| The incidence of tumors has become one of the main causes of death. The research showed that early diagnosis is key to treatment tumors. One of the important means of early detection tumor is high selectivity, high sensitive and fast analysis of tumor markers. Electrogenerated chemiluminescence (ECL) is a technique that combines chemiluminescence and electrochemistry. ECL technique not only has advantages of chemiluminescence analysis such as high sensitivity, wide linear, and simple instrument, but also has the advantages of electrochemical analysis such as easy to controll and good selectivity. ECL assays have attracted considerable interest for tumor markers detection in recent yrears.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 widespread concern in tumor biomarkers detection.The aim of the present work is to design and fabricate peptide-based ECL biosensors for the determination of biological molecule with high sensitivity and selectivity. 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 and an ultrasensitive electrogenerated chemiluminescence peptide-based method for the determination of cardiac Troponin I incorporating amplification of signal reagent-encapsulated liposomes was developed.The major contents in this thesis are described as follows: First part, General introduction. In this chapter, the definition and classification of ECL were briefly introduced. And then the trends in ECL biosensor were reviewed in detail. The classification of the peptide was presented and the research progress of peptide based biosensor was described. 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 biosensor for determination of troponin I with gold nanoparticle amplification. Peptide (FYSHSFHENWPSK) for molecular recognition material, Tnl protein as a target, Ru(bpy)2(dcbpy-NHS)(PF6)2was covalently labeled onto peptide through NH2-containing lysine on the peptide via acylation reaction and was utilized as an ECL probe, combining nanoparticle amplification effect, establish a high sensitivity electrogenerated chemiluminescence method. The ECL intensity versus the concentration of TnI was linear in the range from1.0×10-12g/mL to3.0×10-10g/mL with a detection limit of0.16pg/mL.The proposed method has higher sensitivity and lower detection limit than peptide biosensor for determination of troponin I without using gold nanoparticle. This work demonstrates that high sensitivity of the biosensor can be developed by using gold nanoparticles.Second chapter, ultrasensitive electrogenerated chemiluminescence peptide-based method for the determination of cardiac Troponin I incorporating amplification of signal reagent-encapsulated liposomes. An ultrasensitive electrogenerated chemiluminescence peptide-based (ECL-PB) method for the determination of cardiac troponin I (TnI) incorporating amplification of signal reagent-encapsulated liposome was reported for the first time. A short linear specific binding peptide (FYSHSFHENWPSK) synthesized was employed as molecular recognition element for TnI, which was a reliable biomarker for detecting cardiac injury. Liposomes assembled using a standard sonication procedure were designed as the carrier of ECL signal reagents (bis(2,2’-bipyridine)-4,4’-dicarboxybipyridine ruthenium-di(N-succinimidyl ester) bis(hexafluorophosphate)) for signal amplification. The magnetic capture peptides for the enrichment of the target protein and magnetic separation were synthesized by covalently attaching the peptides to the surface of magnetic beads via acylation reaction, and the liposome peptides were synthesized by covalently attaching the peptides to the signal reagent-encapsulated liposomes. In the presence of TnI, sandwich-type conjugates were generated in incubation of the magnetic capture peptides and the liposome peptides. After a magnetic separation, the sandwich-type conjugates were treated with ethanol and thus a great number of the ECL reagents were released and measured by ECL method at a bare glassy carbon electrode with a potential pulse of+1.15V versus Ag/AgCl in the presence of tri-n-propylamine. The increased ECL intensity has good linearity with the logarithm of the TnI concentration in the range from10pg/mL to5.0ng/mL with an extremely low detection limit of4.5pg/mL. The proposed ECL-PB method was successfully applied to the detection of TnI in human serum samples. This work demonstrated that the employment of the magnetic capture peptides for the enrichment of the target proteins and magnetic separation and the liposome peptides for the signal amplification and polyvalent binding motifs may open a new door to ultrasensitive detection of proteins in clinical analysis.Third chapter, 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. A novel electrogenerated chemiluminescence peptide-based (ECL-PB) with high sensitivity and great selectivity by combining both signal enhancement from gold nanoparticle and signal quenching from ferrocene was demonstrated. A helix peptide (CHSSKLQK) was served as a molecular recognition element and Ru(bpy)32+was used as an ECL signal. The helix peptide was labeled with the ferrocene at NH2-containing lysine and utilized as capture probe (Fc-peptide). Gold nanoaprticles was firstly modified onto Nafion modified glassy carbon electrode by casting the mixture of Nafion and gold nanoaprticles, and then Ru(bpy)32+were effectively incorporated into the composite film of Nafion based on the electrostatic interaction between positively charged Ru(bpy)32+and negatively charged Nafion to form ECL platform(Ru(bpy)32+/AuNPs/Nafion film). The peptide biosensor was fabricated by immobilizing the capture probe tagged with an ECL quencher ferrocene onto gold nanoparticles in Ru(bpy)32+/AuNPs/Nafion film on glassy carbon electrode via self-assembling technique through a thiol-containing cysteine at the end of the peptide. Ferrocene-labeled peptide can move flexibly on the resultant electrode Via Au-S interaction results in a lower ECL background signal, which is fariouable for lower detection limit. The principle of ECL measurement is based on the specific proteolytic cleavage event of Fc-peptide on the gold electrode surface in the presence of PSA. Peptide was recognized and cleaved between the glutamine(Q) and lysine (K) position in the presence of target PSA in the presence of PAS, resulting in the decrease of the amount of Fc on the electrode surface, and thus, resulting in the increase of ECL signal detected. The increased ECL intensity was directly linear to the concentration of PSA in the range from1.0×10-11g/mL to1.0×10-9g/mL with a detection limit of2.3pg/mL. This work demonstrates that the direct transduction of peptide cleavage events into an ECL signal by combining both signal enhancement from gold nanoparticle and signal quenching from ferrocene provides a simple and sensitive method for detecting target protein.In this thesis, we constructed two ECL peptide-based biosensors and one ECL homogeneous method to detect TnI and PSA. 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. |
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