Study On Signal Amplification Strategy Of Electrochemiluminescence Peptide Biosensor

The incidence of tumors is the greatest threat to human health disease in the contemporary ear. With the development of society and economy, and environmental disruption, all kinds of emergency incident increase gradually, including the man-made and the natural. The development of highly sensitive and high-throughout methods for the detection of biomarkers is one of the most rapidly growing research areas in clinical tests for early discovery of heart disease and cancer. Electrogenerated chemiluminescence(also called electrochemiluminescence, abbreviated as ECL) involves the generation of species at electrode surfaces that then undergo electron-transfer reactions to form excited states that emit light. ECL method has many distinct advantages over fluorescence method because it does not involve a light source and avoids the attendant problems of scattered light and impurities luminescent. Moreover, the specificity of the ECL reaction associated with the ECL label and the coreactant species decreases problems with side reactions and is characterized by good spatial and temporal resolution. It has also widely been used in pharmaceutical analysis, bioanalysis, environmental analysis and clinical analysis. ECL analysis is becoming increasingly promising method for biomedical, environmental, clinical analysis owing to its simple instrumentation, high sensitivity, wide dynamic range, reproducibility, simplicity and rapidity analysis, its automation.ECL biosensor is a kind of analysis device based on employing enzyme, antigen/antibody, DNA as molecular recognition element and ECL signals as detection signal. With good selectivity, high sensitivity, fast analysis speed, easy operation and low price, it has been widely used in the life sciences, environmental analysis and drug analysis areas. Short linear binding peptides obtained using phage display, as substitute antibodies, have several advantages as compared to antibodies, including that peptides can be created synthetically in a reliable and cost-effective manner. Peptides are more stable and resistant to harsh environments and peptides are more amenable than antibodies to engineering at the molecular level. Peptide, as a molecular recognition material, has been wide spread concern in tumor biomarkers detection. Functional nanomaterials have excellent chemical and physical properties, high specific surface area, is beneficial to increase the sensitive molecular adsorption ability, and can improve the speed of biochemical reactions. Nanomaterials applied in the electrochemical biosensor can significantly improve the performance of the sensor.The aim of the present work is to develop highly sensitive and selective peptide based electrogenerated chemiluminescence biosensors for the detection of biomarker proteins. Taking advantages of the sensitivity of ECL and the specificity of biological molecular recognition substances, an electrogenerated chemiluminescence peptide biosensor for determination of troponin I with gold nanoparticle amplification and one electrogenerated chemiluminescence peptide-based biosensor for the determination of prostate-specific antigen by combining both signal enhancement from gold nanoparticle and signal quenching from ferrocene were fabricated, respectively, as the electrode modified with gold nanoparticles and DNA materials carriering signal. Exploring with novel properties peptide synthesis and separation methods of electrochemical luminescence probe; Study of peptide sensing interface of new theory and new method; Development the signal enhancement effect of nanomaterials research in electrochemical biosensing applications; Establish a high sensitivity to determine troponin I and prostate specific antigen.The major contents in this thesis are described as follows:First part, General introduction. In this chapter, introduces the definition and characteristics of Electrochemical Luminescence, emphatically expounds the Electrochemical Luminescence signal amplification method based on nanoparticles, and describe the research progress of molecular biology of two-dimensional amplify signals application in detecting tumor labeled objects. Finally, the purpose of this research work was presented. The second part, the research part, is made up of three chapters:First chapter, Electrogenerated Chemiluminescence Peptide-based Biosensor for the Determination of Prostate-specific Antigen Based on Target-Induced Cleavage of Peptide. A novel electrogenerated chemiluminescence peptide-based biosensor (ECL-PB) for the determination of prostate-specific antigen (PSA) was developed on basis of target-induced cleavage of a specific peptide within Nafion film incorporated with gold nanoparticles (AuNPs) and ECL emitting species. A specific peptide (CHSSKLQK) was used as a molecular recognition element, tris(2,2’-ripyridine) dichlororuthenium(II)(Ru(bpy)32+) was used an ECL emitting species as well as ferrocene carboxylic acid (Fc) was done as the ECL quencher. The ECL-PB biosensor was fabricated by casting the mixture of Nafion and AuNPs onto the surface of glassy carbon electrode to form AuNPs/Nafion film, and then electrostaticly adsorbing Ru(bpy)32+into the AuNPs/Nafion film, finaly self-assembling the peptide-tagged with ferrocene carboxylic acid (Fc-peptide) onto the surface of the AuNPs. In the presence of PSA, PSA specifically cleaved the Fc-peptide, led the quencher leave the electrode, and resulted in the increase of the ECL intensity obtained from the resulted electrode in0.1M phosphate buffer saline (pH7.4) containing tri-n-propylamine. The results showed that the increased ECL intensity was directly linear to the logarithm of the concentration of PSA in the range from5.0×10-12g/mL to5.0×10-9g/mL. An extremely low detection limit of8X1(T13g/mL was achieved since the signal amplification through AuNPs and the ECL background suppression through Fc as a ECL quencher. This work demonstrates that combination of the direct transduction of peptide cleavage events with highly sensitive ECL method is promising strategy for design of enzymatic cleavage-based ECL biosensors with highly sensitivity and selectivity.Second chapter, Electrogenerated Chemiluminescence Peptide-Based Biosensing Method for the Determination of Cardiac Troponin I Using Ru(bpy)32+-Functionalized Gold Nanoparticles as Signal-Amplifying Tags. Asensitive electrogenerated chemiluminescence peptide-based (ECL-PB) biosensing method for the determination of protein was developed by employing specific binding peptide(CFYSHSFHENWPS) as molecular recognition element and tris(2,2’-ripyridine) dichlororuthenium(II)(Ru(bpy)32+)-functionalized gold nanoparticles assignal-amplifying tags. Cardiac troponin I(cTnI), a reliable clinical biomarker for the detection of cardiac injury, was chosen as target protein. Gold nanoparticles (AuNPs)were employed as effective tag-carriersnot only for Ru(bpy)32+to form Ru(bpy)32+-AuNPs aggregates (Ru(bpy)32+-AuNPs) via electrostatic interactions, but also for peptide to form peptide labeled Ru(bpy)32+-AuNPs (Ru(bpy)32+-AuNPs-peptide) via thiol-Au bound. The specific peptide as a capture probe is self-assembled on the surface of a gold electrode. The presence of the target cTnI and Ru(bpy)32+-AuNPs-peptide leads to the formation of a sandwich-type peptide/cTnI/Ru(bpy)3+-AuNPs-peptide conjugates on the surface of the electrode, resulting in an ECL signals in the presence of coreactant tri-n-propylamine. The Ru(bpy)32+-AuNPs-peptide not only captures numerous signal-generating molecules, resulting in high ECL signal, but also captures lots of peptide, providing a sensing platform of polyvalent binding motiffor target protein. The results showed that the increased ECL intensity was directly related to the logarithm of the concentration of cTnI in the range from3.0×10-12g/mL to7.0×10-11g/mL with a low detection limit of0.5pg/mL.The proposed ECL-PB method was successfully applied to the detection of cTnI in human serum samples. This work provides a promising strategy for the determination of proteins in clinical analysis with simplicity, high sensitivity andselectivity.Third chapter, Electrogenerated Chemiluminescence Peptide-Based Biosensing Method for the Determination of Cardiac Troponin I based on the supersandwich DNA labeled Ru(bpy)32+. Here a novel strategy for ECL peptide biosensor by a supersandwich DNA is proposed for preparing an electrochemical luminescence probe for the ultrasensitive detection of Cardiac Troponin I. Peptide (FYSHSFHENWPSK) for molecular recognition material and DNA as the signal carrier. First, The specific peptide as a capture probe is self-assembled on the surface of a gold electrode. Electrochemical luminescence probe formed by the use of DNA hybridization to carry signal material, The presence of the target cTnI and electrochemical luminescence probe leads to the formation of a sandwich-type conjugates on the surface of the electrode. Based on the nucleic acid hybridization, more signal materials were carried. The results showed that the increased ECL intensity was directly related to the logarithm of the concentration of cTnI in the range from5.0×10-13g/mL to5.0×10-11g/mL with a low detection limit of0.14pg/mL.The proposed ECL-PB method was successfully applied to the detection of cTnI inhuman serum samples. This work provides a promising strategyfor the determination of proteins in clinical analysis with simplicity, high sensitivity andselectivity.In this thesis, we constructed three ECL peptide-based biosensors. The peptide as molecular recognition material, cardial troponin I and prostate specific antigen as a target analyte and introducing DNA and nanomaterials as a signal amplify material in the electrochemical luminescence analysis. This work provides novel strategy for the fabrication of the ECL biosensor, provides promising analytical devices for the early diagnosis of the clinical disease, and also provides a certain understanding of biosensing signal transduction, recognition reaction and response mode. The achievements in this work will promote the development of ECL biosensor and their applications in analytical, clinical, material and biologicial field.