| Protease is any enzyme that performs proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds.It is closely related to a large number of vital processes in the body’s metabolic processes, including the cell growth, cell death, tissue remodeling, and immune defense, and also used as biomarkers. 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. Based on bioaffinity electrogenerated chemiluminescence (also called electrochemiluminescence and abbreviated ECL) sensors, general biological affinity of ECL analysis was conducted on the electrode surface, with high sensitivity, good selectivity. Commer-cialized Roche (Roche) automatic electrogenerated chemiluminescence Immunoassay analyzer is currently most advanced in the world. It is also based on bioaffinity reaction which is closed cooperation with the analysis of the detection method. The bioaffinity system required little sample size, high sensitivity, on the basis of the chemiluminescen-ce analyzer, with magnetic separation, biotin, affinity and ruthenium markers. However, based on bioaffinity reaction of ECL sensor detection usually is disposable sensor, needs to be fixed electrode, has complex electrode reaction, and several reaction steps, such as washing, separate difficultly. In order to overcome drawbacks from probes directly immobilized on electrodes and commercial ECL biosystems based on bioaffinity reactions and to improve sensitivity for the detection of protease. We put forward a novel bioanalytic systems based on high sensitivity for the quantitative detection of protease.The aim of the present work proposed a novel electrogenerated chemilumine-scence (ECL) bioanalytic system based on bio-cleavage of ECL peptide probe and homogeneous detection and utilized for the first time for highly sensitive quantification proteases to overcome drawbacks from probes directly immobilized on electrodes and commercial ECL biosystems based on bioaffinity reactions. Prostate specific antigen (PSA) as target, specific peptide sequence (CHSSKLQK) as the recognition molecular, ruthenium bipyridine derivatives as ECL signals, established a simple solid phase synthesis with magnetic bead-based electrogenerated chemiluminescence probe (Fe3O4@Au-peptide-Ru1).In order to improve the sensitivity of ECL signal detection of protease, Nafion and the gold nanoparticles modified electrode as signal the material carrier, Established AuNPs/Nafion/GCE detection method for the enrichment of signals to design a high sensitivity bioanalytic system based on bio-cleavage of ECL peptide probe electrogenerated chemiluminescence biosensor, which will be applied to the detection of biomarkers.The major contents in this thesis composed of two parts:introduction and research reports.The first part is the introduction, a brief overview of electrogenerated electrochemiluminescence, electrochemical electrochemiluminescence biosensor and its types, emphatically expounds the Electrochemical Luminescence signal amplification method based on nanoparticles in detecting tumor labeled objects. Summarizing of the peptide was presented and the research progress of peptide based biosensor was described. Finally, the research background, research ideas, research purpose and research contents were presented.The second part is the research report, including the second chapter and the third chapter.Second chapter constructs 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, establish a new enrichment method to detect Ru(bpy)32+, the method of the establishment of the homogeneous solution Ru(bpy)32+ and the detection limit was reduced to 0.85×10-14 mol/L. Building a stable and sensitive electrochemical luminescence analysis method to improve sensitivity of the electrochemical luminescence imaging analysis system for homogeneous detection Ru (bpy)32+. It can provide visual information for clinical diagnosis and pathological research and provide technical support for the electrochemiluminescence biosensor characterization and analysis.Two, this paper mainly design a novel electrogenerated chemiluminescence (ECL) bioanalytic system based on bio-cleavage of ECL peptide probe and homogeneous detection was designed and utilized for the first time for highly sensitive quantification proteases to overcome drawbacks from probes directly immobilized on electrodes and commercial ECL biosystems based on bioaffinity reactions.in this work, prostate specific antigen (PSA) was taken as a model analyte and ruthenium complex-tagged specific peptide (CHSSKLQK) was designed as an ECL probe (peptide-Rul). A simple synthesis method for ECL bioconjugated magnetic beads (Fe3O44@Au-peptide-Rul) was developed through solid-phase synthesis. The ECL bioanalytic system worked currently as:when analyte PSA was introduced into dispersion of Fe3O4@Au-peptide-Rul and caused a bio-cleavage of the peptide, ECL measurement was carried out in the presence of co-reactant tripropylamine using two models. One is homogeneous ECL detection on a bare pencil electrode and the other is ECL detection after the cleaved Rul-part of the peptide was concentrated into surface film of Nafion/gold nanoparticles modified graphite pencil electrode (AuNPs/Nafion/PGE). For bare PGE, the increased ECL intensity was directly linearly proportional to the PSA concentration in the range from 1.0×10-11 g/mL to 1.0×10-10 g/mL. The correlation coefficient was 0.9946. The detection limit was 5.0×10-12g/mL. For modified graphite pencil electrode, the increased ECL intensity was directly linearly proportional to the PSA concentration in the range from 5.0×10-12 g/mL at GCE to 8×10-14 g/mL at AuNPs/Nafion/PGE.The high sensitivity (Slope from 6.3 at GCE to 107 at AuNPs/Nafion/PGE) and low detection limit were obtained at Nafion/AuNPs/PGE. All results indicate that the good performance of the proposed bioanalytical system is achieved using two models on both bare GCE and AuNPs/Nafion/PGE. The proposed ECL bioanalytic system may open a door to ultrasensitive ECL biosensing for other proteases and other detection techniques including optics and electrochemistry. |
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