Electrochemical Biosensor For Detection Of Matrix Metalloproteinase-14 And CD44 Based On Interaction Between Inhibitory Peptide And Its Hemopexin-like Domain

With the development of urbanization,industrialization,aging and the deterioration of ecological environment,the incidence of malignant tumor is increasing in China,It has seriously affected people's quality of life.Some study shows membrane Matrix Metalloproteinase-14?MMP-14?plays an important role in the process of tumor metastasis and invasion in human body,it is also considered as an important indicator of metastasis of some malignant tumors,such as gastric adenocarcinoma,esophageal squamous cell carcinoma and colorectal cancer.The hemopexin-like?PEX-14?is a domain of MMP-14,some study shows that PEX-14 is necessary for MMP-14 homodimerization and heterodimerization with CD44,and activation signaling pathways involved in cell migration,promote the metastasis and invasion of tumor cells in human body.With the increasing demand for diagnostics and therapeutics against MMP-related diseases and other disorders,much effort has been made to develop effective methods to assay MMPs and their inhibitors.Some study shows peptide inhibitor selected by PEX-14 is high specific to MMP-14,it interacts with the PEX-14 and inhibits the activity of MMP-14,significantly inhibited the proliferation of tumor cells.CD44 is a cell surface protein,it is involved in the occurrence,metastasis and invasion of human tumors,and it can be used as a biomarker of some tumors,such as hepatocellular carcinoma,breast cancer,renal cell carcinoma and colon cancer,so it received considerable attention.This study the specific binding of PEX-14 in MMP-14 is used as the recognition system,electrochemical impedance biosensor?EIS?was developed for the detection of MMP-14 and associated surface protein CD44 with high sensitivity and selectivity.Electrochemical Impedance Spectrum?EIS?has been widely used in environmental analysis and medical monitoring due to its simple operation,high sensitivity and fast analysis speed.EIS biosensor is an analytical device which fixes specific recognition substances such as DNA,peptide,antigen/antibody on transducer and uses AC impedance signal as detection signal.Compared with other biometrics,peptide inhibitors have attracted much attention because of their high affinity,high bioactivity,low cost,small molecular weight and easy modification.In this paper,EIS biosensors with high sensitivity and selectivity for detection of MMP-14 and CD44 were developed using peptide inhibitors as molecular recognition substances.This paper consists of two parts:the first part is Chapter I introduction,the second part is research report include Chapter II to Chapter IV.The Chapter I including summary of electrochemical impedance spectroscopy,Matrix Metalloproteinases?MMPs?,cell surface protein CD44 and peptide inhibitors.Finally,the purpose and content of this paper are explained.Chapter II to Chapter IV is research report.Chapter II:Study on labeled and non-labeled PEX-14 Electrochemical Biosensors.Peptide inhibitor selected from PEX-14:IS4,ISS4,IVS4 and IVSS4.This four peptide inhibitor modified with“?CH₂?₆-Cys-OH”,?ISC,ISSC,IVSC,IVSSC?.Four different types of electrochemical biosensors were developed using ISC,ISSC,IVSC and IVSSC as molecular recognition substances.The results show that the electrochemical biosensor developed by using ISC and IVSC as molecular recognition substances has a strong response to PEX-14.We developed non-labled biosensor using ISC as molecular recognition substances and electrochemical impedance technique.Labeling the carboxyl ferrocene derivative?Fc-COOH?at the N-terminal of ISC?CIS-Fc?,and we developed Labeled electrochemical biosensor using CIS-Fc as molecular recognition substances?L-EC biosensor?.The results showed that for EIS biosensor,R_et has a fine linear relationship with the concentration of PEX-14 in the range from 0.1 ng/mL to 7 ng/mL,and the detection limit was 0.03 ng/mL;for L-EC biosensor,the peak current has a fine linear relationship with the concentration of PEX-14 in the range from 1 pg/mL to 10 pg/mL,and the detection limit was 0.3 pg/mL.The results showed that the specific interaction between peptide inhibitor and PEX-14 domain was a recognition system,and the quantitative detection of PEX-14 was realized.And lay a foundation for the development of electrochemical biosensor for MMP-14 detection in the next step.Chapter III:Study on the Electrochemical Impedance Biosensor of MMP-14.We developed direct detection of MMP-14 biosensor based on the specific interaction between peptide inhibitor?IVSC?and PEX-14 in MMP-14structure.We developed indirect detection of MMP-14 biosensor based on the interaction between peptide inhibitor ISC and PEX-14 and MMP-14.The results showed that for direct detect MMP-14 biosensor,R_et has a fine linear relationship with the concentration of MMP-14in the range from 1?g/L to 50?g/L,and the detection limit was 0.19?g/L.For indirect detect MMP-14 biosensor,R_et has a fine linear relationship with the concentration of MMP-14in the range from 0.1 ng/L to 50 ng/L,and the detection limit was 7?g/L.The analytical performance of the two detectors is compared and discussed.The results show that the detection range of the two detectors is complementary,and the detection sensitivity of the indirect sensor is better than that of the direct sensor.Chapter IV:Study on non-labeled CD44Electrochemical Biosensor.We develoed non-labled biosensor for detection of CD44 based on the specific binding of peptide inhibitor?IVSC?to PEX-14 and the formation of heterodimer between PEX-14 and CD44 using electrochemical impedance technique,and the results showed that R_et has a fine linear relationship with the concentration of CD44 in the range from 0.1 ng/mL to 1.0 ng/mL.