Nucleic Acid Signal Amplification Technology For The Construction Of Electrochemiluminescent Biosensor

Nucleic acid signal amplification technology is a series of nucleic acid reaction systems,which transforms the target into a large number of nucleic acid molecular output to achieve the signal of the target molecular amplified on the basis of nucleic acid amplification,ribozyme,deoxyribozyme activity and self-assembly technology.Nucleic acid signal amplification technology is playing an important role in improving the sensitivity of nucleic acid analyses.Moreover,its applications have extended to immunosensors and aptasensors through carefully designing signal transformation strategies.Nucleic acid signal amplification strategy can not only improve the sensitivity of the analysis,but also can simplify the operation of the sensor,saving analysis time,and even improve the selectivity.Electrochemical chemiluminescence(ECL)biosensor is an ideal analysis tool with great potential as its advantages of simple operation,fast analysis,high sensitivity,high accuracy and good selectivity.This paper intended to design nucleic acid signal amplification strategies fot the construction of ECL biosensor with convenient operation,low cost and responsiveness to meet the needs of clinical diagnosis.The work mainly includes the following aspects: 1.Phi29 DNA polymerase-mediated strand displacement amplification for the construction of a signal-off ECL biosensor to detect microRNAPhi29 DNA polymerase is a high-performance DNA polymerase with continuously high-fidelity strand displacement polymerization activity that may be used to mediate strand displacement amplification(SDA)which might overcome the problem that traditional Klenow polymerase can not be used for the amplification of long chain DNA and the replication is error-prone.In this paper,the target-induced strand displacement amplification was mediated by phi29 DNA polymerase and applied in signal-off ECL biosensor for the detection of microRNA.Target microRNA triggered the phi29 DNA polymerase-mediated SDA to produce a large number of nucleotide sequences with accurate complete single-stranded DNA as assisted probes.The assisted probe is capable to hybridize the capture probe and the ferrocene labeled probe(Fc-probe)to form a ternary "Y" junction DNA structure that further quenches the ECL signal by ferrocene.Thus,the ECL intensity will decrease with the increasing concentration of the target microRNA.The biosensor sensitivity is greatly improved due to the efficient signal amplification.In addition,the self-enhanced Ru(II)complex was used to obtain a stable and strong initial signal,which further improves the sensitivity.The ECL sensor has a high sensitivity to response to miRNA-21 with concentrations from 10 amol·L-1 to 1.0 pmol·L-1,and the detection limit was low to 3.3 amol·L-1.2.Target cycling synchronized roll circle amplification strategy applied in ECL biosensor for microRNA detectionRolling circle amplification(RCA)is an efficient isothermal nucleic acid amplification technology widely used for signal amplification in biosensors.In electrochemical or ECL biosensors,it is often necessary to cascade a target cycle to produce RCA primers immobilized on the electrode surface.These cascading signal amplification strategies tends to execute the target cycling and RCA step by step,which not only complicates the operation,but also limits the efficiency of signal amplification.Herein,a novel signal amplification pattern of synchronous target-cycling reaction and RCA was applied in ECL biosensor for ultrasensitive detection of microRNA.It is worth mentioning that a circular template consisting of a guanine-rich(G-rich)domain and a primer binding domain is cleverly designed to achieve the target cyclic synchronous RCA.In the presence of the target miR-21,the primer binding domain of the circular template hybridizes to the primer and miR-21 to form a ternary "P" structure,and then initiates RCA from the 3' terminal of the primer.As the RCA progresses,the target miR-21 is released and participates in the initiation of the next RCA.As G-rich domain of the circular template,the product of the target cycling synchronized RCA contains a series of tandem cytosine-rich(C-rich)sequences,which can act as the ligand for further in situ electrochemical formation of Ag NCs as ECL signal probe.The concentration of target miR-21 was positively correlated with the ECL intensity of Ag NCs.The ECL assay showed excellent linear response in the range of miR-21 concentrations of 100 amol·L-1 to 100 pmol·L-1 and limit of detection as low as 22 amol·L-1.3.Aptamer recognition triggered hairpin assembly enzyme-free signal amplification strategy for the construction of ECL aptasensor for MUC1 detectionAptamer recognition is often along with the transformation of secondary structure,which allows the nucleic acid signal amplification strategy playing a great role in improving the sensitivity of the aptasensor.In enzymatic target cycling signal amplification strategy,in order to achieve the purpose of signal amplification,the polymerization or digestion activity is used to release the target to circularly trigger the aptamer recognition process.However,the introduction of biological enzymes increases the cost of the sensor and limits the applications as the service condition of the sensor should meet the enzyme activity requirements.Based on the aptamer recognition triggered secondary structure transformation and DNA self-assembly technology,target cyclic signal amplification strategy were constructed and initiated by aptamer recognition.Combined with ECL Signal labels of porous self-enhanced Ru(II)polymer hollow nanospheres,an enzyme-free ECL aptasensor were constructed to detect mucin 1(MUC1).The aptamer sequence of MUC1 was embedded in the hairpin DNA recognition probe.When the aptamer is identified,the hairpin DNA opens and exposes the stem domain.This domain can cause the circular assembly of other two hairpin DNAs to achieve signal amplification.The two hairpin DNAs were fixed on the electrode surface and labeled ECL signal label,respectively.As a result,the ECL signal would response to the target.The sensitivity of the biosensor ranges from 1.0 fmol·L-1 to 100 pmol·L-1 and the detection of limit is as low as 0.31 fmol·L-1.