Study On Preparation Of Poly(Thionine)/CNS Photoelectrode And Its Application In Analytical Chemistry

The photoelectrochemical analysis is a detecting method based on energy transfer from light to electrical. The two separate processes in the excitation and detection is a remarkable characteristic of this analytical method. In relation to photometric method, the photoelectrochemical method has the advantages of the simple equipment, low cost, easy miniaturization and integration. It has higher sensitivity and good selectivity compared with generally electrochemical method. The photoelectrochemical sensor is a sensitive devices used in the photoelectrochemical analysis, is generally classified as current-mode of photoelectric or electrochemical sensor. In recent years, the photoelectrochemical sensor has been fast development. Its selectivity, sensitivity, response time and range of applications could be improved by chemical modification to various photoelectrode.In the paper, the poly(thionine)/carbon nanotubes (CNS) photoelectrode with photoelectric activity and electron acceptor was prepared by electropolymerizing thionine (Th) on the surface of CNS-modified ITO electrode. We have found that the prepared photoelectrode can occur to photoelectrochemical reaction with nicotinamide adenine dinucleotide (NADH) as electron donor. Because NADH is an important coenzyme in many biological catalytic process and intermediates of redox reaction in many organisms, the substrate, product, and other related substances could be indirectly determined through detecting NADH in enzymatic reactions. On the basis of the presented photoelectrode, the studied work mainly is as follows:1. A photoelectrochemical system was composed of the photoelectrode and NADH. Through the photoelectrode react with NADH as electron donor, determination of NADH was achieved by monitoring photocurrent. The reaction mechanism and dynamics response between the photosensitive interface and NADH were studied. The preparation of the photoelectrode, and the influence of bias voltage, the light intensity, the solution pH on the photoelectrochemical reaction and detection of NADH were discussed. The preparation mothed of the photoelectrode, the multi-walled carbon nanotubes was firstly immobilized on the surface of ITO electrode, the thionine then was electropolymerized on the surface of CNS electrode by the controlled potential electrolysis were determined. Under the optimized condition of bias voltage0.5V (vs.Ag/AgCl), light intensityl4.3mW/cm2, and pH7.0, the logarithmic response of NADH concentrations from1.00×10-4-1.00×10-8M was obtained with a detection limit of3.00nM. The photoelectrochemical responses have good reproducibility and stability. The photoelectrode still maintain photoelectric activity of more85%than initial response after be used20days.2. Through immobilizing lactate dehydrogenase (LDH) and3a-hydroxysteroid dehydrogenase (3a-HSD) on the surface of photoelectrode by chitosan (CS) and glutaraldehyde, two novel photoelectrochemical sensors were prepared by coupling the photoelectrode with enzymatic reactions. The determination of lactic acid and cholic acid were fulfilled based on the detection of photocurrent from photoelectrochemical effect between Th on the photoelectrode and NADH, which proceed from catalytic reaction of LOD and3α-HSD to lactic acid and cholic acid, respectively. The response mechanism for photosensitive interface of the dehydrogenase/poly(thionine)/CNS were studied. The preparation condition of the photoelectrochemical biosensors, such as enzyme loading, coating fluid volume, the concertration of CS and glutaraldehyde were optimized by experiment. The effect of concentration of NAD+, the bias voltage and the solution pH on the determination of substrate were investigated, and the optimized parameters were selected. The linear response of lactic acid sensors to lactic acid concentrations from1.00×10-6-1.00×10-4M was obtained with a sensitivity of2.05nA/μM, and the detection limit was estimated to0.33μM. The recovery was98%～105%for the determination of lactic acid in Lactated Ringer’s solution. The measuring range of cholic acid sensor for cholic acid was from2.00×10-6to4.00×10-5M with a sensitivity of4.21nA/uM, and the detection limit was estimated to0.67μM. The recovery was in the range of96.4%-103%for the determination of cholic acid in Sodinm Cholate Tablets.3. The repeatedly regenerative ability of poly(thionine)/CNS photoelectrode to NAD+was further verified in the enzymatic reactions by UV spectrophotometry and spectrofluorimetry. The photoelectrode catalyze the oxidation of NADH as electron donor to simulate the role of NADH oxidase successfully. The mutual conversion NADH and NAD+has been completed, establishing a new photoelectrochemical method of regeneration coenzyme. It provides an effective way for coenzyme recycling in the enzymatic reaction system, and has potential applications to improve the use efficiency of coenzyme or reduce the cost of industrial production in the organic synthesis or biological preparation.