In Situ Quantification Of N-cadherin Expression By An Electrochemical Method Based On Gold Clusters Probe

Cancer is the second killer which threats to human health and life worldwide.Tumor metastasis is one of main causes of the death of cancer patients.Epithelial-to-mesenchymal transition（EMT）is the initial step of tumor cells invasion and metastasis,which plays an important role in initiating and promoting tumor cells invasion and metastasis.N-cadherin serves as an important onco-biomarker of EMT progression,whose overexpression can lead to the decrease of cell adhesion function and the metastasis of tumor cells.Pancreatic cancer is a kind of gastrointestinal malignant tumor with characteristics of strong aggression,rapid metastasis,difficult early diagnosis and high mortality.It has been demonstrated that early invasion and metastasis of pancreatic cancer are accompanied by epithelial mesenchymal cell transformation.The expression level and distribution of N-cadherin in pancreatic cancer cells can be determined to provide an important basis for evaluating the malignant degree,differentiation stage and invasion ability of pancreatic cancer cells to adjacent tissues.At present,most of the detection methods rely on the analysis of the tumor biomarkers in body fluids,tissues or cells lysates.The loss of biomarkers and the interference of multiple biomarkers during the analysis is inevitable.Moreover,the techniques normally require expensive equipments,professional operators,and long turnaround times.Electrochemical biosensor is increasingly employed in detection of tumor biomarkers due to its high sensitivity,simple preparation,and quick response.As an emerging novel nanomaterial,metal clusters have aroused great research interests in the field of electrochemistry and electroanalytical chemistry due to their good biocompatibility,unique electronic and molecular structure,as well as low surface coordination number.In recent years,metal clusters have been used as electrochemical sensing materials in the field of biological analysis.This thesis focuses on the EMT process,which is closely related to the invasion and metastasis of malignant tumors.The inherent targeting effect of peptide-gold clusters（Au Cs）was utilized to specifically identify N-cadherin expressed on the cell membrane.Then an electrochemical sensing platform is constructed to accurately quantify the expression of N-cadherin in situ relied on the electrocatalytic property of Au Cs.Thus,different stages of EMT can be accurately distinguished,and early diagnosis and prevention of EMT-related tumor metastasis might be realized in the future.Polypeptide sequences with both N-cadherin targeting and biomineralization cluster ability were designed to biomineralize peptide-protected gold clusters.Au Cs were synthesized by using the polypeptide as template and targeting moiety.Then,an electrochemical sensing platform was constructed based on the Au Cs as a biological probe to achieve in situ,accurate and rapid quantitative analysis of N-cadherin on the surface of cell membranes.The results are as follows.1.A peptide sequence（SWTLYTPSGQSKKKKYCC）was designed to target N-cadherin and mineralize into Au Cs.Au Cs emitted red fluorescence with good dispersion and stability were synthesized by a mild biomineralization method.The precise molecular formula of the clusters,which is Au₂₀Peptide₇,was obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF MS）.Pancreatic cancer cells,PANC-1 and Bx PC-3,with significantly different expression levels of N-cadherin were selected as EMT model cells.Based on the fluorescence characteristics of the prepared Au Cs,it was found that Au Cs could identify the tumor cell membrane N-cadherin observed by laser confocal microscopy.The fluorescence intensity on the cell membrane surface of the two cells was significantly different,which initially confirmed the targeting of Au Cs.The quantitative results of N-cadherin based on mass spectrometry were consistent with the expression level of N-cadherin obtained by Western blot analysis,further confirming the specific targeting of synthesized Au Cs to N-cadherin.2.The in situ electrochemical analysis and sensing platform for N-cadherin on cell membrane surface was constructed based on the targeting property and the inherent electrocatalytic activity of Au Cs.The successful construction of the electrochemical sensor was verified by cyclic voltammetry（CV）,electrochemical impedance spectroscopy（EIS）and differential pulse voltammetry（DPV）.Au Cs can not only accelerate the electron transfer rate on the electrode surface,but also initiate a good electrocatalysis.This indicates that the redox active Au Cs function as both electron transport medium and electrochemically sensing electron conductor.First,the Au Cs were chemically bonded to the surface of the polydopamine modified glassy carbon electrode.The amount of Au Cs fixed on the surface of electrode was linearly analyzed by recording DPV current signals from Au Cs-catalyzed redox reaction between hydroquinone（HQ）and H₂O₂,whereafter the standard curve of electrochemical analysis was established.Subsequently,gradient number of pancreatic cancer cells that recognized Au Cs were modified to the surface of the electrode,and DPV measurement was performed under the similar electrical analytical conditions.The results of DPV peak current of the determined number of cells were substituted into the standard curve for linear regression analysis.After quantitative calculation,the expression number of N-cadherin in each PANC-1 and Bx PC-3 cell is about（6.32±0.22）×10~8and（1.55±0.21）×10~8.These results are very close to that obtained from ICP-MS assay,which is（5.92±0.21）×10~8 and（2.36±0.13）×10~8,respectively.It further indicates that our strategy of in situ quantification of N-cadherin by electrochemical method is reliable.It provides a new way to quantitatively study the changes of EMT phenotype in tumor cells by analyzing the difference of N-cadherin expression in pancreatic cancer cell lines.