Development Of HRP Novel Electrode Based On Self-asembly Method

The construction of ordered enzyme film is vital for enzymatic electrode.Four kinds of horserash peroxidase(HRP) ordered film were prepared using self-assembly method,including(ConA/HRP)n multilayer film,PSS-NB(nile blue)/HRP and PSS-MB(methyl blue)/HRP multilayer film,HRP monolayer film covalently attached on thiols modified gold and HRP-GOD bienzyme multilayer film.The film morphology,deposition mass amount,and the activities of HRP on the multilayer films were studied using atomic force microscopy(AFM),UV-vis spectroscopy,and electrochemical technique.These HRP ordered films were applied to prepare HRP electrodes to determine hydrogen peroxide or aromatic compounds.The(ConA/HRP)_n multilayer films were constructed through biospecific complexation of Con A and HRP on the gold electrodes.AFM and UV-vis spectroscopy were used to follow the layer-by-layer(LBL) assembly process of Con A and HRP.The activities of the multilayer films were also discussed.The size of HRP molecules on the ConA/HRP films correspond to its X-ray diffraction data, which demonstrated the biospecific assembly can control the orientation of deposited HRP.The response of the multilayer electrode increased with the assembled layers of HRP.Under optimized conditions,the linear ranges of the(ConA/HRP)₃ modified electrode for H₂O₂ was 0.8-3.0 mmol·L^-1 with the sensitivities of 10.84μA·mmol^-1·L.The PSS-NB/HRP and PSS-MB/HRP multilayer film were fabricated by the electrostatic LBL assembly of HRP and the cationic dye NB or MB premixed with PSS.UV-vis spectroscopy and cyclic voltammograms(CVs) were used to follow LBL assembly process of PSS-NB/HRP or PSS-MB/HRP multilayer films.The effects of the number of assembled layers of HRP and the salt of PSS solution were investigated.The linear ranges of the(PSS-MB/HRP)₄ or(PSS-NB/HRP)₂ electrode for H₂O₂ was 0.33-8.82 mmol^-1·L or 0.20-7.03 mmol^-1·L,with the sensitivities of 2.04μA·mmol^-1·L and 8.45μA·mmol^-1,respectively.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.The GOD multilayer film and HRP/GOD bienzyme multilayer film were fabricated by the LBL assembly.We employed AFM to investigate the morphology,roughness and uniformity of these multilayer films.The GOD multilayer film modifed electrode was used to determine glucoses at the existence of ferrocenecarboxylic acids.The catalytic activity of the GOD electrode was enhanced with the increase of the number of layers below four and varied in different pH values.The HRP/GOD bienzyme multilayer film modifed electrode was used to determine phenolic compounds and aromatic amines.The linear ranges of catechol and p-phenylediamine was 6.0-60.0μmol^-1·L and 7.6-68.4μmol^-1·L,respectively.The sensitivities was 76.82μA·mmol^-1·L and 131.59μA·mmol^-1·L,respectively.The substances of glucose and ascorbic acid did not cause observable interference in the determination of catechol and p-phenylediamine.HRP electrode was also prepared by immobilizing covalently HRP on gold electrodes modified with self-assembled monolayers(SAMs) of 3-mercaptoproionic acid(MPA),2-aminoethanethiol(AET) or p-aminothiophenol(PATP).Effect of these SAMs on direct electron transfer(direct eT) of HRP electrodes was studied,and the effects of assembly time and pH value were discussed as well.The result indicates that the terminal group of alkanethiols seems to be a key factor determining the orientation of HRP on SAMs.The Au/MPA/HRP electrode got the best effective direct ET from HRP to gold electrode.The Au/MPA/HRP electrode displays an excellent electrocatalytic response to the reduction of H₂O₂ without the aid of any electron mediator.The linear ranges of the Au/MPA/HRP electrode for H₂O₂ was 0.17-2.3 mmol·L^-1 with the sensitivities of 8.51μA·mmol^-1·L.The Michaelis-Menten constant(K_m) was 5.7mmol^-1·L and the response time was less than 3s.