Technical Paper: Optical Method for Detection and Analysis of Biological Molecules
Date: 08/02/2012
Prepared by:
Heather Cooper, University of Cincinnati, Cincinnati, Ohio
Kyle Frank, University of Cincinnati, Cincinnati, Ohio
Submitted to:
Srivasthan Ravi, University of Cincinnati, Cincinnati, Ohio
Dr. Anastasios Angelopoulos, University of Cincinnati, Cincinnati, Ohio
Dr. Anant Kukreti, University of Cincinnati, Cincinnati, Ohio
ABSTRACT
The fundamentals of color play an essential role in our development of a device
to detect bradykinin in human blood. Bradykinin is a key to the regulation of blood
pressure. Unfortunately, this same molecule is associated with a rare but serious
disease—Hereditary Angioedema, known to cause severe and rapid swelling that could
potentially lead to death. Our device permits rapid on-site monitoring of bradykinin
concentrations for the application of appropriate therapies. Such monitoring is
necessary to eliminate time delays associated with off-site instrumental analyses. A
durable polymeric membrane is used for this device to extract and immobilize
bradykinin from blood and permit the use UV spectroscopy to quantify content.
Furthermore, the membrane can act as a catalyst to promote reaction between
bradykinin and other immobilized reagents such as resorcinol to produce a product that
has a unique optical signature in the visible region of the electromagnetic spectrum.
This novel sensing approach allows for the use of light emitting diodes (LEDs) and
photodiode detectors to produce compact and portable sensor devices.
1. INTRODUCTION
A portable instrument that would enable on-site monitoring of bradykinin
concentrations would potentially lead to reduction in the number of people that die each
year from bradykinin related diseases. The application that we have developed uses a
perfluorosulfonic acid (PSA) membrane that offers new technological innovation for the
use of optical detection in medical diagnostic applications. The membrane provides a
durable scaffold to extract and immobilize bradykinin from the human blood for
subsequent spectroscopic analysis in the UV region of the electromagnetic spectrum. In
addition, using the catalytic properties of these membrane scaffolds, we obtain a rapid,
selective, and highly sensitive optical response for biomolecules in the visible region
spectrum for a portable sensor device. This application offers efficient technology to
professionals and enables access to physiological data that can be used to target
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therapies appropriately. Although membrane catalysts have been previously used as
optodes by promoting reactions between various regents (e.g., acid-base condensation
for acetone detection and Freidel-Craft acylation for anhydride detection), our work
represents the first attempt to use this approach for the detection of bradykinin.
Bradykinin is a 9-chain amino acid peptide that causes blood vessels to dilate
and blood pressure to lower. However, bradykinin is also associated with a rare and
severe disease called Hereditary Angioedema. Patients with Hereditary Angioedema
can suffer from rapid swelling of the hands, feet, limbs, face, intestinal tract, larynx
(voice box), or trachea (windpipe). Such symptoms are thought to be caused by the
over activation of bradykinin, the key element of this rare disease. Our aim is to quantify
the level of bradykinin in human blood by using PSA membranes as both durable
supports for UV analysis as well as catalytic optodes that provide a signal in the visible
region of the electromagnetic spectrum. To accomplish this aim, we first determine the
spectrum of immobilized bradykinin within the membrane and then determine the coreagent (resorcinol) concentration required to maximize the visible response that results
from the associated acid-catalyzed reaction at elevated temperature.
Typically, gas chromatography (GC) is the most accurate method to use for this
type of sensing, in addition to flame ionization detection, high performance liquid
chromatography (HPLC), or complex chemical derivation schemes. 3 Unfortunately,
such instrumentation is rarely found in clinical laboratories and substantial time typically
elapses from the time a sample is collected to the time when the results are obtained so
that appropriate interventions could be implemented.
Resorcinol dye has been previously immobilized in perfluorosulfonic acids
membranes to yield an orange brown product through an acid-catalyzed reaction with
various organic anhydrides. The optical response that is obtained is highly sensitive and
is measured to at least 3 parts per billion (ppb). 3
In this work, we demonstrate that the reaction of resorcinol and the biomolecule,
bradykinin, in the presence of a PSA membrane can also produce a highly sensitive and
selective response in the visible of the electromagnetic spectrum.
2. METHODS
PSAs are superacids which have the ability to replace solid acid catalysts in
heterogeneous organic synthesis. Our project uses of PSA membranes to catalyze the
reaction between bradykinin and resorcinol. The membranes contain acid sites capable
of promoting the reaction of immobilized reagents. In the case of immobilized resorcinol
dimerization can also occur to produce an optical response in the visible region
approximately at 430 nanometers.3 We will use immobilized resorcinol to react with
bradykinin following reaction Scheme 1 shown below:
Resorcinol dimer formation within the membrane under identical conditions to that of
reaction Scheme 1 constitutes the control for our experiments.
Our procedure begins by immobilizing bradykinin in a PSA membrane for 31
minutes. The specific PSA used in this study is the trade material, Nafion, produced by
DuPont. After immobilization, the membrane is removed the membrane from the
bradykinin solution and rinsed with DI water for 1 second. The DI water used for this
project was obtained by passing the house water supply through a Millipore Synthesis
unit until 18.2 M cm resistivity was achieved. We allow the immobilized bradykinin
membrane to air dry at room temperature for approximately 1 hour. UV/visible
spectrophotometer (Ocean Optics HR 2000+ CG-UV-NIR High Resolution
Spectrometer, 1 cm path length through a quartz cuvette) is used before and after
immobilization to insure that bradykinin is present in the PSA membrane as well as to
confirm negligible depletion of solution concentration.
The dried membrane containing bradykinin is immersed in resorcinol for 31
minutes and then rinsed with DI water followed by air drying at room temperature for 1
hour or longer until the membrane has become completely dry. We confirm the
presence of both bradykinin and resorcinol using a UV/vis record the spectra of the
membrane. Once bradykinin and resorcinol have both been immobilized within the PSA
membrane, we heat the membrane to 90°C for 15 minutes. After this thermal activation
of the membrane, we record the spectra using a UV/vis and analyze the results.
The control sample is prepared by first diluting the resorcinol stock solution to the
intended concentration that the other experiment used. The control is exposed to the
same experimental conditions as previously outlined, except that bradykinin is not
incorporated into the membrane. We immobilize resorcinol by immersing the PSA
membrane into the intended resorcinol solution for 31 minutes and then rinse with DI
water followed by air drying at room temperature for 1 hour or longer until the
membrane has become completely dry. We confirm the presence of resorcinol in the
PSA membrane using a UV/visible spectrophotometer to record the spectra of the
membrane. When resorcinol is confirmed to be immobilized into the PSA membrane,
we employ for ex situ monitoring by heating the membrane to 90°C for 15 minutes. After
thermo activation of the membrane record the spectra using a UV/visible
spectrophotometer and then to analyze the results. Spectroscopic analysis performed
and the control data compared to the sample data.
3. RESULTS AND DISCUSSION
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Comparison of Immobilized BK Samples
The immobilization of bradykinin into the PSA membrane prior to heating gives a
spectral response, but this response occurs at 225 nm, which is in the ultraviolet region
of the electromagnetic spectrum. This response therefore does not permit the use of
commercial LEDs. However, for ex-situ laboratory analyses, there does exist correlation
between bradykinin concentration and the absorbance value at 225nm that follows the
Beer-Lambert Law.
Figure 1 shows the spectra of various bradykinin concentrations immobilized into the
PSA membrane and recorded using a UV/visible spectrophotometer. The spectra
exhibit a peak in the Ultraviolet region of the electromagnetic spectrum. Peak
absorbance occurs at 225 nm and is correlated to the solution concentration according
to the Beer-Lambert Law. A major challenge that initially occured after immobilizing
bradykinin into the Nafion membrane and then recording the spectrum was that a
significant baseline shift above 500 nm was observed for membranes that were not
previously cleaned in boiling acid. The procedure was adjusted accordingly.
A
B
C
5
D
E
Figure 1. Spectra recorded used to determine if bradykinin was immobilized into the Nafion
membrane to asure to procede further in the experiment, as well as to asure that the BeerLambert Law was obeyed in the fact that bradykinin concentration was derictely proportional to
the absorbance value at 225nm. Labels A through E indicate the various bradykinin
concentrations used in experiments throughout the project. A)Spectra of 5 samples of
bradykinin 10mg per 100mL immobilized into PSA membranes. B) Spectra of 6 samples of
bradykinin 5mg per 100mL immobilized into PSA membranes. C) Spectra of 4 samples of
bradykinin 2mg per 100mL immobilized into PSA membranes. D) Spectra of 1 sample of
bradykinin 1mg per 100mL immobilized into a PSA membrane. E) Spectra of 2 samples of
bradykinin 0.5mg per 100mL immobilized into PSA membranes.
Comparison of Immobilized Resorcinol/Bradykinin Samples
After the immobilization of bradykinin into the Nafion membrane and recording of
spectra, we immersed the membrane into a resorcinol solution of the desired
concentration. The resorcinol solution was dilluted from a stock solution of 10g/L that
was prepared by weighing 0.1000g of resorcinol and measuring 5mL of ethanol and
5mL of DI water. We examined many resorcinol concentrations ranging from 1g/L to as
low as 0.000003g/L.
Figure 2 shows the spectra of various bradykinin concentrations immobilized into the
Nafion membrane, as well as various resorcinol concentrations previously immobilized
in the membrane. The spectra exhibit peaks in the ultraviolet region of the
electromagnetic spectrum that can be correlated to either the bradykinin or resorcinol
concentrations. The variation in peak intensities was recorded.
A
B
7
C
Figure 2. Spectra recorded after resorcinol and bradykinin were immobilized in the membrane.
Absorbance peaks in the UV ensured the presence of these molecules. Labels A through C
indicate the various bradykinin concentrations used in experiments as well as various resorcinol
concentrations used throughout the project. A) Spectra of 10 samples of bradykinin 10mg per
100mL and various resorcinol concentrations ranging from 1g/L to 0.000003g/L immobilized into
PSA membranes. B) Spectra of 6 samples of bradykinin 5mg per 100mL and various resorcinol
concentrations ranging from 0.3g/L to 0.000003g/L immobilized into PSA membranes. C)
Spectra of 4 samples of bradykinin 2mg per 100mL and resorcinol concentrations 0.3g/L and
0.00003g/L immobilized into PSA membranes.
Comparison of Activated Reaction Samples
Thermal activation is used to induce the reaction between immobilized resoricnol and
immobilized bradykinin within a perfluorosulfonic acid (PSA) membrane. The spectra of
this reaction is recorded using a UV/vis spectrophotometer and is analyzed. The
response of this reaction is noted by an absorbance near 430 nanometers.
Figure 3 shows the spectra of various bradykinin concentrations immobilized into the
Nafion membrane, as well as various resorcinol concentrations immobilized into the
previous membranes, then heated at 90⁰C to activate the reaction and recorded using a
UV/visible spectrophotometer. The response shown is in the visible region of the
electromagnetic spectrum at the wavelength of 430nm. This optical response gives the
membrane a brown-yellow appearance which can show that there can be an optical
response from the intended approach.
A
B
C
Figure 3. Spectra recorded after indicated samples were heated to 90⁰C. Peak absorbance at
430 nm was used to monitor reaction. Labels A through C indicate the various bradykinin
concentrations used in experiments as well as various resorcinol concentrations used
throughout the project. A) Spectra of 10 samples of bradykinin 10mg per 100mL and various
resorcinol concentrations ranging from 1g/L to 0.000003g/L immobilized into PSA membranes
heated at 90⁰C. B) Spectra of 6 samples of bradykinin 5mg per 100mL and various resorcinol
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concentrations ranging from 0.3g/L to 0.000003g/L immobilized into PSA membranes heated at
90⁰C. C) Spectra of 4 samples of bradykinin 2mg per 100mL and resorcinol concentrations
0.3g/L and 0.00003g/L immobilized into PSA membranes heated at 90⁰C.
Comparison of Control Samples
The only difference between the control measurements and the sample data previously
summarized was that bradykinin was not immobilized within the PSA membrane in the
former. Only resorcinol at the various concentrations of interest was present in the
membrane. These resorcinol concentrations yielded corresponding peaks in the UV
region of the spectra.
Figure 4 shows the spectra that resulted from the various concentrations of resorcinol
solutions that were immobilized into the Nafion membrane. Resorcinol immobilization
yielded two peaks in the UV region of the electromagnetic spectrum around the
wavelengths 225nm and 275nm, respectively, that correlated to the resorcinol
concentration.
Figure 4. Spectra recorded showing the comparison between various resorcinol concentrations
that were used throughout the project. All background was fitted to 300 nm. Spectra of 4
samples of resorcinol concentrations 1g/L, 0.5g/L, 0.3g/L and 0.2g/L immobilized into PSA
membranes.
Comparison of Heated Control Samples
Control membranes were subsequently heated at 90⁰C. As in the samples containing
bradykinin, an optical response in the visible region of the electromagnetic spectrum
occurred with a peak at 430 nm. This results from resorcinol dimerization and presents
a problem as it convolutes with the bradykinin response. We note from Figure 5 that the
response appears to be independent of resorcinol concentration and in many cases is
of much lower intensity than when bradykinin is present. Such behavior may permit
deconvolution of the two responses.
Figure 5. Spectra recorded for various immobilized resorcinol concentrations. The peak
absorbance in the visible region occurs at about 430 nm. Spectra of 3 samples of resorcinol
concentrations 1g/L, 0.5g/L and 0.3g/L immobilized into PSA membranes heated at 90⁰C.
Calibration Curve
We next attempted to calibrate the peak intensity at 430nm to the bradykinin
concentration by keeping the resorcinol concentration constant at 0.3 g/L. The results
are shown in Figure 6. A statistically significant correlation is indeed observed, despite
the low light absorption intensity. However, the correlation is inverse that predicted by
the Beer-Lambert Law and indicates an alternate mechanism for light absorption. We
hypothesize a site blocking mechanism whereby acid sites within the membrane
become less catalytically active as the reagant concentration increases. Confirmation of
this hypothesis as well as whether the low light absorption is sufficient for commercial
application must wait for further study.
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Figure 6. Shows the calibration curve with respect to the resorcinol concentration 0.3g/L. The
three different bradykinin concentrations that were used in the calibration were 10mg, 5mg and
2mg per 100mL. The error bars show one standard deviation about the mean.
4. CONCLUSIONS
We have observed that it is possible to immobilize, react, and optically differentiate
bradykinin relative to a control at the wavelength 430nm. The UV response to
bradykinin is more intense and may provide an alternate, if less compact and delayed,
sensing approach than a visible response. Either approach would be more cost effective
and provide results for therapeutics more quickly than existing GC/MS methods.
5. ACKNOWLEDGEMENTS
This work was sponsored in part by National Science Foundation Grant # DUE-0756921
for the REU Site on the College of Engineering and Applied Science. A special thanks
to the Graduate Mentor, Srivasthan Ravi, and Faculty Advisor, Dr. Anastasios
Angelopoulos.
References
(1)
Angelopoulos A, Bernstein JA, Kanter D, Ayyadurai S. Optical sensor for
monitoring environmental condition comprises perfluorosulfonate ionomer
membrane comprising solution containing transition metal-free dye component.
University of Cincinnati, 2010.
(2)
Angelopoulos AP, Tremblay MS, Kim YH. Surface and bulk interactions of an
epoxy based azo polymer with a perfluorosulfonate ionomer (Nafion) membrane.
Abstracts of Papers of the American Chemical Society 2000; 220:316-COLL.
(3)
Ayyadurai, S. M., Worrall, A. D., Bernstein, J.A., and Angelopoulos, A.P. (2010).
“Perfluorosulfonic Acid Membrane Catalysts for Optical Sensing of Anhydrides in
the Gas Phase,” Analytical Chemistry, 82, 6265-6272
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