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, Associate Professor, School of Energy, Environmental, Biological and Medical Engineering, College of Engineering and Applied Science, University of Cincinnati 1Thursday, August 9, 2012

2. Introduction Bradykinin Hereditary Angioedema Photo Source: http://api.ning.com/files/lTsGW1TpdpRTPVTM 1gtNqdfyrDJO3PY6rdUnZEM73bZyecxnSzuz2 h73vqVRL90VfdPJ4xMvREjn3Womj6xEGFxGy DtLixf0/MyrtleHAE9108comp.jpg Photo Source: http://upload.wikimedia.org/wikipedia/commons/5/5 1/Bradykinin_updated.png 2

3. Objective Catalytic based sensor to selectively respond to Bradykinin in blood Understand principles of color & UV- Visible Spectroscopy Concentrate on visible region to permit use of inexpensive and portable LED's as a light source Photo Source: http://www.dnr.sc.gov/ael/personals/pjpb/lecture/spectrum.gif Photo Source: http://www.uwgb.edu/heuerc/2D/ColorWheel1.jpg 3

4. Approach Perflurosulfonic acid (Nafion®) membrane as catalysts Specific reaction between analyte and reactive dye molecule within the membrane Optical response monitored using visible light spectroscopy 4 Analyte Reactive dye molecule (resorcinol) Nafion membrane Product Fig 1. Catalytic optodes after exposure to indicated TMA concentrations. Fig 2. Catalytic optode exposed to formaldehyde UV Visible Region Prior Work

5. Approach Cont. Proposed Sensing Reaction: 5 Fig 3. Preparing the 0.5g/L Resorcinol Solution Fig 4. Placing the Membrane in the Solution

6. Methods 6 Sample Preparation ◦ Pretreatment ◦ PSA Membranes ◦ Immobilize BK ◦ Immobilize Resorcinol Thermo activation ◦ Reaction at 90 ⁰C ◦ UV-Visible Spectrophotometer Fig 5. Conducting the Reaction at 90 ⁰C on a Hot Plate

7. Materials & Instrumentation Fig 6. UV-Visible Spectrophotometer Fig 7. Bradykinin 10mg in 100mL Stock Solution Fig 8. Resorcinol Solutions Fig 9. Light Path from UV-Visible Spectrophotometer 7

8. Procedure Pretreatment of Nafion Membrane Prepare Stock Solutions Immobilize Bradykinin Record Spectra Immobilize Resorcinol Heat at 90 ⁰ C Compare/Analyze Graphs Immobilize Resorcinol Heat at 90 ⁰ C 8

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 ◦ 0.00003 g/L ◦ 0.000003 g/L 9 Fig 10. Nafion Membrane Immersed in a Resorcinol Solution

10. Results - Control 10

11. Results - Control 11

12. Results - Reaction 12

13. Results - Reaction 13

14. Results - Reaction 14

15. Results 15

16. Calibration Curve 16

17. Conclusions Bradykinin Yields UV Signal Catalytic Approach can Detect Biomolecules Response can be Calibrated to Bradykinin Concentration Blocked Active Sites: 17

18. References 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, 6265-6272 18

19. Questions? 19