The Construction Of HRP Multilayer Enzyme Electrode And Its Application

The construction of mulayer ordered enzyme electrode is the important method with the achievement of fabricating stable enzyme electrode and deep developments. Layer-by-layer assembly (LBL assembly) is a kind of effective approach to fabricate organic nano-thin films. LBL assembly has important research significance in the construction of multilayer enzyme film and multilayer enzyme electrode because of its versatility, simplicity, easy operation and its controlling the components and thickness of the film.The multilayer horserash peroxidase (HRP) electrodes were constructed with electrostatic and bio-specific LBL assembly technique, including poly (allylamine hydrochloride) (PAH)/HRP multilayer electrode, monolayer or multilayer concanavalin A (Con A)/HRP electrode based on the precursor of PAH/poly (sodium-p-styrene-sulfonate) (PSS)/PAH, PSS-NB (nile blue)/HRP multilayer enzyme electrode based on NB premixed with PSS, and HRP/GOx (glucose oxidase) multilayer enzyme electrode in the dissertation. These electrodes were applied to determine hydrogen peroxide, phenolic compounds and aromatic amines with the features of high sensitivities, good stability and anti-interference.Through electrostatic LBL assembly, PAH/HRP multilayer enzyme electrode was fabricated. We employed AFM to investigate the morphology, roughness and uniformity of the PAH/HRP multilayer films. The results showed the response of the multilayer electrode increased with the assembled layers of HRP within 1-5 layers using catechol as substrate. Under the optimized experimental conditions and electrode preparation, the linear range of catechol was 6.0-120.0μmol l^-1 with the (PAH/HRP)₅ modified electrode and the sensitivity was 48.91 nA 1μmol^-1 . The interference effects were investigated by detecting the response of the enzyme electrode to catechol in the presence of glucose and ascorbic acid. It was found that these species do not interference at the concentrations given. The recovery of the (PAH/HRP)₅ electrode was 98.7-109% for the cresolis saponatus sample. For the determination of phenolic compounds, the trend of the sensitivity is consistent with the ability of the electron-donor conjugation of the substiruents in the phenolic compounds.Through biospecific complexation of Con A and sugar residues in the glycoenzymes, monolayer and multilayer HRP electrodes were constructed on the gold electrodes based on the precursor film of PAH/PSS/PAH. UV-vis spectroscopy was used to follow the layer-by-layer self-assembly process of FITC (fluorescein isothiocyanante) labeled Con A and HRP. The effects of pH and salt of the precursor film on the response of the assembled HRP electrode were investigated using catechol as substrate. The results indicated that the catalytic currents of the HRP electrode were optimized while the pH and the concentration of NaCl in the precursor film were 12.0 and 0.4 mol l^-1, respectively. The response of the multilayer electrode increased with the assembled layers of HRP within 1-4 layers. Under the optimized experimental condition and electrode preparation, the linear range of catechol was 6.0-48.0μmol l^-1 with the (Con A/HRP)₄ modified electrode and the sensitivity was 162.79 nA 1μmol^-1. The sensitivity of (Con A/HRP)₄ modified electrode was much higher than that of (PAH/HRP)₅ modified electrode.Through biospecific complexation of Con A and sugar residues in the glycoenzymes, HRP/GOx multilayer electrode was constructed on the gold electrode. We employed AFM to investigate the morphology, roughness and uniformity of the multilayer films. Under the optimized experimental condition and electrode preparation, (Con A/HRP)₂-(Con A/GOx)₂ modifed electrodes were used to determine phenolic compounds and aromatic amines. The linear ranges of catechol and p-phenylediamine were 6.0-60.0 and 7.6-68.4μmol l^-1, respectively. The sensitivities were 76.82 and 131.59 nA 1μmol^-1, respectively. The substances of glucose and ascorbic acid did not cause observable interference in the determination of catechol and p-phenylediamine. The stability and repeatability were improved because H₂O₂ needed for the enzyme reaction generated 'in situ' led to the reduction of inhibition of H₂O₂ to assembled enzymes.The multilayer PSS-NB/HRP electrode was fabricated by the electrostatic LBL assembly of HRP and the low molecule dye NB premixed with PSS. UV-vis spectroscopy was used to follow the layer-by-layer self-assembly process of PSS-NB/HRP. The effects of the number of assembled layers of PSS-NB and the salt of PSS-NB solution were investigated. While the electrode modified by two bilayers of PSS-NB/HRP (PSS-NB assembly solution containing 0.3 mol l^-1 NaCl), the linear range of H₂O₂of the electrode was 0.20-7.03 mmol l^-1, the sensitivity was 8.45μA 1 mmol^-1. The influence of glucose and ascorbic acid was examined in the determination of H₂O₂. The results showed that these substances did not cause observable interference in the determination of H₂O₂.