Electrochemilu Minescence Cytosensor And Single-cell Imaging Analysis

Electrochemiluminescence(ECL)is a kind of chemiluminescence relied on the high-energy electron-transfer triggered by electrochemical reactions.It is a technology coupled with electrochemistry and chemiluminescence.ECL is a powerful technology in analytical research due to its wide linear range,low background and high controllability.When we carried our cell analysis or cell imaging using ECL technology,it was difficult to realize the ECL reaction in the cell covered area because of the steric hindrance of cells on electrodes,which limited the application of ECL in cell-based analysis.Therefore,we established new ECL methods based on permeable substrates to achieved the sensitive cell sensing and the ECL imaging at single-cell level without cells' steric hindrance.The main research contents were described as follows:1.Direct electrochemiluminescence imaging of a single cell on a chitosan film modified electrodeSingle-cell imaging is essential for elucidating the biological mechanism of cell function because it accurately reveals the heterogeneity among cells.The electrochemiluminescence(ECL)microscopy technique has been considered a powerful tool to study cells because of its high throughput and zero cellular background light.However,since cells are immobilized on the electrode surface,the steric hindrance and the insulation from the cells make it difficult to obtain a luminous cell ECL image.To solve this problem,direct ECL imaging of a single cell was investigated and achieved on chitosan and nano-TiO2 modified fluoride-doped tin oxide conductive glass(FTO/TiO2/CS).The permeable chitosan film is not only favorable for cell immobilization but also increases the space between the bottom of cells and the electrode,thus,more ECL reagent can exist below the cells compared with the cells on a bare electrode,which guarantees the high sensitivity of quantitative analysis.The modification of nano-TiO2 strengthens the ECL visual signal in luminol solution and effectively improves the signal-to-noise ratio.The light intensity is correlated with the H2O2concentration on FTO/TiO2/CS,which can be applied to analyze the H2O2 released from cells at the single-cell level.As far as we know,this is the first work to achieve cell ECL imaging without the steric hindrance effect of the cell,and it expands the applications of a modified electrode in visualization study.2.Electrochemiluminescence Investigation of Glucose Transporter 4 Expression at Skeletal Muscle Cells Surface Based on a Graphene Hydrogel ElectrodeIn situ detection of the expression level of cell-surface receptors has become a hotspot study in recent years.We propose in this manuscript a novel strategy for sensitive electrochemiluminescence(ECL)detection of glucose transporter 4(GLUT4)on human skeletal muscle cells(HSMC),Graphene hydrogel(GH)was selected to fabricate a permeable electrode with purpose of overcoming the steric hindrance of cells on electrode,which leads errors in the detection of cell-surface receptors.GLUT4 was labeled with carbon dots(CDs),which generate ECL emission at the interface between GH and cells,so about half amount of GLUT4 expressed at cell-surface could be determined,which provided an accurate GLUT4 expression quantification.The prepared cytosensor exhibited a good analytical performance for HSMC cells,ranging from 500 to 1.0 × 106 cells·mL-1,with a detection limit of 200 cells·mL-1.The average amount of GLUT4 on per HSMC cell was calculated to be 1.88 ×105.Furthermore,GLUT4 on HSMC surface had a 2.3-fold increase under the action of insulin.This strategy is capable of evaluating the receptors on cell-surface,which may push the application of ECL for the disease diagnosis.3.Potential-Resolved Electrochemiluminescence for Simultaneous Evaluation of Two Cell-Surface Receptors and Visual Diagnosis of Single Apoptotic CellsSingle-cell analysis provides insights into biological and medical sciences and enhances the understanding of cell behavior and intercellular communication.In this work,a potential-resolved electrochemiluminescence(ECL)method was developed and further used for apoptotic cells diagnosis.Under the treatment of resveratrol(RVL),the expression of epidermal growth factor receptor(EGFR)on MCF-7 cell-surface was decreased.Meanwhile,RVL induced apoptosis and promoted phosphatidylserine(PS)eversion in the cytomembrane.Here,Au@L012 and g-C3N4 as ECL probes were functionalized with epidermal gro,wth factor(EGF)and peptide(PSBP)to recognize the EGFR and PS on cell-surface,respectively,showing two well-separated ECL signals during a potential scanning.The relative ECL change of g-C3N4 and Au@L012 correlated with the degree of apoptosis,which provided a new way to investigate apoptosis.Importantly,the proposed method succeeded to simultaneously evaluate the EGFR and PS expression and achieved the visualization analysis of ECL probes labeled cells on an ECL microscopy.This might be the first time to diagnose single apoptotic cell using ECL imaging technique.Notably,this strategy contributed to visually studying tumor markers and pushing the application of ECL imaging for the disease diagnosis at single-cell level.