Study On Electrochemiluminescence Sensor Based On The Nanomaterials And Enzymes Assisted Signal Amplification Technology

ECL biological biosensor is a way combined ECL technology and biology analysis with high sensitivity, high selectivity, low background and other characteristics. Construction of the interface of biosensor is a key to converte biometric event to detected physical and chemical signals. In recent years, nanotechnology biosensor has attracted wide attention in the field of biomedicine and chemistry, and has been a major research of analytical chemistry. For this purpose, this research focus on the nanomaterials and enzyme assisted signal amplification for sensitive biological detection, and has obtained a series of effective results, specific contents are as follows:Part 1 In situ energy transfer quenching of quantum dot electrochemiluminescence for sensitive detection of cancer biomarkersIn this work, we develop a sensitive and selective method for the detection of a cancer biomarker (carcinoembryonic antigen, CEA) based on a new electrochemiluminescence (ECL) energy transfer mechanism, in which the energy transfer occurs from the excited quantum dots (QDs) to the in situ electro-generated quenchers. A CdTe QD-containing composite film is first deposited on the electrode followed by the conjugation of the primary antibody (Abl) on the film. Subsequent incubation of the modified sensing electrode with CEA and the secondary antibody-alkaline phosphatase-gold nanoparticle labels (Ab2-ALP-AuNP) leads to the formation of the Abl/CEA/Ab2-ALP-AuNPs immunocomplexes on the electrode surface. The captured ALP catalyzes the p-nitrophenyl phosphate disodium salt (p-NPP) substrate in the ECL detection buffer to p-nitrophenol (p-NP). The potential sweep on the electrode results in the oxidation of p-NP to p-benzoquinone (p-BQ) and the generation of excited QDs. The ECL emission of the excited QDs is therefore quenched through direct energy transfer from the excited QDs to p-BQ. This ECL quenching effect is significantly amplified because of the numerous ALP enzymes involved in each antibody-antigen recognition event. This proposed method of amplified quenching of QD ECL emission offers a low detection limit of 1.67 pg mL-1 for CEA. In addition, this method exhibits high reproducibility and selectivity and can also be applied to serum samples. Given these advantages, this new ECL energy transfer approach holds great promise for the detection of other biological targets and has potential applications in clinical diagnoses.Part 2 Electrochemiluminescence recovery-based aptasensor for sensitive Ochratoxin A detection via exonuclease-catalyzed target recycling amplificationBased on the recovery of the quantum dot (QD) electrochemiluminescence (ECL) and exonuclease-catalyzed target recycling amplification, the development of a highly sensitive aptasensor for Ochratoxin A (OTA) detection is described. The duplex DNA probes containing the biotin-modified aptamer are immobilized on a CdTe QD composite film-coated electrode. The presence of the OTA target leads to effective removal of the biotin-aptamers from the electrode surface via exonuclease-catalyzed recycling and reuse of OTA, which prevents the attachment of streptavidin-alkaline phosphatase (STV-ALP) through biotin-STV interaction. The electron transfer (ET) from the excited state CdTe QD ([CdTe]*) to the electro-oxidized species of the enzymatic product of ALP during the potential scan is thus inhibited and the QD ECL emission is restored for quantitative OTA detection. Due to the exonuclease-catalyzed target recycling amplification, the inhibition effect of ET is significantly enhanced to achieve sensitive detection of OTA down to 0.64 pg mL-1. The proposed method is selective for OTA and can be used to monitor OTA in real red wine samples. Our developed ECL recovery-based aptasensor thus offers great potential for the development of new ECL sensing platforms for various target analytes.Part 3 Target-induced structure switching of DNA for label-free and ultrasensitive electrochemiluminescent detection of proteinsAlthough nuclease-assisted target recycling amplification has been increasingly used in sensitive detection of nucleic acid targets, the application of this signal amplification approach for protein detection is of particular challenge because of the restricted enzymatic activity of nuclease on the nucleic acid sequences. Based on a new target-triggered DNA structure switching strategy, we report herein a highly sensitive and label-free method for electrochemiluminescent detection of thrombin via Exo Ⅲ-assisted recycling amplification. The target thrombin binds with and releases the corresponding aptamer of the dsDNA probes on the sensing surface, which switches the dsDNA strands from 3’-protruding termini to 3’-recessing termini and creates nicking sites for Exo Ⅲ. Subsequently, Exo Ⅲ cleaves these dsDNA strands with 3’-recessing termini and initiates cyclic cleavage of the dsDNA probes, leading to highly reduced intercalation of the electrochemiluminescent indicators into the grooves of the dsDNA strands and significantly inhibited electrochemiluminescence emission for label-free detection of thrombin down to 45 fM. Besides, the proposed method is highly selective and can be expanded for the monitoring of trace amounts of other target analytes when coupled with appropriate target binding aptamers.