Construction Of Chemiluminescence/Electroluminescence Visual Sensing Platform And Its Immunoassay

In recent years,the incidence of malignant tumors and myocardial diseases has increased year by year,which seriously endangers human life and health.People pay more and more attention to their own health and safety,and the application of analytical chemistry in life science shows more and more important value and significance.However,in the early stage of disease,the content of some bioactive molecules in human body is often very low,which makes it difficult to quantitatively detect some trace bioactive molecules.Therefore,it is very important to explore new signal amplification strategies combined with advanced detection techniques to achieve high sensitivity/specificity immunoassay.Chemiluminescence（CL）/electrochemiluminescence（ECL）technology is widely used in immunoassay because of its high sensitivity,wide linear range and fast analytical speed.Furthermore,imaging,as a new technology rising rapidly,combined with CL/ECL technology can effectively realize the visual detection of bioactive molecules.Of course,while pursuing advanced and effective detection technology,it is also urgent to further improve the sensitivity of immunoassay to achieve high sensitivity detection of trace bioactive molecules in human body.This paper constructs a series of new CL/ECL imaging biosensing platforms by combining CL/ECL imaging technology with different signal amplification strategies to realize sensitive detection and biochemical analysis of bioactive molecules.The main innovations and research contents are as follows.1.A novel chemiluminescence（CL）imaging platform was constructed for prostate specific antigen（PSA）detection in a multiple signal amplifying manner.To construct the platform,the primary antibody for PSA was firstly immobilized on a O-ring area of a glass slide for recognizing the PSA.The horseradish peroxidase（HRP）and the secondary antibody of PSA（Ab₂）functionalized Au NPs（HRP-Au NPs-Ab₂）were modified on the platform through immunoreaction between PSA and Ab₂.The excellent catalytic effect of Au NPs and HRP on the HRP-Au NPs-Ab₂ to the luminol-H₂O₂ CL system provided the dual-signal amplification for PSA detection.To further enhance the sensitivity,tyramine signal amplification（TSA）strategy was introduced:tyramine-HRP conjugates were added into the O-ring reservoir and thus tyramine-HRP repeats formed in the presence of H₂O₂,generating a multiple signal amplification because of the large amounts of HRP on the sensing interface.The excellent performance of HRP-Au NPs-Ab₂ and TSA strategy endows the CL platform with high sensitivity.The PSA was detected with a photomultiplier tube（PMT）and visually analyzed by a charge coupled device（CCD）,respectively.The linear ranges of PMT and CCD for PSA are 0.1–100.0 ng m L^-1 with a detection limit of 0.05 pg m L^-1 and 0.5–100.0 ng m L^-1 with a detection limit of 0.1 pg m L^-1,respectively.The levels of PSA in several human serum samples were determined and the recoveries are ranged from 82.5%–117.0%.This CL immunosensing platform holds great potential for bioactive molecules detection visually and sensitively.2.With the development of micromachining technology,microelectrode chip has entered people’s vision,among which bipolar electrode has attracted people’s attention.Therefore,a novel closed bipolar electrode-based electrochemiluminescence（BPE-ECL）imaging platform for visual detection of cardiac troponin I（c Tn I）was constructed using tyramine signal amplification（TSA）strategy and enzyme-catalyzed precipitation techniques（BCP）.In this platform,tyramine-HRP repeats formation and the BCP were executed on the cathodic pole in the presence of H₂O₂,which the ECL from Ru（bpy）₃²⁺/TPA system was used as signal reporter on the anodic pole.In the presence of c Tn I,a large number of HRP from HRP-Au NPs-Ab compounds and tyramine-HRP repeats catalyze the formation of insoluble precipitation on the cathodic pole of the BPE,resulting in the decreased ECL from Ru（bpy）₃²⁺/TPA system on the anodic pole.Thanks to the TSA and BCP on the cathodic pole of the BPE platform,the method for c Tn I detection exhibits the detection limits of 5.0×10^-13 g m L^-1 by a photomultiplier tube and5.0×10^-12 g m L^-1 by a charge-coupled device imaging.Furthermore,the content of c Tn I in human serum samples was determined and the recovery was between 90.0%and112.0%.This multiple signal amplification strategy provides new perspectives for the BPE-ECL imaging platform in the biochemical analysis of biomarkers with low abundance.3.A highly sensitive split ECL immunosensing platform was constructed to improve the surface conductivity of the electrode by in situ generating Au NPs at the cathode pole of the BPE and enhance the ECL signal of the Ru（bpy）₃²⁺/TPA system.The target myoglobin（Myo）can be detected with high sensitivity.The alkaline phosphatase（ALP）loaded on the sensor catalyze AAP produce a certain amount of AA,transfer the AA to the BPE cathode electrode and then the in situ formation of Au NPs.With the increase of target concentration,the AA concentration was increased,and the amount of Au NPs generated on BPE increases.The change of anode ECL signal was detected by different Au NPs generated on the electrode surface,and the highly sensitive split ECL immunoassay was realized.The results show that the detection limit is 5.0×10^-13–5.0×10^-8 g m L^-1,the detection limit is 3.0×10^-13 g m L^-1（PMT）,and the logarithm of gray value and target concentration between 1.0×10^-12–1.0×10^-8 g m L^-1 shows a good linear response.The detection limit is 5.0×10^-13 g m L^-1（CCD）.