Study On Photoctrochemical Analysis For Glucose Oxidase As Label

The immunoassay is a method of analyzing various substances?such as drugs,hormones,proteins,microorganisms,etc.?based on antigen and antibody-specific reactions.Because the specific binding of antigen and antibody cannot give obvious pHysical signals,most of the immunoassay methods use labeling technology.Among them,the enzyme-linked immunoassay method is the most widely applied.However,in the electrochemical enzyme-linked immunoassay,the redox mediators of the enzymes have the disadvantages of high cost,potential cytotoxicity,and low selectivity.And hydrogen peroxide,the commonly used substrate also reduced the selectivity due to its high redox potential.In this paper,based on the direct electron transfer of glucose oxidase?GOD?,and combined with photoelectrochemical?PEC?detection technology,instead of using traditional electronic media,a PEC immunoassay method has been developed with GOD as a marker.First,ZnO was electrodeposited on the ITO electrode to form a nano-ZnO photoelectrode.After the coated antibody was assembled on the surface of the photoelectrode to construct an immunoprobe,the immune reaction was completed by sandwiching antigen and glucose oxidase-labeled secondary antibody on the immunoprobe.Finally,the direct electron transfer from coenzyme-flavin adenine dinucleotide?FAD?of GOD to the nano-ZnO photoelectrode under light irradiation was achieved by using chitosan,and the sample was detected from the given photocurrent.Compared with the traditional electrochemical enzyme-linked immunoassay,the proposed method has the advantages of good selectivity,high sensitivity and wide detection range.The research work is mainly divided into the following three parts:1.The photosensitive material ZnO was electrodeposited on the surface of ITO electrode to form a nano-ZnO photoelectrode.FADH₂ was obtained through chemical reduction of FAD?using Na₂SO₃?or electrochemical reduction?using the second working electrode?,and it was found that it can generate photoelectrochemical reaction with nano-ZnO photoelectrode under illumination to produce photocurrent.And photocurrent and FAD have a good linear relationship at a certain concentration.The direct electron transfer between GOD?FAD?and photoelectrode was validated by using chitosan as an electronic conductor,which laid a theoretical foundation for PEC immunoassay with GOD as a marker.2.Previously,GOD and detection antibody?secondary antibody?were immobilized on gold nanoparticles as enzyme-labeled antibodies.Then the multi-walled carbon nanotubes?MWCNTs?were immobilized on the surface of nano-ZnO photoelectrode.The CEA?carcinoembryonic antigen?monoclonal antibody was coated on the photoelectrode surface to assemble an immunoprobe by means of rich carboxyl groups as binding sites on the surface of MWCNTs.After completing the immunoreaction with the double-antibody sandwich method,the electron donor FADH₂ was generated from reduction of GOD?FAD?with glucose.When FADH₂ transfer electrons to the nano-ZnO photoelectrode under light irradiation through chitosan,CEA was quantitated by the obtained photocurrent.In this paper,immunoprobes,enzyme-labeled antibodies and immunoreaction states were characterized by electrochemical impedance spectroscopy?EIS?,scanning electron microscopy,ultraviolet and transmission electron microscopy.The effects of bias voltage,electrolyte pH,incubation time and temperature of the immune reaction on photocurrent response were optimized.The results showed that with the increase of CEA concentration,the photocurrent response appeared an exponential increase,and the photocurrent intensity was proportional to the logarithm of CEA concentration with a correlation coefficient?R²?of 0.9983.The detection range was 0.001-100 ng/m L,and the detection limit was 0.3 pg/mL.By comparing PEC immunosensors with and without electronic wires?chitosan?,it was found that PEC immunoassay with direct electron transfer has higher detection sensitivity and wider detection range.3.Altering the antigen-antibody pair,using the CA199 antigen as a sample,another PEC immunosensor was constructed in the same way.After optimizing preparation and measurement conditions of the immunosensor,the photocurrent intensity is proportional to the logarithm of CA199 concentration,R² was 0.9987,the detection range was0.001-100 ng/mL,and the detection limit was 0.3 pg/m L.The effect of chitosan or ionic solution as two different electronic wires on the detection of CA199 was tested.It was found that PEC immunosensor has better performance when using chitosan as an electronic wire.