Fabrication And Application Of Novel Nanomaterial Modified Electrodes For Electrochemical Biosensor

Electrochemical biosensor is developed by the combination of electrochemical analytical method with biosensing technology.Electrochemical biosensor have received considerable attentions in different fields including industry,agriculture,clinical medicine,environmental protection and so on because of their excellent properties such as easy operation,good selectivity,remarkably high sensitivity,fast response,low cost.Nanomaterials have extensive applications in electrochemical biosensing field due to their large specific area,strong adsorption capability,efficient electrochemical properties,excellent biological compatibility and good stability.The construction of nanointerface has accelerated the development of electrochemical biosensing.This dissertation focuses on fabricating a series of electrochemical biosensors with rapid response,high sensitivity,low detection limit,good stability and reproducibility by combination the electrochemical biosensing and several nanomaterial-modified electrodes,which can be summarized as following section.1.The mixture of Co₃O₄-graphene nanocomposite and horseradish peroxidase?HRP?was spread on the surface of carbon ionic liquid electrode?CILE?.Then Nafion film was used for the immobilization.The results of spectroscopy proved that HRP kept up its native structure in the complex material.Direct electrochemistry of HRP resulted in a couple of quasi-reversible redox waves on cyclic voltammograms,reflecting the realization of direct electron transfer of HRP with electrode.The improvement of electrochemical responses was due to the usage of highly conductive Co₃O₄-graphene nanocomposite with biocompatible interface.Moreover,the immobilized HRP showed remarkable electrochemical catalytic capacity for the reduction of trichloroacetic acid and NaNO2 with high sensitivity,low detection limit,wide linear range,good reproducibility and long-term stability.2.Molybdenum disulfide?MoS₂?nanosheets and horseradish peroxidase?HRP?modified electrode was prepared with ionic liquid 1-hexylpyridinium hexafluorophosphate based carbon ionic liquid electrode?CILE?.UV-Vis and FT-IR spectra showed that the native framework of HRP was retained after mixed with MoS₂ nanosheet.A pair of well-defined redox peaks of HRP appeared on MoS₂/CILE,implying the realization of direct electron transfer.Electrochemical parameters of HRP were calculated according to the cyclic voltammetric data with the electron transfer coefficient and electron transfer rate constant as 0.51 and 1.65 s-1.The HRP modified electrode exhibited a prominent electrocatalytic behavior towards the reduction of trichloroacetic acid?TCA?.The linear range for TCA detection was in the range from10.0 to 63.0 mmol L-1 with the detection limit of 0.67 mmol L-1?3??.3.The effect of carboxyl grapheme?G-COOH?on the direct electrochemistry reaction of myoglobin?Mb?was checked by using carbon ionic liquid electrode?CILE?as the substrate electrode.The modifier was prepared by mixing Mb and G-COOH solution homogeneously.Then Mb-G-COOH mixture was casted on the surface of CILE.Nafion was acted as a film to increase stability of the composite on the electrode surface.In the phosphate buffer solution with pH value of 3.0,Mb showed a pair of well-defined redox peaks with the formal peak potential?E0??of-0.231V?vs.SCE?.The immobilized Mb in the film maintained its biological activity and showed a surface controlled process with the heterogeneous electron transfer rate constant?ks?of1.33 s-1.The Mb modified electrode exhibited good electrocatalytic ability to the reduction of trichloroacetic acid?TCA?with wider dynamic range of 5.0 to 57.0 mmol L-1,lower detection limit of 1.0 mmol L-1?3??and the Michaelis–Menten constant?KMapp?of 1.30 mmol L-1.4.A new electrochemical DNA sensor was constructed for the detection of Staphylococcus aureus nuc gene sequence by the application of chitosan-graphene?CTS-GR?and gold nanoparticles?AuNPs?modified electrode.AuNPs were electrodeposited on the surface of carbon ionic liquid electrode?CILE?to get AuNPs/CILE.Then CTS-GR nanocomposite was casted onto the surface of AuNPs/CILE to obtain CTS-GR/AuNPs/CILE.The presence of nanocomposite on the electrode surface could not only increase the surface area,but also be benefit for immobilization of the probe ssDNA sequence by electrostatic attraction.Hybridization were took place with the complementary ssDNA sequences and assessed by usingmethylene blue?MB?as an electrochemical indicator.The reduction peak current of MB were in line with the concentration of the specific Staphylococcus aureus nuc gene sequences in the range from 1.0×10-13 to 1.0×10-6mol L-1with a detection limit of3.33×10-14 mol L-1?3??.This electrochemical DNA biosensor displyed prominent selectivity to distinct one-base and three-base mismatched ssDNA sequences,and the PCR products were further detected with favourable results.5.A simple,effective and convenient method was reported to detect Listeria monocytogenes hly gene sequence by using an electrochemical DNA biosensor based on a nanocomposite consisting of gold nanoparticles?AuNPs?and partially reduced graphene oxide?p-RGO?.Gold nanoparticles was electrodeposited on the CILE surface,and then p-RGO was formed on the surface of AuNPs/CILE by controlling the electroreduction conditions.NH2 modified ssDNA probe sequences were immobilized on the surface of modified electrode via covalent bonds between the unreduced carboxyl groups on the surface of p-RGO/AuNPs/CILE and the amine group at the5'-end of ssDNA.Differential pulse voltammetry was also applied to monitor DNA hybridization with methylene blue?MB?as an electrochemical indicator.Under optimal conditions the biosensor is highly sensitive with a low detection limit of3.17×10-14 mol/L?3??.Also,the developed biosensor showed a good linear relationship between the current value and logarithm of the target DNA concentration ranging from 1.0×10-13¹.0×10-6 mol/L.The sensor had good stability and selectivity,and could discriminate one-or three-base mismatched ssDNA sequences.PCR samples of Listeria monocytogenes hly gene sequence was successfully detected,indicating potential application of the biosensor in real sample.