Beruflich Dokumente
Kultur Dokumente
Prepared by
VILLANUEVA, Matthew L.
October 4, 2016
Department of Chemistry
University of Santo Tomas
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
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
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.
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.
Reaction constant
Effective maximum
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.
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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