Research On Construction Of Electrochemiluminescence MicroRNA Biosensors Based On Novel Coreaction Accelerator

Electrochemiluminescence（ECL）refers to an analytical technique that combines electrochemistry and chemiluminescence.It has the advantages of both electrochemical and luminescence analysis techniques,and also possesses the merits of high sensitivity,easy operation,good selectivity and high reproducibility.As a rule,the individual ECL luminophore has a relatively weak ECL intensity due to its annihilation effect,which cannot meet the requirements of the analytical determinations.Researchers often improve the luminophor intensity through introducing a coreactant or coreaction accelerator into the detection process,building an ECL system with high luminous efficiency to achieve the purpose of improving detection sensitivity.In consequence,finding and designing a new type of coreactant or coreaction accelerator with good biocompatibility and high promotion efficiency is one of the dominating methods to improve the detection sensitivity.In addition,nucleic acid signal amplification technology is utilized to convert a small number of targets into a large number of nucleic acid molecules,and achieving circular amplification of targets,which is another way to increase the detection sensitivity.Based on the introduction of coreaction accelerator in the ECL system combined with certain nucleic acid amplification technology,this work constructs high efficient,good biocompatibility and low cost biosensing platforms to achieve ultrasensitive detection of tumor marker micro RNA 21（mi R-21）.This article mainly is carried out from the following aspects:1.An ultrasensitive electrochemiluminescence biosensor for detection of Micro RNA by in-situ electrochemically generated copper nanoclusters as luminophore and Ti O₂ as coreaction acceleratorCopper nanoclusters（Cu NCs）with low-cost and high-efficiency had gained growing popularity in various areas of biosensing and bioimaging and thus the synthetic methods of Cu NCs had gained attractive attentions and improvements.Most of the methods for preparing stable Cu NCs such as chemosynthesis and solvent reduction,sank into the problems of size control and irreversible aggregation.In this work,numerous AT-rich double-stranded DNA（ds DNA）was firstly produced from the conversion of a small amount of target mi R-21 via the combination of exonuclease III（Exo III）assisted amplification and hybridization chain reaction（HCR）,which could reduce the aggregation caused self-etching effect of Cu NCs and improve the emitting of Cu NCs.Simultaneously,the introduction of Ti O₂ in the sensing interface not just acted as the immobilizer of ds DNA stabilized Cu NCs,more than acted as the coreaction accelerator to accelerate the reduction of the coreaction reagent（S2O82-）for significantly enhancing the ECL efficiency of Cu NCs.The biosensor showed an excellent linear relationship in the concentration range from 100 amol/L to 100 pmol/L with the detection limit of 19.05 amol/L and expanded the application of Cu NCs in biological detection.2.Novel ABEI/dissolved O₂/Ag₃BiO₃ nanocrystals ECL ternary system with high luminous efficiency for ultrasensitive determination of micro RNAAs the coreaction accelerator of N-（aminobutyl）-N-（ethylisoluminol）（ABEI）,dissolved oxygen has the merits of simplicity and availability and has attracted widespread attention of researchers.However,the ECL intensity of ABEI/dissolved O₂ system was poor and cannot meet the needs of detection.Herein,a ternary ECL sensing platform with high luminous efficiency was established for ultrasensitive bioanalysis of the mi R-21 from cancer cell based on introducing the silver bismuth oxide nanocrystals（Ag₃Bi O₃ NCs）as coreaction accelerator into the ABEI/dissolved O₂ system.Specifically,the Ag₃ Bi O₃ NCs could promote the reduction of dissolved O₂ to generate great deal of superoxide anion radical（O₂·-）,reacting with the ABEI to dramatically enhance the ECL response.Simultaneously,a small amount of target mi R-21 could trigger a one-step DNA self-assembly to form the DNA nanonet for embedding abundance ABEI with the assistance of doxorubicin hydrochloride（Dox）,realizing the high-intense ECL response.Furthermore,owing to the thin layer and porous structure,the obtained DNA nanonet could shorten the electron-transfer path of the ternary ECL system to reduce the energy loss,further improving the sensitivity of the sensing platform.As expected,the developed ECL sensing platform of mi R-21 detection manifested prominent sensitivity with a low detection limit of 7.1 amol/L in the concentration of mi R-21 range from 10 amol/L to 100 pmol/L.3.A novel electrochemiluminescence biosensor based on the biocompatible laccase as coreaction accelerator and ameliorative strand displacement amplification for micro RNA detectingThe ternary ECL system formed by introducing a coreaction accelerator into the binary system has aroused great interest among researches due to its high luminous efficiency.However,the previously reported coreaction accelerators such as organic compounds,metals,and metal oxides etc.have poor biocompatibility,limiting the application of the ternary system in bioanalysis.Herein,a biocompatible enzyme,laccase,was employed for the first time as a coreaction accelerator of ABEI/dissolved O₂ to form a high-efficiency ternary system,and the probable luminescent mechanism of the ternary system was researched in detail.Simply,the laccase boosted the reduction of O₂ to generate messes of superoxide radical（O₂·-）and then reacting with ABEI radical（ABEI·-）to produce more excited state ABEI*,acquiring high-intense ECL response.Concurrently,combining with the improved strand displacement amplification（SDA）reaction trigged by target mi R-21,an ultrasensitive sensing platform was constructed for detecting micro RNA.As expected,the proposed biosensor of mi R-21 determination has preeminent selectivity and favourable sensitivity with the limit of detection was 80.8 amol/L（S/N = 3）,and makes the ternary ECL system have greater application prospects in the field of biological analysis and clinical diagnosis.