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
Saturday, February 23, 2019

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
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:
Preparing the 0.5 Resorcinol Solution
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
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
UV-Visible Spectrometer
Bradykinin 10mg Stock Solution
Resorcinol Solutions
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 BK in 100 mL Water 4 Resorcinol Solutions: 1 g/L
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

11. Results

12. Results

13. Results

14. Results
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. Goals
Obtain Maximum Absorbance Point

16. Tasks Low Concentrations Preparing a Resorcinol Solution
Placing a Membrane in a Resorcinol Solution
Observe low concentrations of Resorcinol (0.003, , )
Heat the background as well at 90C

17. Timeline – Week 1 Monday Tuesday Wednesday Thursday Friday
Research Training
Literature Research
Background Briefing
Progress Meeting
Workshop
Enrichment Seminar
Progress Report

18. Timeline – Week 2 Monday Tuesday Wednesday Thursday Friday
Research Training
Literature Research
Background Briefing
Progress Meeting
Workshop
Presentation Preparation
Progress Report
Field Trip
Conducting Research
Biweekly Presentation
Technical Paper

19. Timeline – Week 3 Monday Tuesday Wednesday Thursday Friday
Literature Research
Workshop
Progress Meeting
Field Trip
Conducting Research
Progress Report
Presentation Preparation
Technical Paper
Poster Preparation

20. Timeline – Week 4 Monday Tuesday Wednesday Thursday Friday
Literature Research
Progress Meeting
Field Trip
Conducting Research
Progress Report
Presentation Preparation
Technical Paper
Poster Preparation
Enrichment Seminar
Biweekly Presentation

21. Timeline – Week 5 Monday Tuesday Wednesday Thursday Friday
Literature Research
Progress Meeting
Conducting Research
Progress Report
Presentation Preparation
Technical Paper
Poster Preparation

22. Timeline – Week 6 Monday Tuesday Wednesday Thursday Friday
Literature Research
Progress Meeting
Conducting Research
Progress Report
Presentation Preparation
Technical Paper
Poster Preparation
Field Trip
Biweekly Presentation
Workshop

23. Timeline – Week 7 DRAFT Monday Tuesday Wednesday Thursday Friday
Literature Research
Progress Meeting
Conducting Research
Progress Report
Presentation Preparation
Technical Paper
DRAFT
Poster Preparation
Enrichment Seminar

24. Timeline – Week 8 FINAL PRESENT Monday Tuesday Wednesday Thursday
Friday
Presentation Preparation
Technical Paper
FINAL
Poster Preparation
Progress Meeting
Final Presentation
PRESENT

25. Questions?