Nano-structured platform for highly selective, sensitive and quantitative electrochemical detection of small redox-active cellular messengers in aqueous buffer solution with biological interferrents

The objective of this thesis is to develop a robust, flexible and versatile nano-structuredplatform for the detection of small redox-active biomessengers and demonstrate the platform by applying it to the detection of H2O2, CO and H2S. Endogenously generated small redox-active molecules represent a unique class of cellular messengers in biological systems. Examples include reactive-oxygen-species (ROS) such as hydrogen peroxide (H2O2) and superoxide O2^-, reactive-nitrogen-species (RNS) such as NO, HNO and ONOO-, as well as carbon monoxide (CO) and hydrogen sulfide (H2S), among others. Their generation, targeting and regulation are associated with diverse yet critically important biological, physiological and pathological functions. For the study of their biological functions as well as the elucidation of their working mechanisms, it is highly desirable to have methodologies and techniques for their detection in a selective, sensitive and quantitative manner and in the presence of many interfering chemical species. Chapter 1 gives a concise introduction and overview of the importance of detecting small cellular messengers, the advantages of using electrochemical method as a sensing technique and the reasons for using nanotechnology. The objective of this thesis is also included. Chapters 2 reports our effort in the development of a nano-structured electrochemical platform and its usage in the detection of H2O2 and glucose in aqueous buffer solution with biological interferrents. This platform contained three functional layers: an analyte-dependent catalyst, a nano-structured filter and a substrate surface for immobilization of biomolecules or other desirable macromolecules. By using nano-structured platinum (Pt) as the catalyst, we were able to measure H2O2 by electrochemical oxidation in a broad concentration range, and in a sensitive, quantitative, and selective manner. As a natural extension to the detection of H2O2, we developed a glucose sensor by combining the H2O2-sensing platform with the immobilization of glucose oxidase (GOD) on the nano-structured surface spatially separated from that of the H2O2-catalysts surface. The selective detection of H2O2 and glucose in the presence of common interfering chemical species was also studied. The linear range of detecting H2O2 we achieved was from 10μM to 5mM. The linear range of detecting glucose we achieved was from 50μM to 5mM. The detection limit was 40μM. The Km of the glucose oxidase we found on GOx - Pt nano-frame electrode was 24.02mM. The interference coefficient of ascorbic acid with respect to H2O2 detection by using the DPA method we developed on Au nano-frame electrode was 0.114. The measurable range of glucose without normal level (100μM) ascorbic acid significant interference (20%) was 500μM to 5mM. Chapters 3 reports a method developed recently in our group for the detection of CO in aqueous buffer solution. The method was built on the similar nano-structured electrochemical platform that we developed for the detection of H2O2 and glucose but with one important difference: we chose a nano-structured electro-active pseudo-catalyst (electrochemical mediator), Cu2O, for the detection of CO. The reason was that CO is an electronically very inert molecule toward either catalytic oxidation or reduction in aqueous medium and in ambient conditions. By selecting Cu2O as a pseudo-catalyst (electrochemical mediator), we employed a non-redox reaction, yet electrochemical active approach for the detection of CO in aqueous buffer solution selectively and quantitatively. This approach has an intrinsic advantage of immune to many interfering species. The pros and cons of our method will be discussed. The detection limit of CO on Cu sputtering electrode was found to be 4.60μM using ΔIpc and 19.54 μM using ΔIpa. The detection limit of CO on Cu deposition electrode was found to be 4.95μM using ΔIpc and 21.27μM using ΔIpa. For low concentration range from 5 to 50μM, using ΔIpc, the sensitivity of Cu sputtering electrode was about 3 times higher than that of the Cu deposition electrode, hence the Cu sputtering fabrication method was better than the Cu deposition fabrication method. Chapters 4 reports a very promising method for the highly selective, sensitive and quantitative detection of H2S in aqueous buffer solution and in the presence of the most common interfering species including cysteine, methionine, and glutathione. Our method was built on the nano-structured electrochemical platform we developed for the detection of H2O2 and glucose, but with the selection of nano-structured gold (Au) catalyst uniquely suited for thiol-containing compounds such as H2S. Special effort was spent on solving the interfering problems caused by cysteine, methionine and glutathione. The linear range and detection limit of detecting H2S by desorption method we developed were from 5μM to 200μM and 2μM respectively. The linear range and detection limit of detecting H2S by adsorption method were from 10μM to 3mM and 10μM respectively. The interference coefficient of cysteine with respect to H2S detection by using the DPA desorption method on nafion - Au nano-frame electrode was 0.00915. There was no interference found from methionine and glutathione by using the DPA desorption method on nafion - Au nano-frame electrode. This thesis reports the development of a new, robust and flexible electrochemical platform for the detection of various redox-active gaseous molecules in aqueous media. This platform has a unique advantage of integrating and supporting three nano-structured components: an analyte-dependent catalyst, a screening or filtering structure, and a substrate-surface for immobilization of biomolecules (or other desirable macromolecules). A brief summary and future perspectives are given at the end of the thesis