Technical Paper: Optical Method for Detection and Analysis of Biological Molecules

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 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 co-reagent (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.

1. 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.

1. RESULTS AND DISCUSSION

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 225nm, 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 thatfollows 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 500nm was observed for membranes that were not previously cleaned in boiling acid. The procedure was adjusted accordingly.

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 Beer-Lambert 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 thebradykinin or resorcinol concentrations. The variation in peak intensities was recorded.

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.

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 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 immobilizedresorcinol concentrations. The peak absorbance in the visible region occurs at about 430nm.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.

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 deviationabout the mean.

1. 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 monitoringenvironmental condition comprises perfluorosulfonateionomer membrane comprising solutioncontaining 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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