| Summary: | 碩士 === 明志科技大學 === 材料工程系碩士班 === 107 === In this study, a novel electrochemical biosensor was prepared. High-quality single-layer graphene, grown by chemical vapor deposition, was wet-transferred onto a substrate, followed by thermal annealing to obtain bilayer graphene, which was then subjected to oxygen low-damage plasma treatment (O-LDPT), to obtain the target electrodes. While the top layer of graphene was oxidized, the bottom layer remained a transmission layer. Till date, this electrode structure had never been used for electrochemical detection of miRNA-21. Here, the miRNA-21 probe was immobilized on the upper graphene oxide layer by the formation of a covalent amino bond, and hybridized with the target miRNA-21 after immobilization. Chronocoulometry was used determime the surface density of the probe; the latter was found to increases with increasing processing time. The probe density was estimated to be 1 × 1012 molecules/cm2. Differential pulse voltammetry (DPV) measurements have been performed to evaluate the sensitivity of this biosensor. Peak currents obtained from the DPV spectra increased linearly as the target RNA concentration decreased from 100 nM to 1 pM; the limit of detection (LOD) of this biosensor was calculated to be 236 fM (R2=0.9822). We had previously confirmed using X-ray photoelectron spectroscopic analysis that addition of hydrogen during the oxygen plasma treatment (O/H-LDPT) can increase the carboxyl group content. We observed that probe density can be effectively increased to 1.42 × 1012 molecules/cm2. We also compared the LOD values under two different plasma conditions (O-LDPT and O/H LDPT). Preparation of biosensor electrodes by O/H-LDPT was found to effectively reduce the LOD to 24.6 fM (R2=0.9742). In order to achieve specificity of the biosensor we added a blocking step after immobilization to remove the active site, and then hybridized with different concentrations of miRNA-21 in BSA. The measured LOD was 19.83 fM (R2=0.981). Graphene oxide has excellent hydrophilicity and biocompatibility, along with the advantages of rapid detection, high sensitivity and environmental friendliness; therefore, graphene oxide/graphene structure has great potential for use in electrochemical biosensor applications.
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