The objective of this thesis is to develop a robust, flexible and versatile nano-structured
platform for the detection of small redox-active biomessengers and demonstrate the platform
by applying it to the detection of H
2O
2, CO and H
2S.
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 (H
2O
2) and superoxide O
2-, reactive-nitrogen-species (RNS) such as NO,
HNO and ONOO
-, as well as carbon monoxide (CO) and hydrogen sulfide (H
2S), 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 H
2O
2 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 H
2O
2 by electrochemical oxidation in a broad concentration range, and
in a sensitive, quantitative, and selective manner. As a natural extension to the detection of
H
2O
2, we developed a glucose sensor by combining the H
2O
2-sensing platform with the
immobilization of glucose oxidase (GOD) on the nano-structured surface spatially separated
from that of the H
2O
2-catalysts surface. The selective detection of H
2O
2 and glucose in the
presence of common interfering chemical species was also studied.
The linear range of detecting H
2O
2 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 H
2O
2 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 H
2O
2 and glucose but with one important
difference: we chose a nano-structured electro-active pseudo-catalyst (electrochemical
mediator), Cu
2O, 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 Cu
2O 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 H
2S 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 H
2O
2
and glucose, but with the selection of nano-structured gold (Au) catalyst uniquely suited for
thiol-containing compounds such as H
2S. Special effort was spent on solving the interfering
problems caused by cysteine, methionine and glutathione.
The linear range and detection limit of detecting H
2S by desorption method we
developed were from 5μM to 200μM and 2μM respectively. The linear range and detection
limit of detecting H
2S by adsorption method were from 10μM to 3mM and 10μM
respectively. The interference coefficient of cysteine with respect to H
2S 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.
Post a Comment