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

Introduction Bradykinin Hereditary Angioedema Photo Source: http://api.ning.com/files/lTsGW1TpdpRTPVTM1gtNqdfyrDJO3PY6rdUnZEM73bZyecxnSzuz2h73vqVRL90VfdPJ4xMvREjn3Womj6xEGFxGyDtLixf0/MyrtleHAE9108comp.jpg 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. Photo Source: http://upload.wikimedia.org/wikipedia/commons/5/51/Bradykinin_updated.png

Objective Use optical spectroscopy to detect and quantify Bradykinin Photo Source: http://www.uwgb.edu/heuerc/2D/ColorWheel1.jpg 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 Photo Source: http://www.dnr.sc.gov/ael/personals/pjpb/lecture/spectrum.gif

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

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

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

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

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

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

Results

Results

Results

Results

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

Goals Obtain Maximum Absorbance Point

Tasks Low Concentrations Preparing a Resorcinol Solution Placing a Membrane in a Resorcinol Solution Observe low concentrations of Resorcinol (0.003, 0.0003, 0.00003) Heat the background as well at 90C

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

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

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

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

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

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

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

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

Questions?