Development of a Novel Nitric Oxide Sensor Using Nitrophorin 4 on an Attenuated Total Reflectance Platform

Persistent Link:
http://hdl.handle.net/10150/268592
Title:
Development of a Novel Nitric Oxide Sensor Using Nitrophorin 4 on an Attenuated Total Reflectance Platform
Author:
Lemon, John
Issue Date:
2012
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Nitric oxide plays a major role in physiology and disease pathology. There are many available methods for the detection of NO; however, these techniques typically detect products of nitric oxide decomposition. Herein, I present a novel method of direct nitric oxide detection using a nitrophorin mutant to capture NO on an optical waveguide platform. Nitrophorins are unique among ferric proteins for their ability to bind NO strongly. The spectral shift of the Soret band of the Nitrophorin was used to monitor NO concentration. The limit of detection was found to be 18 nM, and a linear response to 225 nM. The sensor is highly specific, non-destructive and reusable. Detection of NO was demonstrated in a solution of BSA at serum level concentration, and cell culture solution containing 10% FBS. This method allows for direct NO with high specificity, low detection limit and good temporal resolution. Also described herein are the investigations of the structure of the NO receptor, soluble guanylate cyclase (sGC). sGC is a 150 kDa heterodimeric protein that catalyzes the production of cyclic guanosine monophosphate from guanosine triphosphate, which leads to many downstream affects such as vasodilation. The structural analysis was performed with transmission electron microscopy (TEM). Data presented indicate that the protein is too heterogeneous to be reconstructed with TEM. This is either the result of the sample preparations examined, the purity of the sample, the inherent flexibility or conformational heterogeneity of the protein after applying the sample to the TEM grid.The final project presented describes the use of silica colloidal crystals for the enhancement of the sensitivity of protein microarrays as a function of silica particle size. Protein microarrays are a tool used to discover biomarkers of diseases, and increasing the sensitivity lowers the limit of detection of the method. This is accomplished by an increase of the surface area available for proteins to bind, and by using silica so that the surface chemistry of the microarray is maintained. It was concluded that 510 nm colloids provide the greatest enhancement of the fluorescence of a BSA ant-BSA-FITC system, providing a 17-fold enhancement over the control.
Type:
text; Electronic Dissertation
Keywords:
Nitrophorin; Chemistry
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Montfort, William R.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleDevelopment of a Novel Nitric Oxide Sensor Using Nitrophorin 4 on an Attenuated Total Reflectance Platformen_US
dc.creatorLemon, Johnen_US
dc.contributor.authorLemon, Johnen_US
dc.date.issued2012-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractNitric oxide plays a major role in physiology and disease pathology. There are many available methods for the detection of NO; however, these techniques typically detect products of nitric oxide decomposition. Herein, I present a novel method of direct nitric oxide detection using a nitrophorin mutant to capture NO on an optical waveguide platform. Nitrophorins are unique among ferric proteins for their ability to bind NO strongly. The spectral shift of the Soret band of the Nitrophorin was used to monitor NO concentration. The limit of detection was found to be 18 nM, and a linear response to 225 nM. The sensor is highly specific, non-destructive and reusable. Detection of NO was demonstrated in a solution of BSA at serum level concentration, and cell culture solution containing 10% FBS. This method allows for direct NO with high specificity, low detection limit and good temporal resolution. Also described herein are the investigations of the structure of the NO receptor, soluble guanylate cyclase (sGC). sGC is a 150 kDa heterodimeric protein that catalyzes the production of cyclic guanosine monophosphate from guanosine triphosphate, which leads to many downstream affects such as vasodilation. The structural analysis was performed with transmission electron microscopy (TEM). Data presented indicate that the protein is too heterogeneous to be reconstructed with TEM. This is either the result of the sample preparations examined, the purity of the sample, the inherent flexibility or conformational heterogeneity of the protein after applying the sample to the TEM grid.The final project presented describes the use of silica colloidal crystals for the enhancement of the sensitivity of protein microarrays as a function of silica particle size. Protein microarrays are a tool used to discover biomarkers of diseases, and increasing the sensitivity lowers the limit of detection of the method. This is accomplished by an increase of the surface area available for proteins to bind, and by using silica so that the surface chemistry of the microarray is maintained. It was concluded that 510 nm colloids provide the greatest enhancement of the fluorescence of a BSA ant-BSA-FITC system, providing a 17-fold enhancement over the control.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectNitrophorinen_US
dc.subjectChemistryen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMontfort, William R.en_US
dc.contributor.committeememberSaavedra, S. Scotten_US
dc.contributor.committeememberMiranda, Katrina M.en_US
dc.contributor.committeememberHeien, Michael L.en_US
dc.contributor.committeememberMontfort, William R.en_US
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