an optical ber gradually decreasing in diameter, after which it reaches a constant-diameter waist region, and then gradually increases back to the original diameter. Light enters a continuous taper from one end and is transmitted through the taper to the detector.

Principles of detection in tapered ber optic sensors Changes of sensor response arising from the evanescent eld are very versatile because they can be detected alone, amplied, or detected in conjunction with various optical methods. The use of evanescent sensing has been previously investigated with many different sensing principles, and includes: Changes in output power due to refractive index changes alone (intensity-based TFOBS), Evanescent eld absorption (absorption TFOBS), Fluorescence (uorescence TFOBS), Surface Plasmon Resonance (SPR). Changes in output power when refractive index alone changes In intensity based sensors, changes in the magnitude of the evanescent eld are detected by measuring the changes in the output power in a de-cladded uniform fibers or TFOBS. Factors which inuence the sensitivity of an intensity-based TFOBS include bending, radius, length, and taper ratio. Smaller diameters increase the sensitivity as they cause a larger evanescent field. In TFOBS it was shown that the longest taper (10 cm) gave the largest dp and with shorter tapers the penetration depth depended strongly on launch angle and a clear trend between the angle and dp was not observed. Taper ratio also affects the sensitivity of a sensor which is the ratio of the waist diameter of a tapered fiber to the total diameter of a uniform fiber. Evanescent eld absorption In order to use tapers for absorption measurements, the light source must be at a suitable wavelength which is absorbed by the sample analyte. In a uniform-core optical ber stripped of cladding, absorbance is governed by the LambertBeers Law:

Where is the absorption coefficient and L is the length of interaction and is the fraction of light in the evanescent domain.

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

In a uniform-core optical ber, is quite low. In the tapered ber, radius changes as a function of the distance along the axis of the ber, such that r = r(z), where z is the distance along the axis. The angle of each guided rate which is is also not constant. The taper can be considered as a sequence of uniform sensing regions with,

It was found that reducing the core radius enhances absorbance by increasing and increasing the integration range of the angle of ray propagation. Tapered fibers have higher average value of . Radius affects the sensitivity in an absorption sensor as it influences the number of ray reflections per unit length. In the case of intensity-based FOBS, taper ratio also determines the sensitivity of absorption FOBS. Bending also increases the penetration depth and consequently an increase in absorption occurs.

Evanescent wave uorescence There are two general methods for using uorescence with TFOBS. In the rst method, the sandwich assay is covalently bonded to the surface of the ber. Then, the target biomolecule is introduced that binds to the antibody. In the second method which is the direct method, a uorescent dye is rst attached on the surface of the taper. When a molecule is near the surface of the taper, the fluorescence quenching is measured. Research was conducted and concluded that for uorescence sensing, a probe of constant diameter is not ideal. When an evanescent fluorescent sensor was designed it was found that due to V number mismatch, the return of uorescent signal was inefficient in this design. Other geometric factors which inuence the sensitivity of uorescence TFOBS include length, angle of incidence, bending and surface area. Surface plasmon resonance (SPR) SPR is the variation of evanescent wave phenomenon. The geometry for a SPR sensor is a metal coated dielectric in which light propagates. Instead of having the evanescent eld interact directly with the sample or excite uorescent molecules, optical energy is transferred to the surface of the metal layer as packets of electrons called plasmons. When SPR occurs, the intensity of reected 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. From the resonance angle or wavelength one can obtain the RI of the analyte. Gold or silver is used as the coating for SPR sensors depending on the alteration required in the resonance parameter. As silver has a low stability 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. Silver decreases the width of the resonance curve whereas gold increases the separation between resonance wavelength, thus increases sensitivity. Geometric factors which affect the performance of a SPR sensor include the metal thickness, length, waist diameter, and launch angle.

The tapered ber optic sensors (TFOBS) have been used for the measurement of cells, proteins, and DNA. 1. 2. Cell concentration Pathogen detection

Ferreira et al. developed an intensity-based evanescent sensor to detect the growth of Escherichia coli which is a rod shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. The sensor was fabricated by chemically etching a multimode ber. Sensing is based on the interaction of the bacteria with the evanescent eld as well as attenuation. Maraldo et al. used TFOBS to detect the growth of E. coli JM 101. Growth was monitored by light transmission through the tapered ber. Different other researchers also worked with the pathogen detection and the corresponding responses were obtained. Fluorescence resonance energy transfer (FRET) involves the non-radiative energy transfer from a uorescent donor molecule to an acceptor molecule due to the dipoledipole interactions when the two are in close proximity. Ko et al. used the FRET principle with an optical ber tip sensor to detect S. Typhimurium. Kramer et al. studied the detection of S. typhimurium in sprout rinse water using the uorescence sandwich method. Zhou et al. is another author who used a sandwich immunoassay to detect Salmonella. An antibody-based sandwich uorescence FOBS was developed by Geng et al. to detect L. Monocytogenes. Detection of L. monocytogenes was performed again by Nanduri et al. in phosphate buffered saline (PBS) to evaluate the effect of ow on antibody immobilization. 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 tapered ber sensors drawn by a ame. The power output of the ber depends on the amount of light absorbed by the solute, which depends on the concentration. An important parameter in evanescent absorption is the product of the extinction coefcient and light path, and is used to characterize sensitivity. In a tapered ber, the interaction length and extinction coefcient cannot be separated. Chen et al. developed a chemiluminescent-based ber optic sensor to detect a multilayer of enzyme alkaline phosphatase. Kishen et al. developed FOBS to monitor Mutans streptococci activity in human saliva. Kapoor et al. used a uorescent sandwich FOBS to detect trophic factor activated signaling molecules in cells.

Toxins measurement Staphylococcal enterotoxins are a major cause of food poisoning. Tempelman et al. quantied Staphyloccoccal enterotoxin B (SEB) in a ber optic biosensor using a uorescent sandwich immunoassay. Shriver-Lake et al. used an array biosensor to detect SEB in buffer and six different types of food samples. Staphylococcus aureus is the only species which produces protein A, and was detected by Chang et al. using a uorescent sandwich FOBS. Narang et al. reported uorescent TFOBS for the detection of ricin. Antibody to ricin was immobilized onto tapered ber surface using two methods: silanization and avidinbiotin linkage. James et al. developed a method to detect lipopolysaccharide (LPS) endotoxin, which is the most powerful immune stimulant and causes sepsis. Thompson et al. developed an evanescent ber optic immunosensor to detect fumonisin B-1. Thus, FOBS have many applications such as pathogen detection, medical diagnosis and real time detection of DNA hybridization. The new effort in the future scope of the FOBS is to improve its sensitivity and selectivity. Conclusion - Future of Fiber Optic Bio Sensors In the past 2 decades, there has been a significant evolution in the design of FOBS from the use of simple de-cladded fibers to 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. 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 micro arrays. Microarrays are solid phase-based assay systems consisting of an array of miniaturized test sites arranged in rows and columns. Protein micro arrays generate enormous quantitative information which saves on the labor costs. They are robust, reliable research tools and they are a useful screening tool in biomarker screening programs. In protein arrays, capture molecules needs to be immobilized on a solid support. In arraying technology, different methods are used where nanoliter and picoliter sample volumes can be directly deposited onto the surface and thus enhancing the sensitivity of the biosensor. There are different detection technologies employed for microarray experiments. CCD cameras or laser scanners with confonal detection, planar waveguide excitation devices combined with CCD cameras or photomultipliers as detectors, proprietary combination of electrochemical luminescence (ECL) detection and patterned arrays are some of the detection techniques used. Bead based arrays were a type which was used mainly when the number of parameters to be analyzed simultaneously was comparatively low and they analyze the samples in a very short span of time. There are two types of array formats, the forward-phase arrays which are the most frequently used microarray assay formats and the second type is the reverse-phase arrays. Protein expression analysis can be carried out using different techniques. Two color labeling approach can be used wherein the difference in target protein concentration between two different tissues can be revealed immediately. The main disadvantage in direct labeling approach is that low abundance proteins may not be detectable and in contrast to it are the miniaturized, multiplexed sandwich immunoassays which provide feasibility for simultaneous analysis of

increasing number of target proteins. Reverse phase micro assays are an assay format for protein profiling approach where low amount of sample is only required and the possibility of screening large number of samples is feasible simultaneously. Thus, microarrays are robust, reliable which requires only minimal amount of sample for screening. Protein microarrays are constantly growing and have become useful sensing tools in biomarker screening programs. They have also been introduced into clinical trials to screen the adverse effects of potential drug candidates. The demands in the medical field and the aim to find out alternatives to cut down on the costs will be the driving forces for protein arrays to gain a substantial role in the diagnostic market. As was shown in this review, there is a solid foundation of work to support the use of TFOBS in a wide variety of applications. Given its promising advantages, it is likely that TFOBS will remain a popular choice among researchers and practitioners for detection of biological agents.

Reference [1] Optical Biosensors: Today and Tomorrow (2 nd Edition) Frances S. Ligler and Chris Rowe Taitt [2] A review of fiber-optic biosensors Angela Leunga, P. Mohana Shankarb, Raj Mutharasana [3] Review of the use of biosensors as analytical tools in the food and drink industries Lucilene Dornelles Mello, Lauro Tatsuo Kubota [4] Sensitive optical biosensors for unlabeled targets: A review Xudong Fan, Ian M. White, Siyka I. Shopova, Hongying Zhu, Jonathan D. Suter, Yuze Sun