Research On Electrochemiluminescence Biosensor Based On Multiple Signal Amplification Strategies

In recent years,the researchers make efforts to develop efficient biosensors for the sensitive detection of multiple biomarkers in the field of surface enhanced Raman spectroscopy,electrochemiluminescence,fluorescence,electrochemistry and photoelectrochemistry.As powerful analytical methods,ECL and electrochemical analysis has gained special attention of the researchers due to their low background signals,wide linear range,high sensitivity,good selectivity,simple operation and low cost and so on.Due to the extremely low concentration of tumor markers,which is not conducive to the early diagnosis of the disease,a variety of amplification techniques including chain hybridization,DNA nanostructures,and DNAzyme have been applied to amplify the target concentration and reduce the detection limit.This paper mainly explores new electrochemiluminescence biosensing methods based on a variety of amplification strategies to achieve highly sensitive detection of Dam MTase,AFB1,TB,miRNA and GSH.1.In this study,a new multifunctional DNA nanotube?DNANT?was self-assembled and used to load Ru?phen?₃²⁺and methylene blue?MB?as amplified signal probes for versatile electrochemiluminescence?ECL?and electrochemical?EC?“on-off”assays of Dam methylase?MTase?and aflatoxin B1?AFB1?.The target Dam MTase first catalyzed the methylation of hairpin DNA?H1?,and then the methylated DNA was cleaved by endonuclease DpnI to expose a single-strand DNA.After the Ru?phen?₃²⁺-DNANTs or MB-DNANTs signal probes were assembled to the electrode by hybridization,remarkable“signal on”states for amplified ECL or EC assays of MTase were obtained.Furthermore,in the presence of the target AFB1,the structure of DNANTs collapsed due to the specific binding of AFB1 to aptamer S2 in NTs,which led to the release of signal probes(Ru?phen?₃²⁺or MB)from the electrode to achieve“signal off”state for dual detection of AFB1.Taking advantage of the multifunctional DNANTs amplification signal probes,the versatile biosensors showed good analytical performance with very wide linear ranges(0.001-100 U·m L^-1 and 0.0001-100 ng·mL^-1for MTase and AFB1 assay by DPV)and lower detection limits(2.1×10^-4 U·mL^-1 and0.018 pg·m L^-1 for MTase and AFB1 by DPV).This is the first time that ECL and EC“on-off”methods have been achieved separately for dual target assays,which opens a new avenue of DNANT-based signal amplification strategy for the versatile design of biosensors in various biological detections.2.A novel biosensing platform based on the Ag?I?ion-enhanced or Ag nanocluster?NC?-quenched electrochemiluminescence?ECL?of CdSe quantum dots?QDs?was designed for versatile‘‘on–off''assays of thrombin?TB?and mi RNA.A surface-programmed chain reaction?SPCR?triggered by proximity-dependent hybridization?PDH?and bipedal molecular machinery?BMM?was proposed through a precisely designed hairpin DNA-modified sensing surface.On the one hand,Ag?I?was captured by C-C mismatch to form a stable Ag?I?-chimeric DNA complex.Ag?I?as an effective co-reactant greatly improves the ECL signal strength of CdSe QDs,remarkable“signal on”states for amplified ECL assays of TB were obtained.On the other hand,the C-rich chain was used as a template to capture the AgNO₃ released from the mesoporous silica spherules,and to generate Ag NCs by in situ reduction,quenching the ECL intensity of CdSe QDs to achieve“signal off”state for dual detection of miRNA-21.For the first time,this method combines Ag?I?enhancement and Ag NCs quenching to detect dual targets in the same sensing system,and the detection limits of TB and miRNA-21 can reach 0.165 fM and 4.97 aM,respectively.3.In this work,a versatile electrochemiluminence?ECL?and a photoelectrochemical?PEC?“signal on”biosensing platform were developed for a sensitive assay of GSH by a Mn²⁺-powered DNAzyme amplification strategy combined with DNA-Walker-triggered allosteric conversion.First,MnO₂ nanosheets were reduced to Mn²⁺by GSH;then,Mn²⁺as a substitute target triggered DNAzyme assisted cleavage-cycling amplification to generate numerous DNA output?s3?.Meanwhile,the DNA molecular machine was introduced to bridge signal probes for versatile biosensing,which included hairpin DNA as a track and an arm as a walker.The presence of DNA output?s3?activated the swing arm to hybridize with hairpin DNA and then cut it by Nt.BbvCI,which initiated autonomous walking of the arm for forming a large number of streptavidin?SA?aptamers.Thus,a large number of CdS:Mn-SA tags as versatile signal probes was linked to the electrode by specific SA aptamer binding,generating highly enhanced ECL and PEC signals for sensitive detection of the target.The present biosensing system take advantage of metal ion based DNAzyme amplification,a DNA walker machine,multi-signals of QDs,and specificity of aptamers,which can provide a universal and efficient biosensing method for detecting various targets.The designed strategy demonstrated good performance for a GSH assay in human serum samples,showing more promising applications than other reported methods.