Visualization Of Electrofluorochromic Switches Based On Bipolar Electrode Array

Bipolar electrodes（BPE）have received extensive attention from researchers in the screening of electrocatalysts,the synthesis of functional materials and the detection of biomarkers.Particularly in the field of bioanalysis,BPE arrays and electrofluorochromic（EFC）imaging are combined to convert the electrical signal from the sensing pole of interest into a visually readable fluorescent signal at the reporting pole.High-sensitivity electrochemical signals are obtained by applying high-resolution optical imaging techniques to achieve high-content,high-sensitivity and high-resolution detection of non-electrochemically active proteins on the cell surface or released electroactive molecules.In view of this,based on the visualization method of BPE arrays and electrofluorescence switches,this doctoral is dedicates to the optimization of electrode arrays,signal amplification,and device design.Three experimental studies have achieved the detection of cell surface mucin（MUC1）,glycan and the electroactive molecule dopamine（DA）released from cells,respectively.The details are as follows:（1）A screen-printed conductive carbon electrode array containing five 500μm BPEs,which are connected to an anodic sensing cell and a cathodic reporter cell was constructed.Based on the BPE array and EFC switch visualization method,the detection of the cell surface biomarker MUC1 is achieved.Firstly,chitosan-multiwall carbon nanotubes（CS-MWCNTs）are modified at the anodic pole（sensing pole）of BPE to capture antibodies,and electroactive molecules ferrocene（Fc）-labeled aptamers are targeted on the cell surface due to the specific binding between antigens and antibodies or between antigen and aptamer.Then,the electroactive substance p-benzoquinone（BQ）and p H-sensitive fluorescent probe（2-（2-（4-hydroxystyryl）-6-methyl-4H-pyran-4-ylidene）malononitrile）（SPM）in acetonitrile was added to the cathodic pole（reporter pole）of the BPE.Under an applied voltage,the Fc attached to the electrode at the anodic pole is oxidized,while BQ at the cathodic pole is reduced,producing a strongly alkaline p-benzoquinone anion radicals(BQ^·-).BQ^·-reacts with the SPM in solution,causing the SPM to lose hydrogen proton turned into SPM^-,so that the fluorescence signal changes from"OFF"to"ON".The results showed that the fluorescence intensity was linearly correlated with the logarithm of the cell concentration in the range of 100 to 1.0×10⁶ cells/m L,with a detection limit of 3 cells.The amount of MUC1 on a single breast cancer（MCF-7）cell was calculated to be 6.02×10⁴ molecules/cell.Here,the closed BPE isolates the aqueous with oil phases and is available for two-phase reactions.By skillfully utilizing the EFC imaging principle of separated electroactive groups and fluorophores,with the coupled BPE technique,this experiment achieves the transformation of electrical signal to fluorescent signals.At the same time,the method displays the advantages of low background signal,high sensitivity,short reaction time,and high contrast.However,due to the large electrode size and distance between electrodes（500μm）with the electrode array placed in a reporter cell,the diffusion of BQ from the solution to the electrode surface and the electrochemically generated BQ^·-species diffusion into the solution or even the diffusion of SPM^-throughout the reporter reservoir were dominate,thus reducing the detection sensitivity.（2）To study cellular heterogeneity at the single-cell level,an in-situ analysis of galactosyl groups on the surface of a single cell is established utilizing a laser-etched indium tin oxide（ITO）as BPE arrays integrated with microfluidic chips with a single-cell capture structure.Inspired by the chip design in the second chapter,a single ITO electrode is placed in a 20μm reporter channel,which reduces the longitudinal diffusion of electrofluorescent molecules between the electrodes and increases the sensitivity significantly.Taking the human liver cancer cells Hep G2 cells as a model,galactose oxidase（GAO）can selectively oxidize the hydroxyl group at the C6 position of galactosyl group on the cells surface to an aldehyde group,and aniline catalyzed the reaction of biotin hydrazide with the aldehyde group to form a hydrazone complex to introduce biotin targets to the cell surface.Furthermore,through the specific binding of biotin and avidin,ferrocene-labeled DNA（Fc-DNA）and biotin-labeled DNA（Bio-DNA）functionalized gold（Au）nanoprobes are targeted to galactosyl group on the cell surface to achieve the site-specific labeling of glycans and signal amplification.A single cell pretreated with sequential labeling is trapped in the trap at the anode of the BPEs under a certain flow rate,and the remaining cells pass through the two boundaries of the channel and outflow the channel.Fc modified at the anodic pole is oxidized,while non-fluorescent resazurin at the cathodic pole is reduced to generate the highly fluorescent resorufin at a suitable potential.The cathode reporter signal is visualized using a fluorescent confocal microscope to reflect the progress of the anode non-fluorescent redox reaction of interest.The coupling of BPE and EFC imaging realize the detection of galactosyl groups on the surface of a single cell,and the limit of detection（LOD）is as low as 5.0×10⁸ molecules of galactosyl expression per Hep G2 cell,providing a simple and easy method for single cell analysis.In order to further improve the spatial resolution,conducting electrochemical analysis at the nanoscale has become the next focus of much attention in this topic.（3）Nanoelectrodes have distinctive electrochemical properties,involving rapid or selective mass transport and fast response times.When individual nanoelectrodes are assembly into large-area,highly ordered arrays,they allow for nanoscale spatial resolution imaging.Based on this unique advantage,gold（Au）nanowires electrodeposited in a highly ordered anodic aluminum oxide（AAO）template are served as bipolar nanoelectrodes（BPn E）.EFC molecule poly[9-hexyl-2,7-di（2-thienyl）carbazole]（PTCz6）is electropolymerized on one side of the AAO-supported Au nanoelectrode arrays which avoids the diffusion of the EFC molecules.High-resolution imaging of heterogeneous electrochemical process is conducted by coupling BPn E and the immobilization strategy of EFC molecules.Dopamine（DA）released from rat pheochromocytoma（PC12）cells under high K⁺stimulation is used as the analytical model.At an applied voltage of 1.6 V,DA is oxidized,and the oxidized non-fluorescent PTCz6_ox at the cathodic pole is correspondingly reduced to the fluorescent reduced state PTCz6_red,which is collected by a fluorescent confocal microscope.Based on the superiority of BPn E and the immobilization strategy of EFC molecules,high spatial resolution imaging of single nanoelectrodes and highly sensitive detection of DA were achieved with a detection limit as low as 0.45 n M,and the amount of DA released from a single PC12 cell was calculated to be 0.13 p M.In addition,the effects of the dopamine drugs L-3,4-dihydroxyphenylalanine（L-Dopa）and reserpine on DA release from live PC12 cells can be monitored dynamically.This work open a new avenue for monitoring the effects of dopamine drugs at high spatial resolution and provide a high-throughput in vitro drug screening platform for DA-related psychiatric disorders.In summary,based on BPE array and electrofluorescence switch visualization method,three biosensors have been constructed in this thesis.The specific binding of electrochemically active probe molecules to the cell surface by molecular recognition enables sensitive detection of non-electrochemically active protein expression levels or glycans on the cell surface and the electroactive molecule dopamine（DA）released from cells.To characterize the differences between populations of cells and to discern subpopulations of cells,molecular analysis at the single cell level is required.The optimized BPE array,combined with the microfluidic chip for single cell capture,realizes single cell detection,which is of great significance to the study the heterogeneity of single-cell.Eventually,in order to further improve the spatial resolution of imaging,a new mechanism of using gold nano-BPE arrays combined with confocal microscopy for cellular active molecule detection was designed and proposed.And the effect of drug stimulation on the release of electroactive molecules from cells was explored.In conclusion,these studies provide new ideas and strategies for the highly sensitive detection of active molecules,surface proteins or glycans released from cells,and also reveal the mechanisms of cell-drug interactions.