Study on Phenols Sensing by Electrochemical Methods

Novel electrochemical sensing methods for phenolic compounds have been developed for the quality control in tea industry (Part 1 & 2) and for the detection of phenolic pollutants based on substrate recycling amplification strategy (Part 3). Electrodeposition techniques of proteins on a charged bio-matrix were designed to immobilize analytical enzymes on a micro-pattern (Part 4), localized substrate recycling amplification may be investigated on the micro-fabricated bio-patterns.art 1: Redox potential of tea infusion as an index for the degree of fermentation Redox potential of tea infusion was suggested as a working index for estimating the extent of tea fermentation. The redox potential of tea infusion was measured between platinum and Ag/AgCl electrode pair with a voltmeter with a high input impedance (>1000 GΩ). The reliability of redox potential measurement was verified with a standard redox couple, ferricyanide / ferrocyanide. Effect of tannic acid concentration and ascorbic acid to the redox potential of tannic acid were discussed. Redox potential showed a high correlation with the fermentation degree and the indoor withering time (fermentation time). Several kinds of tea with different fermentation extents including Japanese Sencha (unfermented), Chinese Pilochun (slightly fermented), Taiwanese Pouchong (mild fermented), Taiwanese Oolong (partial-fermented), Western black tea (fully fermented) were measured and compared.art 2: Determining the levels of tannin in tea by ferricyanide pre-reaction in an amperometric flow-injection systemn amperometric flow-injection assay for tea tannin was proposed, which was based on ferricyanide pre-oxidation to prevent the electrode fouling problems frequently encountered in oxidation of phenolic compounds. The working potential and flow rate were optimized to be -0.1 V vs. Ag/AgCl and 2.28 mL min-1, respectively. The linear dynamic range of tannic acid (10-50 μg mL-1 and 100-500 μg mL-1) can be adjusted using 1 mM and 10 mM ferricyanide, respectively. The proposed method was verified with Folin-Ciocalteau method (r = 0.9808), and the ferrous tartrate method (r = 0.9899). Tea with various fermentation degrees were successfully measured with a sample throughput of 15 samples h−1.art 3: Amperometric flow-injection determination of catechol, phenol and bisphenol A by ferrocyanide substrate recycling amplification within an immobilized tyronsiansen on-line phenols sensing flow-injection analysis (FIA) system was proposed, which was based on the broad phenolic oxidation capacity of mushroom tyrosinase (EC 1.14.18.1). The enzyme was densely immobilized on controlled pore-size aminopropoyl glass beads packed within a mini-reactor, and the reactor was installed in the flow stream of an amperometric FIA system. By reacting with 10 mM ferrocyanide (in pH 7.0, 10 mM phosphate buffer) supplied from the side stream of the flow system, substrate recycling amplification was conducted to regenerate the reduced phenols for the subsequent oxidation process catalyzed by the immobilized tyrosinase. The signals of catechol and phenol were amplified 100 fold; bisphenol A, an important endocrine distruptor, can be detected at 2.5 ×10-6 M (S/N>3; working potential: +0 V vs. Ag/AgCl; flow rate: 1.0 mL min−1).art 4: Localized Electrodeposition of Chitosan as Matrix for Enzyme Immobilizationased on the pH gradient electrochemically generated between electrodes, two localized chitosan electrodeposition methods were developed by either scanning electrochemical microscope (SECM) direct mode or microfluidic electrodeposition. With SECM direct mode, a localized pH gradient was induced between planar Au working electrode (the substrate) and the Pt auxiliary microelectrode by potential pulses (1 s, 1.0 V vs. Ag/AgCl). An array of chitosan spots with hundreds of µm in diameter can be directly deposited on the Au substrate sequentially. The procedure could be carried out within a microfluidic channel in order to restrict the spreading of chitosan solution. Chitosan was gradually deposited (10 min, 3.0V Au substrate vs. Pt wires) along the channels with defined micro-scaled geometry (line width = 100 µm). The surface amino groups labeled with NHS-fluorescein were observed with confocal laser scanning microscopy. The deposition method using microchannels demonstrated a more reproducible and well-defined surface. Both methods can also be used to entrap glucose oxidase (GOx) during the deposition process. SECM in the generation-collection mode was used to detect the activity of GOx within the chitosan micro-patterns by oxidation of the formed hydrogen peroxide. The activity distribution of the immobilized GOx on the deposited chitosan layer was more uniform with the microfludic channel method as compared to that obtained by SECM direct mode. The roughness was investigated with non-contact atomic force microscopy.