Detection of Bradykinin with Reactive Dye Molecule Approach Heather K. Cooper, Chemistry, University...
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Detection of Bradykinin with Reactive Dye Molecule Approach Heather K. Cooper, Chemistry, University of Cincinnati Kyle A. Frank, Chemical Engineering, University of Cincinnati Graduate 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 Introduction Methods & Materials Results Acknowledgements References Conclusions 1. 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. 2. 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. 3. 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. • The use of catalytic membranes as optodes offers a new technological approach in medical diagnostics. • The feasibility of the approach has been demonstrated with various low-molecular weight reagents (anhydrides, acetone, and formaldehyde), but has never been previously attempted with biomolecules. • The approach enables access to prompt and rapid data that can be used to target therapies appropriately. 0 2 4 6 8 10 12 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 f(x) = − 0.00847448979591837 x + 0.141188775510204 R² = 0.994849639622617 Calibration Curve for Resorcinol 0.3 g/L Resorcinol 0.3 g/L Linear (Resorcinol 0.3 g/L) Control Bradykinin Concentration, mg/L per 100mL Absorbance (430nm) 200 300 400 500 600 700 800 0 0.5 1 Bradykinin 10mg Samples Sample 1 Sample 2 Sample 3 Sample 4 Wavelength, nm Absorbance 200 300 400 500 600 700 800 0 0.5 1 Bradykinin 10mg Samples with Resorcinol heated at 90⁰C Sample 1 with 1 g/L Res Sample 2 with 0.5 g/L Res Sample 3 with 0.3 g/L Res Wavelength, nm Absorbance 200 300 400 500 600 700 800 0 0.5 1 Resorcinol Membranes heated at 90⁰C 1 g/L 0.5 g/L 0.3 g/L Wavelength, nm Absorbance • Immobilizatio n of bradykinin into the Nafion Membrane giving a response in the UV region of the electromagnet ic spectrum. • Control: Only resorcinol was immobilized into the PSA Membrane and then heated to obtain optical response in the visible region of the electromagnet ic spectrum (dimerization ). • Sample: 10 mg bradykinin was first immobilized with various resorcinol concentration s nd then heated to obtain an optical response in the visible region of the electromagnet ic spectrum (proposed addition reaction). • The calibration curve taken at various Bradykinin concentration s and at Resorcinol 0.3 g/L . Research Experiences for Undergraduates (REU) Program, College of Engineering and Applied Science (CEAS), University of Cincinnati, Project #3: Optical Method for Detection and Analysis of Biological Molecules, NSF Grant # DUE-0756921. A special thanks to the Graduate Mentor, Srivasthan Ravi, and Faculty Advisor, Dr. Anastasios Angelopoulos. • UV signal is sufficient to permit spectroscopic analysis of biomolecules to provide more cost- effective alternative to Gas Chromatography. • It possible to optically detect biomolecules in the visible region of the electromagnetic spectrum using a catalytic membrane approach. • Product signal after bradykinin reaction with resorcinol can be calibrated to concentration. • Calibration is inverse of the Beer-Lambert law relationship, suggesting that bradykinin may be impeding catalytic activity at acid sites in the PSA membrane. Activation Sites Bradykinin • Proposed reaction between bradykinin and resorcinol to give an optical response in the visible region of the electromagnetic spectrum: • Procedural diagram to react bradykinin with resorcinol and assess optical response. Heating at 90 C is used to activate the reaction. Control consisted of resorcinol immobilized in PSA membrane without bradykinin and exposed to identical reaction conditions as the bradykinin samples. Membrane heating at 90 degrees Celsius on a hot plate. A row of 0.3g/L Resorcinol solutions with immersed already immobilized BK membranes for the Calibration Curve. Absorbance = 0.066 Wavelength = 428.34 nm Absorbance = 0.088 Wavelength = 432.02 nm O H OH N afio n-H + N H O NH NH 2 NH OH R 1 OH N H O NH NH 2 NH O R 1 + O H 2
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Transcript of Detection of Bradykinin with Reactive Dye Molecule Approach Heather K. Cooper, Chemistry, University...
Detection of Bradykinin with Reactive Dye Molecule ApproachHeather K. Cooper, Chemistry, University of Cincinnati
Kyle A. Frank, Chemical Engineering, University of CincinnatiGraduate 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
Introduction
Methods & Materials
Results
Acknowledgements
References
Conclusions
1. 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, 2010.2. 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.3. 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.
• The use of catalytic membranes as optodes offers a new technological approach in medical diagnostics.
• The feasibility of the approach has been demonstrated with various low-molecular weight reagents (anhydrides, acetone, and formaldehyde), but has never been previously attempted with biomolecules.
• The approach enables access to prompt and rapid data that can be used to target therapies appropriately.
0 2 4 6 8 10 120
0.02
0.04
0.06
0.08
0.1
0.12
0.14
f(x) = − 0.00847448979591837 x + 0.141188775510204R² = 0.994849639622617
Calibration Curve for Resorcinol 0.3 g/L
Resorcinol 0.3 g/LLinear (Resorcinol 0.3 g/L)Control
Bradykinin Concentration, mg/L per 100mL
Abso
rban
ce (4
30nm
)
200 300 400 500 600 700 8000
0.5
1
Bradykinin 10mg Samples
Sample 1Sample 2Sample 3Sample 4
Wavelength, nm
Abso
rban
ce
200 300 400 500 600 700 8000
0.5
1 Bradykinin 10mg Samples with Resorcinol heated at 90⁰C
Sample 1 with 1 g/L Res
Sample 2 with 0.5 g/L Res
Sample 3 with 0.3 g/L Res
Wavelength, nm
Abso
rban
ce
200 300 400 500 600 700 8000
0.5
1 Resorcinol Membranes heated at 90⁰C
1 g/L
0.5 g/L
0.3 g/L
Wavelength, nm
Ab
sorb
an
ce
• Immobilization of bradykinin into the Nafion Membrane giving a response in the UV region of the electromagnetic spectrum.
• Control: Only resorcinol was immobilized into the PSA Membrane and then heated to obtain optical response in the visible region of the electromagnetic spectrum (dimerization).
• Sample: 10 mg bradykinin was first immobilized with various resorcinol concentrations nd then heated to obtain an optical response in the visible region of the electromagnetic spectrum (proposed addition reaction).
• The calibration curve taken at various Bradykinin concentrations and at Resorcinol 0.3 g/L .
Research Experiences for Undergraduates (REU) Program, College of Engineering and Applied Science (CEAS), University of Cincinnati, Project #3: Optical Method for Detection and Analysis of Biological Molecules, NSF Grant # DUE-0756921. A special thanks to the Graduate Mentor, Srivasthan Ravi, and Faculty Advisor, Dr. Anastasios Angelopoulos.
• UV signal is sufficient to permit spectroscopic analysis of biomolecules to provide more cost-effective alternative to Gas Chromatography.
• It possible to optically detect biomolecules in the visible region of the electromagnetic spectrum using a catalytic membrane approach.
• Product signal after bradykinin reaction with resorcinol can be calibrated to concentration.
• Calibration is inverse of the Beer-Lambert law relationship, suggesting that bradykinin may be impeding catalytic activity at acid sites in the PSA membrane.
Activation Sites Bradykinin
OH OH N afio n -H+NH
O
NH
NH2
NH
OH
R1
OH
NH
O
NH
NH2
NH
O
R1
+ OH2
• Proposed reaction between bradykinin and resorcinol to give an optical response in the visible region of the electromagnetic spectrum:
• Procedural diagram to react bradykinin with resorcinol and assess optical response. Heating at 90 C is used to activate the reaction. Control consisted of resorcinol immobilized in PSA membrane without bradykinin and exposed to identical reaction conditions as the bradykinin samples.
Membrane heating at 90 degrees Celsius on a hot plate.
A row of 0.3g/L Resorcinol solutions with immersed already immobilized BK membranes for the Calibration Curve.
Absorbance = 0.066Wavelength = 428.34 nm
Absorbance = 0.088Wavelength = 432.02 nm
