Project #3: Optical Method of Detection and Analysis of Biological Molecules

1. Project #3: Optical Method of 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 Faculty Mentor: Dr. Anastasios Angelopoulos

2. Purpose • 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 • To develop catalytic base sensor to selectively respond to Bradykinin

3. Background • 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 • Performed reactions by immobilizing BK and then immobilizing Resorcinol into the Nafion membrane

4. Background (cont’d) • However, the spectra response showed that the absorbance of BK gets convoluted with the absorbance of Resorcinol which makes it difficult to quantify BK • To quantify BK, BK was immobilized into the membrane before Resorcinol • The reaction between BK and resorcinol inside the membrane is done by heating the membrane on a hot plate at 90⁰C • However, the optical response of the reaction interferes with the response of the control (resorcinol) at 90⁰C • Series of experiments were done to assist the interference of BK reaction with control • Data showed that optimal responsiveness to BK can be achieved by keeping resorcinol concentration as low as possible

5. Materials • Pipet • Pipet Tips (Resorcinol, Water, Ethanol, BK) • Petri Dishes • UV-Visible Spectrometer • Hot Plate • Tweezers • Program – SpectraSuite • Nafion Membrane • Gloves • Thermometer

6. Elements • Bradykinin • Resorcinol • Water • Ethanol

7. Methods & Procedures • 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

8. Methods & Procedures (cont’d) • 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

9. Experiment • 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 • Analysis of the spectra show that the baselines of all the peaks were shifted due to possible contamination of the Nafion membrane

13. Goal • To get the baseline closer to the origin to obtain better quality of data

14. Tasks • Pretreatment of the Nafion membrane with sulfuric acid before the immobilizing of BK

15. Timeline