1. Project #3: Optical Method for Detection and Analysis of Biological Molecules
Participants:
Heather K. Cooper, Chemistry, University of Cincinnati, Senior
Kyle A. Frank, Chemical Engineering, University of Cincinnati, Sophomore
Graduate Student Mentor:
Srivasthan Ravi, Chemical Engineering, University of Cincinnati
Faculty Mentor:
Dr. Anastasios Angelopoulos, Chemical Engineering, University of Cincinnati
Thursday, September 20, 2018Thursday, September 20, 2018

2. Introduction Bradykinin Hereditary Angioedema
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Bradykinin is a peptide that causes blood vessels to dilate and therefore causes blood pressure to lower
Overactivation of Bradykinin is thought to play a role in a rare disease called Hereditary Angioedema
Studies indicate a need to directly monitor BK in circulation to target therapies appropriately
Unfortunately direct monitoring of BK is found to be difficult due its very low concentrations in blood.
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3. Objective Use optical spectroscopy to detect and quantify Bradykinin
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Objective
Use optical spectroscopy to detect and quantify Bradykinin
Understand principles of color
Learn to use UV-Visible Spectrophotometer
Concentrate on visible region to permit use of inexpensive and portable LED's as a light source
The objective is to develop a catalytic based sensor to selectively respond to Bradykinin in blood
The color perceived is the complementary color of the color absorbed by the object
3 factors can influence the perception of color: light source, object being viewed, and observer (person)
When light passes through a compound, some of the energy in the light kicks an electron from one of the bonding or non-bonding orbitals into one of the anti-bonding ones
The energy gaps between these levels determine the frequency (or wavelength) of the light absorbed
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4. Approach Analyte Prior Work
Perflurosulfonic acid (Nafion®) membrane as catalysts
Specific reaction between analyte and reactive dye molecule within the membrane
Optical response monitored using visible light spectroscopy
Analyte
Reactive dye molecule
(resorcinol)
Nafion
membrane
Product UV
Visible Region UV
PSA membranes are previously shown to catalyze reactions between a reagent and analyte.
Prior Work
Fig 1. Catalytic optodes after exposure to indicated TMA concentrations.
Fig 2. Catalytic optode exposed to formaldehyde

5. Approach Cont. Proposed Sensing Reaction:
Fig 3. Preparing the 0.5 Resorcinol Solution
Fig 4. Placing the Membrane in the Solution
Diffuse Resorcinol and then BK into the membrane
If the membrane doesn’t show a colored response at room temperature, we heat the membrane at 90C to get a colored response
Why 90 – membrane starts to show a colored response at that temperature through past experiments (has been done for less temperature)
High molecular weight compound = high activation energy
Can go to higher temperatures but not beyond 120, membrane starts to deteriorate

6. Methods Sample Preparation Thermo activation PSA Membranes
Immobilize BK
Immobilize Resorcinol
Thermo activation
Reaction at 90⁰C
UV-Visible Spectrometer
Fig 5. Conducting the Reaction at 90⁰C on a Hot Plate
PSA membrane in the form of thin films (.5cm X 2cm)
Soak and dry into BK Solution to immobilize Resorcinol into the membrane
Soak and dry into Resorcinol Solution to immobilize Resorcinol into the membrane
Heat membrane at 90C
A colored response is observed using UV-Visible Spectrometer

7. Materials & Instrumentation
Fig 6. UV-Visible Spectrometer
Fig 7. Bradykinin 10mg Stock Solution
Fig 8. Resorcinol Solutions
Fig 9. Light Path from UV-Visible Spectrometer
Pipet
Pipet Tips (Resorcinol, Water, Ethanol, BK)
Petri Dishes
UV-Visible Spectrometer
Hot Plate
Tweezers
Program – SpectraSuite
Nafion Membrane
Gloves
Thermometer
Bradykinin
Resorcinol
Water
Ethanol

8. Procedure R E C A O C N T T I R O N L Pretreat the Nafion Membrane
Pretreatment of Nafion Membrane R E A C T I O N
Prepare Stock Solutions C O N T R L
Immobilize Bradykinin
Immobilize Resorcinol
Immobilize Resorcinol
Record Spectra
Pretreat the Nafion Membrane
BK Stock Solution
Immobilize BK
Resorcinol Solutions
Immobilize Resorcinol
Activate the Reaction
Prepare BK Stock Solution
Prepare Resorcinol Stock Solution
Immobilize BK into a Nafion Membrane
Record the Spectra of the BK Membrane
Dilute the Resorcinol Solution to very low concentration
Immobilize Resorcinol into the BK Membrane
Record the Spectra of the BK/Resorcinol Membrane
Heat the BK/Resorcinol Membrane at 90⁰C
Record the Spectra of the Reaction
Prepare the Control Membranes of the Previous Reaction
Record the Spectra of the Control Membranes
Heat the Control Membranes at 90⁰C
Record the Spectra of the Heated Control Membranes
Compare graphs to analyze the responses from both the reaction and the control
Heat at 90⁰C
Heat at 90⁰C
Compare/Analyze Graphs

9. Procedure 10mg, 5mg, 2mg, 1mg and 0.5mg BK in 100 mL Water
7 Resorcinol Solutions:
1 g/L
0.5 g/L
0.3 g/L
0.03 g/L
0.003 g/L
g/L
g/L
Fig 10. Nafion Membrane Immersed in a Resorcinol Solution
BK stock solution was prepared by mixing 10mg of BK in 100mL water
BK was immobilized into 3 membranes and spectra of the membranes were recorded
Different concentrations of Resorcinol were prepared: 1g/L, 0.5 g/L, 0.2 g/L
These 3 concentrations were immobilized in the 3 BK membranes
These a spectra of these 3 membranes were recorded

10. Results - Control

11. Results - Control

12. Results - Reaction

13. Results - Reaction

14. Results - Reaction
Background and sample membranes pretreated in boiling acid to ensure cleanliness
Baselines corrected to background signal at 300 nm (no reaction)
Statistics of sample variation assessed

15. Results

16. Results

17. Project Goals Maximum Absolute Difference Absorbance MAIN GOAL:
-To use these understandings to identify and quantify Bradykinin (BK) at very low concentrations

18. Student Goals Research Processes Teamwork Technical Language
Learn the processes of Research and what the environment is like to work in.
Learn to work as a efficient team working to each individuals strengths and helping each others weaknesses.
Learn the technical language in presenting your research and how to present it effeciently.
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19. Tasks Observe Various BK Concentrations
Fig 12. Preparing a Resorcinol Solution
Fig 11. Placing a Membrane in a Resorcinol Solution

20. Timeline Progress Meeting = ~2 per week Progress Reports = 1 per week
Equipment Training
Background Briefing
Literature Research
Conducting Research
Data Analysis
Technical Paper
Poster
Presentation
Progress Meeting = ~2 per week
Progress Reports = 1 per week
Bi-weekly Presentations = 1 per 2 weeks

21. Deliverables

22. References http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0002427/
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.
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.
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,

23. Questions?