A New Method For Bioanalysis Based On Bipolar Electrode Array And Electro-optical Conversion Imaging

Nanoelectrodes are widely used in the field of electroanalytical chemistry due to their advantages such as low background current,high current density,and fast transmission rate.The study of charge transfer and material transport in electrochemical reactions using nanoelectrode is of great significance for understanding the reaction mechanism and improving the spatial resolution of imaging.As a"wireless"technology,bipolar electrodes（BPEs）not only realize a simplified device that connects countless nanoelectrodes to the external power supply in a simple way,but also solve the problem of low current at a single nanoelectrode.The preparation of high-density nanoelectrode arrays by simple and economical methods is of great significance to improve the analytical performance and spatial resolution of electrochemical imaging.Based on the above,the gold nanoelectrode array was prepared by the template-assisted electrochemical deposition method,and a closed bipolar electrodes（c-BPEs）array based on the gold nanoelectrode array was constructed combined with an electro-optical conversion system,to realize high-sensitivity analytical performance and high-spatial-resolution electrochemical imaging.The main research contents are as follows:1.Construction of a c-BPEs system based on the high-density gold nanoelectrode arrayA high-density gold nanoelectrode array with a diameter of～200 nm and spacing of～450 nm was prepared by electrochemical deposition using porous anodic alumina（AAO）as a template.Based on the above gold nanoelectrode array,the c-BPEs system was constructed.Combined with ECL technology,the low-abundance protein on the cell membrane was used as the model to verify the high sensitivity of the c-BPEs system and the feasibility of electric-optical conversion.The"antigen-antibody"sandwich immune assembly was used to couple the silica-coated thionine（Ab2-Th@Si O₂）as the electrochemical probe and biometric element in the sensing pole.When sufficient voltage was applied,oxidation and reduction reactions occur simultaneously at both poles of c-BPEs,and the conversion of the electrochemical signal to the optical signal and high-sensitivity detection of alpha-fetoprotein in the cell membrane or the real sample was achieved.Meanwhile,the"antigen-antibody"specific recognition improved the detection selectivity.The results showed that the constructed c-BPEs system based on a gold nanoelectrode array has good analytical performance,which lays a good foundation for subsequent electrochemical imaging.2.In situ ECL imaging of H₂O₂ in living cells was achieved by the c-BPEs system based on a gold nanoelectrode arrayA label-free and noninvasive method was developed for the rapid,high-throughput,and in-situ imaging analysis of intracellular hydrogen peroxide（H₂O₂）.The above-constructed c-BPEs system combined with an electrochemiluminescence microscope（ECLM）was used to achieve highly selective imaging of endogenous H₂O₂ content in cells by potential resolution.Ru（bpy）₃²⁺was used as the ECL luminant,and dimethylethanolamine（DBAE）was used as the coreactant in the reporting pole.When sufficient voltage was applied,a reduction reaction of H₂O₂ occurs at the sensing pole,and an oxidation reaction of Ru（bpy）₃²⁺/DBAE occurs at the reporting pole.According to the principle of electric neutrality,the content of endogenous H₂O₂in living cells can be monitored according to the ECL signal.The system can be used to distinguish between different cancer cells,as well as between cancer cells and normal cells.To achieve parallel,high-throughput,and in situ imaging analysis of endogenous H₂O₂ in multiple cells intuitively and visually.It provides a new platform for the imaging of label-free single cells or intracellular biomolecules.3.The c-BPEs system based on a gold nanoelectrode array achieves high spatial resolution fluorescence imaging of single cells and single particlesThe main factors affecting the spatial resolution of nanoelectrode array electrochemical imaging are the electrode size,diffusion of luminescent molecules,and medium viscosity.To further improve the spatial resolution of electrochemical imaging,a high-density gold nanoelectrode array with a diameter of～140 nm was prepared by template-assisted electrochemical deposition.And the c-BPEs system was constructed based on this nanoelectrode array,and electrofluorochromism（EFC）was used as the output signal.The electrochemical process of single Pt NPs catalysis or the adhesion characteristics of a single cell at the sensing pole was monitored by recording the intensity of the fluorescence signal at the reporting pole.The fluorescent molecules were covalently coupled to the surface of an independent gold nanoelectrode using a conductive polymeric film carrier at the reporting pole,and ionic liquid with high viscosity and good conductivity was used as an electrolyte,which significantly improves the spatial resolution of imaging,up to submicron level.The results show that the c-BPEs system successfully realizes the fluorescence imaging of single-cell adhesion and the monitoring of the oxygen reduction reaction process catalyzed by a single platinum nanoparticle on the surface of a single gold nanoelectrode.It is expected to be used in the study of single-molecule imaging.In conclusion,the high-density gold nanoelectrode arrays were prepared by template-assisted electrochemical deposition.Combined with the"wireless"characteristics of BPEs,numerous gold nanoelectrodes were coupled to an external power source by a pair of driving electrodes,and a c-BPEs system was successfully constructed for highly sensitive electrochemical analysis and rapid and high-throughput electrochemical imaging.These studies provide new ideas for the establishment of high-throughput,highly selective,and highly sensitive methods for the detection of endogenous small molecules and cell membrane proteins.Based on the characteristics of high spatial resolution and high throughput,the gold nanoelectrode array has broad application prospects in the study of single-particle electrocatalytic heterogeneity.Compared with the traditional electrochemical scanning microscope（SECM）,the combined use of BPEs array and microscope improves the temporal resolution of electrochemical imaging,which has guiding significance for subsequent biological imaging.