Author: Sun, Cheng
Title: Biosensor based on decorated reduced graphene oxide Au electrode for the electrochemical detection of light chain Botulinum neurotoxin A
Degree: M.Sc.
Year: 2014
Subject: Electrochemical sensors.
Botulinum toxin.
Hong Kong Polytechnic University -- Dissertations
Department: Interdisciplinary Division of Biomedical Engineering
Pages: xi, 53 leaves : color illustrations. ; 30 cm
Language: English
Abstract: Botulinum neurotoxin (BoNT) was considered to be among most lethal toxic proteins known to humans in nature; it suppresses the release process of neurotransmitter at neuromuscular junctions and causes disease botulism. Synaptosomal-associated protein 25 kDa (SNAP-25), as one of the SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), relates to the both verbal and performance intelligence quotient, as well as ability of learning and memory. The Light chain BoNT/A (LcA) is released as a protease in vivo exposure therapy and it specifically cleaves at the SNAP-25 at the nerve terminal, thus interfering with neural transmission by blocking the release procedure. As a result, there is great interest in developing simple novel materials modified biosensor and rapid methods to complete electrochemical measurement of toxin with high sensitivity and specificity. In this project, a novel nanomaterial based biosensor was applied to detect the Light chain BoNT/A (LcA) by immobilized with SNAP-25. First, reduced graphene oxide (rGO) formed by reduction of graphene oxide and observed under spectroscopy, then dropped rGO on the bare gold electrode surface to make rGO modified electrode, the electrochemical property of this electrode was characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 5.0 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] solution. Second, Pyrenebutyric acid (PBA) was added to fabricate rGO-PBA/Au electrode, which can be worked as a suitable platform for protein immobilization. Third, synaptosomal-associated protein 25 Green Fluorescence Protein (SNAP-25 GFP) was immobilized to the composite rGOPBA surface through EDC/NHS intermediated condensation reaction. This estimates result was also determined by CV and EIS technologies in 5.0 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] solution. Finally, BoNT LcA was then dropped on the protein-coated electrode and specifically cleaved SNAP-25 to change the exposure surface so that impact the electrochemical characteristic. However, the other group of experiment was added Light chain BoNT/B (LcB), which should not have reaction with SNAP 25 and could not lead the change of electrochemical parameters in measurement. In the final stage, the electrochemical property and the electrocatalytic activity were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), as well as differential pulse voltammetry (DPV). A good linear relationship would be observed by using a logarithmic scale for LcA concentrations in the range from 5 pg/ml to 100 pg/ml with the detection limit of 5 pg/ml. The selectivity property of this novel biosensor could be illustrated by control experiment of detecting LcB, which cannot bring about an electrochemical change in LcB Toxin-contained sample. The good analytical performance and simplicity of this biosensor can be attributed to the electrode coated with reduced graphene oxide which opens new way to be used as botulinum neurotoxins sensor for the analysis of the toxin-contained samples with high sensitivity and good selectivity.
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