Detection Of Matrix Metalloproteinases By An Electrochemiluminescence Peptide Biosensor Based On A Cleavage Reaction

Matrix metalloproteinases (MMPs) constitute a family of zinc dependent endopeptidases capable of degrading multiple extracellular matrix components. These enzymes play important roles in regulating cell apoptpsis, proliferation, tumor matastasis and invasion. MMPs have been found to be closely associated with matastasis and invasion of tumor, which makes the MMPs biomarkers for early cancer diagnosis and prognosis tests.For example, the over-expression of MMP2 and MMP9 in prostate and breast cancer,they can be used as biomarkers in early-stage prostate and breast cancer diagnosis. In addition, some protease, enzyme activator or enzyme inhibitors can accelerate the process of apoptosis of tumor cells, so that they can be as drugs for the treatment of tumors.For example, through chemical connections of the substrate of protease and classical anti-cancer drugs, we can get anticancer with low toxicity and targeting. Therefore, the development of ultrasensitive, fast, simple and multiplexed method of joint detection for matrix metalloproteinases is of great significance for early diagnosis of primary tumor and metastatic disease as well as drug screening.Electrogenerated chemiluminescence also called Electrochemiluminescence (ECL) involves the generation of species at electrode surfaces that then undergo electrochemical reactions to form excited states that emit light.The ECL singal mainly detected by PMT and CCD. Due to its many advantages, such as high sensitivity, excellent repeatability, wide linear range and amenable miniaturization. ECL method is becoming increasingly promising method in food inspection, bio-medicine, environment monitoring and clinical analysis. As molecular recognition materials, peptides have many advantages, including that peptides have small molecular weight, easy to synthesize, cost-effective, high biological activity and good environmental resistance compared with proteins, so they have been widely employed in tumor biomarkers detection.The aim of this work is to develop simple and ultrasensitive electrogenerated chemiluminescence peptide-based biosensors for the detection of matrix metalloproteinases and inhibitors by employing peptides as molecular recognition elements, (Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2and(dfppy)2Ir(dcbpy)(PF6)2 was used ECL signals.Multiplexed ECL biosensor was proposed for the simultaneous determination of matrix metalloproteinase-2 and matrix metalloproteinase-7.The major contents in this thesis are described as follows:First part, General introduction. In this chapter, we give a brief introduction of the definition, classification, effect and function of matrix metalloproteinases and then summarized the applications of electrogenerated chemiluminescence peptide-based bioassay in biological analysis; Then several detection methods of matrix metalloproteinases were reviewed and the trends in ECL biosensor were in detail. Finally, the idea, methods and purpose of this research work was presented.The second part, the research part, is made up of two chapters:First chapter, A simple and sensitive electrogenerated chemiluminescence biosensor was developed to monitor matrix metalloproteinase 2 (MMP-2) by employing a specific peptide (CGPLGVRGK) as a molecular recognition substrate. Bis(2,2’-bipyridine)-4’-methyl-4-carboxybipyridine-rutheniumN-succinimidyl ester-bis(hexafluorophosphate)(Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2 (Rul)was usedan ECL emitting species and covalently labeled onto the peptide through NH2-containing lysine on the peptide via acylation reaction to form Rul-peptide as the ECL probe.An ECL peptide based biosensor was fabricated by self-assembling of the ECL probe onto the surface of gold electrode. MMP-2 can specifically cleave the Rul-peptide on the electrode surface, led the partly Rul-peptide leave the electrode surface, and then resulted in the decrease of the ECL intensity obtained from the resulted electrode in 0.1 M phosphate buffersaline (pH 7.4) containing tri-n-propylamine.The decreased ECL intensity was piecewise linear to the concentration of MMP-2 in the range from 1 ng/mL to 500ng/mL. Moveover, the ECL biosensor is successfully applied to detection of MMP-2 secreted by living cell, such as HeLa cells. Additionally, the biosensor was also applied to the evaluation of matrix metalloproteinase inhibitors. The strategy presented here is promising for other disease-related matrix metalloproteinase assay and matrix metalloproteinase inhibitor profiling with sensitivity and simplicity.Second chapter, A dual-potential multiplexed electrogenerated chemiluminescence (ECL) peptide-based biosensor is developed to simultaneous detect matrix metalloproteinase using two potential separation ECL emitters. (dfppy)2Ir(dcbpy)(PF6)2 (Ir1, in which dfppy is 2-(2,4-difluorophenyl)pyridine and dcbpyis 2,2’-bipyridine-4,4’-dicarboxylate) and bis(2,2’-bipyridine)-4’-methyl-4-carboxybipyridine-rutheniumN-succinimidyl ester-bis(hexafluorophosphate)(Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2 (Rul) were used as multiple ECL signals.An ECL emission with potential separation of Irl and Rul at gold electrode was observed at 0.9 V and 1.4 V vs Ag/AgCl, which is promising for simultaneous determination of multiple targets. Moreover, a multiplexed ECL biosensor was proposed for the simultaneous determination of matrix metalloproteinase-2 and matrix metalloproteinase-7 as a "proof-of-principle"concept. Based on the decrease of ECL intensity at different potential, the multiplexed ECL bioassay enabled the simultaneous monitoring of MMP-2 and MMP-7 in a single run.A detection limit as low as 5 ng/mL and 10 pg/mL for MMP-2 and MMP-7 can be achieved by this sensing approach, with a linear relationship between decreased ECL intensity and the concentration from 10ng/mL to 300ng/mL for MMP-2 and 0.05ng/mL to lng/mLfor MMP-7.The dual potential response ECL biosensor endows a feasibility tool for clinical diagnosis in complex biological systems.This novel multiplex ECL bioassay provided a simple, sensitive, specific and reliable alternative for the simultaneous detection of tumor markers in clinical laboratory.