Analysis of ascorbic acid in human blood serum with improved

Ultraviolet/Visible specstroscopic PDMS microfluidic sensor

Prepared by
VILLANUEVA, Matthew L.

October 4, 2016

Department of Chemistry
University of Santo Tomas

In partial fulfillment of the requirements for

First Term AY 2016-2017

Course Instructors:
Christina A. Binag, Ph. D.
Jose H. Bergantin, Jr., Ph.D.

Abstract
The article is about the analysis of amino acid in the blood and the
improvement of the microfluidic sensor to quantify the nutrient. The blood
sample was prepared for separation. The microfluidic sensor was further
developed with alumina zerogel and PDMS to obtain the optimum enzyme
catalysis

for

the

analysis.

The

enhanced

technique

offers

low

consumption of samples, simplified process, and straightforward amino

acid detection.
Introduction
Ascorbic acid, commonly identified and available generically as vitamin C,
belongs to a group of natural products that is of great importance in our body. The
chemical formula of the compound is C 6H8O6; having a structure shown in figure 1. It is
a vital antioxidant in the body. The nutrient can be found in the fruits and vegetables.
Ascorbic acid can help in the maintenance and construction of the blood vessels,
bones, and skin. It endorses the restoration of the muscles from the abrasions, cuts
and wounds; help fight against infections; inhibition of the change of many dangerous
irritants like tobacco smoke, smog into cancer-producing substances; regulation of
cholesterol absorption resulting to less high blood pressure and low chance of getting a
heart disease. It helps in the absorption of iron in the blood and is the common aid in
scurvy. Vitamin C is an all-around helpful vitamin that is available in drug stores in
specified doses.

Figure 1. Ascorbic Acid

Among the many benefits of the ascorbic acid, it has also its side effects if not
taken in regulation. Over dosage of vitamin C can lead to gastrointestinal upsets like
nausea, vomiting, stomach, and diarrhea. The other side effects include temporary
faintness, dizziness, headache, and heartburn. Meanwhile, the deficiency of the nutrient
in the body leads to a lower immune system that can make the body vulnerable against
diseases and can lead to death in 5-6 months of deficiency (Bsoul and Terezhalmy,
2004). Low levels of ascorbic acid lead to diseases of the bones and mouth. The mouth
can induce tooth problems, gingivitis, and bleeding gums. For the bones, defective
development of it can be the result of deficit ascorbic acid in the body. The study of Bi,
Duarte, Brito, Vilas-Boas, Cardoso and Frietas focuses in the enhancement of the
spectrochemical method Ultraviolet-Visible (UV-Vis) spectroscopy to make the
quantification of ascorbic acid in the blood of a person. With the help of UV-Vis with
improved method of adsorption, the researchers aims to have a faster and rapid
measurement of ascorbic acid due to the increase of the population.
New Developments and State-of-the-Art
UV-Vis, in the recent years, adapted to the new technology and scientists needs
making it more useful, more accessible and much more user-friendly. It makes analysis
the various samples ranging from cationic metal solutions to more biologically organic
compounds possible. The latest developments in the UV-Vis caters primarily in the
biological samples. New improved spectrometers are available in the market.

The company Thermo Fisher, a household name in terms of scientific services,

released their new line of UV-Vis spectrometers in October 2015. The innovatory
instruments was called the Thermo Scientific NanoDrop One and NanoDrop One c UVVis microvolume spectrometers. These are architected to meet the researchers need for
understanding the sample quality with no expensive interruptions due to troubleshooting
and repetition of experiments. The new spectrometers claim to detect and find the
sample contaminants and more accurate concentration results, faster feedback time
with quality technical support, improved sensors and analysis through digital imagery in
form of graphs. It is streamlined to analyze protein and nucleic acid samples. With
features like touchscreen interface of high resolution, the instrument is easy to use.
Meanwhile its innovative technology, auto-range pathlength, brings a new method of
UV-Vis Spectroscopy without diluting the concentrated samples. It also features costeffective measurements, also eliminating the use of cuvettes, requiring samples of 1-2
L to be analyzed in just matter of seconds. With it adapting to modern technology, it
features sharing and archiving via Ethernet, USB, and Wi-Fi.

Figure 2. NanoDrop One (left) LAMBDA 1050 (right)

Another state-of-the-art product available in the market is the Perkin-Elmers
LAMBDA UV/Vis/NIR Spectrophotometer 1050. The instrument is the new model of the
Perkin-Elmer company which promises results that arent seen before. Perkin Elmer is
also known for providing high-end instruments in the field of spectroscopy. The
instrument features the range of UV to NIR (near infrared) that is 175 nm to 3300 nm
respectively. Featuring better intensity of sensitivity, higher resolution, and faster

speeds, applications to quantitative and qualitative analysis are wider and more
informative. The instrument boasts its resolution and sensitivity. The said features were
claimed unmatched and faster scanning without erroneous results. It also contains the
biggest sample compartments. Aside the size of compartments, the instrument features
amazing sample control that includes integrating spheres that diffuse and specular
reflectance, detectors like the Universal Reflectance Accessory improved with patented
InGaAs and dual Si detectors, drive units Pol/ depol that provides automatized control of
depolarized and polarized light using a PC. The capability of analysis in nanomaterials
of the instrument can help in the rising interest in nanomaterials used in polymer, optical
and non-optical.
Generally, UV-Vis evolved in new heights dating back to its conception at early
1940s to the present day. Back in the older days, the samples are limited to inorganic
compounds. Along the way, the technology upgraded paving the way of other samples
like bio-organic compounds to be analyzed. From using the a single beam light source,
it was further improved by making the dual beam spectrometers by using prism to bend
light into two separate paths. Diodes of the spectrometers are improved every year.
Recent developments in UV-Vis includes the kinetics of enzymes, photoacoustic
spectroscopy,

derivative

spectroscopy

and

many

more.

Other

experimental

developments of the spectroscopy includes quantification of ascorbic acid in human

blood and microwave-assisted UV-Vis in pharmaceutics.
History or Background Literature
Spectroscopy, in general, was discovered as early as 1600s. Back then, people
wondered at the multicolored curvature of light and the rainbow effect in the early
telescopes. Isaac Newton searches answers in these questions by further studying the
topic of light itself, wanting to find the origin of colors. Newton performed a simple
experiment by making sunlight go into through a tiny hole in the shutter window then
passes through a prism (Thomas). He then discovered a spectrum of colors or spectra
from the end of the prism as seen in Figure 3. He hypothesized that the sunlight was a
form of white light when passed through a prism can be separated into different colors.
To further expound the idea, he reversed engineered the previous experiment making

the individual colors to pass through the prism in reversed position. The experiment was
successful in the means that the individual colors formed a white light. It was therefore
concluded that white light is combination of different colors. William Wollaston improved
the experiment in year 1802 by using a thin slit, not a round aperture, then it produced
spectrum of visible lines. Each line shows the image of a slit and represented by a
different color in the spectrum. Wollaston observed interrupting dark lines that are
parallel to the slit. Joseph van Fraunhofer studied the dark lines about a decade later.
He discovered that there are 500 dark lines in the spectrum and assigned the lines
accordingly. He assigned by A-H, A for the red and H for the violet region. He is also the
one who measured the D lines wavelength.

Figure 3. Newtons prism experiment

Gustav Kirchoff in 1859 theorized that a substance with a good emittance of a
specific wavelength will absorb light of the same specific wavelength. Theory was to
further explain the relationship of absorption and emission in order to describe the
Fraunhofer lines. The study concluded that the dark lines were caused by gases in the
atmosphere of the sun. Kirchoff saw the potential for chemical analysis. He then
discovered thallium and rubidium with the help of Bunsen. They detected new colors in
the spectra. The elements of thallium and helium were discovered with the help of
spectroscopy by Sir William Crookes, and astronomer Sir Norman Lockyer and chemist
Edward Frankland.

Niels Bohr in 1913 embarked an innovative way in the interpretation of the

spectra. He proposed that the ideas brought up by Planck and Einstein are to be used
in the analysis of the spectra. Bohr stated that electrons only exist in constant energy
and a change of energy is by the means of transition states. In the transition states, the
electron will either absorb or emit energy that is the difference energy between the two
states. The characteristic is reflected in the spectral lines.
This lead the instrumental use of emission and absorbance spectroscopy in the
observance of outer electron transitions and electronic examination of matter structure.
The German physicist August Beer determined the relationship between the
concentration and the absorption of light then Beers Law come into being. The color
comparator, one of first instruments to quantify concentration by the absorption of light.
The transmitted light of the sample was manually compared to a standard solution and
the path length was adjusted until the two solutions project the same light intensity. The
instrument was like a spectrometer. Along came the 1940s, the first UV-Vis
spectrophotometers are commercially introduced. The spectrophotometers are replaced
the inaccurate photodetectors. Spectrophotometers also use the fundamental equation
of Beers Law. The Cary 11 was the first UV-Vis absorption spectrophotometer. Since
then, the UV-Vis spectroscopy improved many ways. Photodiode arrays are being
created and developed to collect the wavelengths concurrently, reducing the analysis
time. To having more user-friendly instruments that provides graphical data onto
screens. The spectrometers of today can analyze blood samples, bioorganic samples
and a lot more just into solution forms. The wavelength of the instruments can be
adjusted digitally. The developments of UV-Vis dont stop. It will just be continually
improved. There are many researches that further increase the sensitivity of UV-Vis
spectrophotometer like amino acid quantification (Bi et. al.) that will be further dissected
in the critique
Discussion and Critical Review
Ascorbic acid is vital nutrient of the body. Quantification of amino acid in the
blood requires many steps. The goal of the experiment is to enhance the microfluidic
sensor in the UV/Vis spectrophotometer for ease in determining the amount of ascorbic
acid in the human blood. The experiment proper is divided into four major steps: the

construction of the microfluidic chips, immobilization of proteins using modified PDMS

channels, the ascorbate oxidase activity study in microfluidic biosensor, and the blood
sample analysis.
The first step in the procedure is the construction of the microfluidic chips. The
PDMS microfluidic straight channel was created with the lithography method. PDMS or
Polydimethylsiloxane is a compound used in various ways from water-repellent
materials to anticaking agents or antifoaming agents. It is a colorless, viscous liquid. It
can be used for the fabrication of microfluidic systems due to its optically transparent
characteristic and its thermal stability is suitable for optical datacom communication.
(Cai et. al., 2010). Specification of the microfluidic chip to be attached in the UV/Vis is a
length if 4cm and cross section of 100m x 100m. A diameter of 1mm reservoirs were
attached to the ends of the channel. Then a glass substrate was used to bond the
channels. The specific glass used is the Sigma-Aldrich Corning microscope slides with
length and width of 75mm x 50 mm.

Figure 4. PDMS installation

The PDMS channels was then used for the immobilization of proteins. Proteins
help. The gel used for the immobilization was Al 2O3 sol-gel. Sol-gel refers to the
materialization of network of oxides by polycondensation reactions of a precursor in a
liquid. The gel was synthesized by following a method previously published by Liu et.
al., 2000. The channels was then filled with the specific Al 2O3 sol-gel then it was
incubated and used directly for protein absorption. Al 2O3 xerogel was obtained from the

incubated Al2O3.Otherwise, the proteins was mixed in the sol-gel and it was injected to
the microchannel for immobilization.
The study of ascorbate oxidase activity was carried out. It was performed by
injecting 100M AA in 50 mM KH 2PO4 NaOH with a pH 6.2 into the microfluidic
channel containing ascorbate oxidase. For reference, another was performed without
containg the ascorbate oxidase. The time of reaction was attuned by manipulating the
substrate flow at different rates. UV/Vis spectroscopy provided the AA concentration
decline during the reaction by monitoring it online. The measurements were performed
in 3 times.
The blood sample was provided by a volunteer that was healthy and was at her
30s. The sample was collected at the Laboratorio de Patologica Clinica Hilario de Lima
in Unilabs, Braga, Portugal. The blood sample was only used for this specific
experiment and not used in other types of experiment. The collected sample was
treated for partial protein isolation and stored at -20C. When the storage is already
28H, the AA of the sample was ready to be analyzed by the microfluidic sensor with the
protein ascorbic acid oxidase. The specified senor was formed with 0.1 mg/mL ascorbic
oxidase in the channel with xerogel modified PDS The formation was carried out by
physisorption (define). The analysis proper was done by diluting the blood sample 1:1
volume by volume with the phosphate buffer of pH 7.4 with 0.15M NaCl. Then it was
pumped through the modified microfluidic channel. The analysis consumed about L of
sample that matches to 10L serum or 20-25L of whole blood sample.
Ascorbic oxidase, the enzyme used for quantification of AA, is a very unstable
enzyme. Unlike from other stable enzymes that can last up to months, ascorbic oxidase
can be difficult to be maintained. The experiment needs the enzyme to be stable and
maintained to be properly used and attached to the microfluidic sensor. Three different
protocols where carried out in the enzyme immobilization. The types are PDMS
channels with and without the alumina xerogel modification, and alumina sol-gel
encapsulation. The effectiveness of the protocols were compared. Scheme 1 shows
how the different protocols were carried out.

Figure 5. Representation of physisorption of different protocols. a) direct adsorption of

proteins in PDMS surface; b.) protein encapsulation by sol-gel onto the PDMS inner
surface; c.) sinanolized inner microfluidic channel surface and placed one layer of Al 2O3
xerogel, and then utilized for protein physisorption.
Even though these three protocols for protein immobilization can load the
proteins, only one is efficient and applicable for an ease protein immobilization. The
three are compared by analyzing the photos from the confocal laser scanner
microscope. The images suggests that all can load proteins but microfluidic PDMS with
adsorption by Al2O3 zerogel can provide good results while requiring low loading
amount. The data collected gave results that the modified PDMS can give a plateau
value within 1 hour of incubation.

Figure 6.a) First protocol adsorption b) Sol-gel encapsulation adsorption c) Modified

PDMS adsorption
The table 1 summarized the calculated values that gives the necessary data in
comparing for the efficiency of the three protocols. Clearly the data points out that the
modified PDMS with Al2O3 is the best fit microfluidic sensor for the analysis. It displays
that it is the one with the maximum enzymatic activity or catalysis in the three protocols.
With the optimum catalysis, time of immobilization can be reduced greatly. The large
surface area brings the credit of having shortened diffusional effect. Consequently,
using it as the main biosensor.
Enzymes were immobilized by

Reaction constant

Effective maximum

Physisorption to PDMS surface

Sol-gel
8.5 days of

rate
0.025 0.002s-1
0.047 0.005s-1

rate
19.6 1.6M/s
36.8 3.9M/s

0.017 0.001s-1

13.3 0.8M/s

0.080 0.001s-1

62.6 0.8M/s

encapsulation

drying
Overnight

drying
Physisoroption to Al2O3 xerogel

surface
Figure 7. Measured performance of the three protocols
Now that the main biosensor was chosen, the quantification proper of the
experiment will be performed. To obtain the AA concentration, a calibration method was
used. The graph was prepared by plotting the concentration of AA in samples/L vs.
absorbance change (a.u.). After the plotting, the slope was calculated bearing the value

of 0.0114 and linearity of 0.9893. The higher sensitivity can be possible through
increase of channel length and decrease of infusion rate of the sample. In the other
hand, there is a limit of detection of 3 to be considered in the UV/Vis
spectrophotometer. After all the computations, data gathering and the consideration of
reference range of concentration of AA at 23-85, the AA concentration in the serum can
be calculated as 2x(22.91.4) = 45.8 2.8M.

Figure 8. Calibration graph of AA concentration

The experiment proved that techniques can be further improved depending on
the type of analyte and analysis that is needed. In the particular study, biosensor
enhancement is applied to test for AA in blood serum. Quantification of AA requires
ascorbic oxidase enzyme for detection. The pure blood sample undergone separation
for the reason that AA is only required in the analysis for the UV/Vis spectrophotometry
analysis. The experiment used a modified PDMS for the reasons stated above.
Advantages of the techniques are the simplicity of quantifying AA in blood serum and
the consumption of L of samples.
Applications and Societal Impact
The human body needs different types of vitamin in order to function normally.
Vitamins A, C, B, and E, just to name a few, are essential for daily living. Deficiency of
these vitamins may lead to poor maintenance and function of the body from day to day
basis. According to the study of Chien et al, vitamin A deficiency during maternity may

lead to complications of the fetus. The experiment was performed in mice and the
findings show that deficiency affected the development of the fetal pancreas.
The role played of the ascorbic acid in the human body is very important. It
present in the different parts of the body but in various concentrations. Thus in order to
replenish bodily functions, adequate amount of ascorbic acid must be consume in a day.
Ascorbic acid can be found in citrusy fruits and vegetables. AA acts as an antioxidant in
many ways like for cancer prevention, cardiovascular diseases to cataract and ocular
diseases. Many studies reported the different effects of the AA in the body. From the
study of Cocchi et al, it was discovered that Vitamin C contains antidepressant effect.
Given the many functions of AA, one of its great contribution on the body is collagen
synthesis. Collagen synthesis requires AA as a cofactor in the conversion of procollagen
to collagen (Bikker et. al). Vitamin C deficiency also gives corresponding defect in the
regulation of the body. The study of Bikker et al, compared 4 case reports of surgical
patients. It was tackled that ascorbic acid deficiency weakens the wound healing factor
of patients that underwent surgery. They also reported that AA treatment to patients
lead to faster healing process.
Quantification of ascorbic acid in the human blood can prevent the over dosage
or deficiency of the antioxidant in a patient. To this day, optimum amount is debated for
consumption of the nutrient. With new developments and study about ascorbic acid, it
can be quantified easily using a small amount of sample from the patient. The efficiency
and precision of the technique enhanced using UV/VIS can lead to more
groundbreaking discoveries. This is may lead to quantification of other essential
vitamins in the body like ferulic acid and folic acid in the blood.
Conclusion
The researchers were able to develop new strategy to quantify the ascorbic acid
present in the human blood. The ground breaking technique was performed with the
most optimized protocol: the formed PDMS chip with the alumina xerogel through
physisorption. It was prepared to be used in the microfluidic sensor of the UV/Vis for AA
analysis. As a result of the modification of the PDMS chip, the unstable enzyme
ascorbic oxidase was used properly and quantification of AA in the blood was a

success. The technique was also efficient in determining the amount with small volume
of the blood sample. The method used can be a guide to modify a PDMS channel for
other quantification and using a corresponding unstable enzyme. In conclusion, the
objectives set are achieved and the experiment was an accomplishment for biosensors.
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