| Summary: | 碩士 === 義守大學 === 生物技術與化學工程研究所碩士班 === 94 === In order to shorten the distances between active sites and the electrode surfaces for enhancing electron transfer rate and increasing sensing sensitivity, many investigations have aimed at the assemblies of electron
transfer media on enzyme electrode surfaces. In this study, ferrocenyl derivatives were used to modify the electrode surfaces. By forming a self-assembled monolayer at first, adsorbing ferrocenyl poly(ethyleneimine) (Fc-PEI), and immobilizing glucose oxidase layer, the
ferrocene-modified enzyme electrode was fabricated layer-by-layer.Three designs of modified electrodes were evaluated for theirelectroactivity to develop the third generational, reagentless biosensors--Fc/GOD/NH2-(CH2)2-S/Au system, Fc-PEI/PSS/COOH-(CH2)10-S/Au system, and GOD/Fc-PEI/ PEG/ NH2-(CH2)2- S /Au system. In Fc/GOD/NH2-(CH2)2-S/Au system, the redox peak currents on the
electrode were weaken in phosphate buffer solution (PBS, pH 7.4) and even kept decaying as increasing the scanning numbers in cyclic voltammetric measurement (the oxidation peak current was reduced from 4 μA to 0.06 μA in PBS after 400 cycles of scan), whereas kept electroactivity well in ClO4- solution. Thus, the detection failed for that
the peak currents were decreasing as increasing glucose concentration. It revealed that ferrocene-derivatives lost their electroactivity as scanning in PBS. Therefore, the activity-preserving was a main issue in material
design. The following design was a test of electroactivity of ferrocenyl derivatives, using LbL technique to fabricate the Fc-PEI/PSS/COOH-(CH2)10-S/Au system. The peak currents increased as increasing the bilayers of polyelectrolytes, however, they still decayed during repeated measurement (the oxidation peak current was reduced from 61.9 μA to 29.9 μA in PBS after 50 cycles of scan). It was observed that a IV deep-brown spot on the sensing area due to the deposition of reduced
ferrocenyl compounds, revealing the hydrophobicity of reduced ferrocenyl derivatives might decrease their mobility in water and thus eliminate the electron transfer in cyclic voltammetric (CV) measurement.
In order to increase the mobility of Fc group, the GOD/Fc-PEI/ PEG/NH2-(CH2)2- S /Au system was developed with the epoxyl chain-ended poly(ethylene glycol) as a spacer to improve the flexibility and hydrophilicity of the grafted chain. In CV measurement, the redox peak currents were no longer declining as increasing scanning numbers,
displaying the oxidative peak at 0.4 V. Consequently, the electrode was used to detect the glucose concentration at the oxidative potential of ferrocenyl derivatives. The response current was linear proportional to the
glucose concentration in the range of 50-70 mg/dL of glucose concentration, with a regression of 0.995 and a sensitivity of 0.662±0.142 μA(dL/mg)mm-2 and a RSD of 19.8 %. As increasing the enzyme coat, the charged enzyme was adsorbed onto the cationic Fc-PEI surface
in LbL method to form the two-bilayered system ((GOD/Fc-PEI)2/ PEG/NH2-(CH2)2- S /Au). The results showed that the linear detection limit was enlarged to 60-200 mg/dL of glucose, with a regression of 0.97 and a sensitivity of 0.329±0.011 μA(dL/mg)mm-2 and a RSD of 3.30 %. To
increase the numbers of enzyme layers seemed increase the thickness of multilayer, leading to the decreased sensitivity and the increased detection limit.
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