Highly Sensitive Electrochemiluminescent Biosensing Analysis Based On Multiple Signal Amplification Strategies

Highly sensitive and accurate detection of disease markers is important for early diagnosis and treatment of diseases.Electrochemiluminescence（ECL）biosensors are favored by researchers because of their near-zero detection background and fast analysis speed.However,its sensitivity is still not sufficient to meet the needs of early screening,early diagnosis and early detection of some major diseases such as cancer,so the development of new ECL sensing methods based on efficient signal amplification strategies is crucial to achieve sensitive and accurate detection of low abundance disease markers.Based on this,this thesis utilizes various signal amplification strategies such as aggregation-induced electrochemiluminescence（AIECL）,resonance energy transfer（RET）,Fenton-like reactions and enzymatic reactions to take full advantage of triple and bipolar electrodes（BPE）.The research aims to build a new platform for high performance ECL biosensing to achieve sensitive and accurate detection of disease markers.The main research and technological innovations are as follows:1.Gold nanoclusters（APO@Au NCs）encapsulated in desferrin（APO）were used as ECL luminophores and Zn²⁺as inducer to trigger AIECL effect in the restricted domain space of APO to construct a new platform for the sensitive detection of prostate-specific antigen（PSA）by cleaved ECL sensing.Firstly,a sandwich-type immunosensor was established by immobilising the target protein PSA and Zn O nanoflowers（NFs）probes.The nanoflower structure was disrupted by the addition of HCl to release large amounts of Zn²⁺;then,it was transferred to an electrochemical detection cell containing APO@Au NCs,and Zn²⁺passed through the ion channels on the surface of the protein cage into the APO cavity,which induced the aggregation of Au NCs and hindered the ligand free vibration and rotation-induced energy dissipation,generating a strong ECL signal.Compared to Au NCs dispersed in solution,the ECL intensity of Au NCs in the aggregated state within the APO cavity was increased by approximately 6-fold.The new platform constructed was used for the sensitive detection of PSA under optimised conditions with a linear range of 5.0×10^-14-1.0×10^-7 g/m L and a detection limit as low as 2.6×10^-15 g/m L.In addition,an application study was carried out for the determination of PSA in human serum with recoveries ranging from 93.5%to 119%.This work is a novel and efficient AIECL strategy using the restricted space of APO to effectively improve the ECL efficiency of metal nanoclusters,providing a new method for the sensitive detection of disease markers and a new idea for the design of AIE detection systems.2.Sensitive and accurate detection of myoglobin（Myo）ratios on home-made double electrodes using sulphur quantum dots（S QDs）and ruthenium-coated ruthenium silicide(Ru（bpy）₃²⁺@Ru Si NPs)as cathodic ECL emitters,combined with spatially and potentially resolved techniques,and with the RET effect of Au@Ag₂S NPs and Ru（bpy）₃²⁺@Ru Si NPs A new platform for ECL sensing.Firstly,S QDs and Ru（bpy）₃²⁺@Ru Si NPs were modified at the dual electrode interface;then,a fixed concentration of Myo was modified at the cathode interface as the reference electrode by layer modification;the Au@Ag₂S NPs probe and a series of concentrations of Myo were modified at the anode interface as the working electrode.NPs have a broad light absorption range and can serve as a perfect acceptor for Ru（bpy）₃²⁺@Ru Si NPs,with the RET effect occurring between the two to achieve signal amplification.When a cyclic scanning voltage is applied,a constant cathode signal and a concentration-dependent variation of the anode signal are obtained,and the ratio of the two signals is used as the basis for quantitative analysis,effectively avoiding the interference caused by system and other errors and improving the reliability of the analysis results.The sensor enables sensitive detection of Myo at concentrations of 1.0×10^-13-1.0×10^-7 g/m L with a detection limit as low as 5.0×10^-14 g/m L.It has also been successfully applied to the analysis of Myo in human serum samples.3.A new cathode-catalyzed bipolar electrochemiluminescence（BP-ECL）sensing platform for sensitive detection of PSA was designed by combining the advantages of physical separation of closed BPE cathodes and anodes with signal amplification strategies of Fenton-like reactions and redox-active species replacement.The high catalytic activity of Cu²⁺,the Fenton-like reaction with H₂O₂,promotes the conversion of H₂O₂ into reactive intermediates and enhances the ECL intensity of the luminol L-012.The cathode end uses K₃Fe（CN）₆ with low reduction potential instead of dissolved oxygen with high reduction potential to significantly reduce the driving voltage and accelerate the reaction rate,further enhancing the ECL intensity.The synergistic amplification effect at both ends of the BPE was used to achieve highly sensitive detection of PSA with a linear range of 1.0×10^-13-1.0×10^-7 g/m L and a detection limit as low as 5.0×10^-14 g/m L.4.A novel dual-mode sensing platform was developed for the sensitive detection of PSA based on the advantages of physical separation of the yin and yang extremes of BPE,combined with metal peroxide hydrolysis and enzymatic reaction signal amplification strategies,using Prussian blue（PB）and L-012 as colorimetric and ECL indicators,respectively.First,glucose oxidase-loaded Mg O₂ nanoflower complexes（GOD@Mg O₂NFs）were used as signal probes.The sandwich immunosensor was constructed by immobilizing the target protein PSA and the signal probe in a 96-well plate with the aid of specific recognition between antigen-antibodies.When glucose is added,GOD catalyzes the oxidation of glucose to produce gluconic acid and H₂O₂.The acidic environment generated by gluconic acid promotes the hydrolysis of Mg O₂ NFs and further production of H₂O₂,which is transferred to the BPE anode cell of the pre-modified luminescent reagent L-012 for testing.Under a single electrochemical scan,L-012 at the anodic end emits an ECL signal while PB（blue）at the cathodic end is converted to Prussian white（PW,white）,enabling dual mode output of ECL and colorimetry.Optimised for highly sensitive,visualised detection of PSA,the detection limits for the ECL and colorimetric methods were 2.7×10^-12 g/m L and 3.1×10^-9g/m L,respectively,with linear ranges of 5.0×10^-12-1.0×10^-7 g/m L and 5.0×10^-9-1.0×10^-7 g/m L.The constructed dual-mode detection platform was successfully applied to the analysis of PSA in real samples of human serum.By fusing different sensing principles and achieving dual-mode co-configuration on a single sensor device,the traditional limitation of separate dual-mode assays is overcome.