Novel Electrochemical DNA Biosensing Strategy Based On Hybridization Chain Reaction

Electrochemical biosensor is a kind of highly sensitive and selective detection device that combines specific biorecognition with electrochemical analysis.It has shown great advantages in biological analysis,clinical diagnosis and environmental monitoring due to its characteristics of low cost,simple construction and quick response.The application of electrochemical biosensors to sensitive detection of disease-related biomarkers is of great significance for early diagnosis and prognosis of diseases.However,the sensitivity of electrochemical biosensors still needs to be improved due to low level of biomarkers in the early stage of disease in organism.In order to achieve ultrasensitive detection of trace biomarkers,various signal amplification techniques are emerging.Herein,in order to improve the sensitivity of electrochemical biosensors,three novel electrochemical DNA biosensing strategies were proposed based on hybridization chain reaction coupled multiple biological amplification strategies for the detection of disease-related biomarkers.The main work contents are as follows:A novel multiple strand displacement reaction(MSDR)coupled hybridization chain reaction(HCR)was proposed for label-free and ultrasensitive Type b3a2 assay.Primarily,two kinds of messenger DNAs(mDNAs)with a portion of same sequence were introduced to hybridize with the long substrate strand DNA on magnetic beads to form bioconjugates.In the presence of target and a hairpin DNA,MSDR would be initiated and achieved target recycling as well as the release of two mDNAs.One target input converted into multiple mDNAs,which greatly increased the utility ratio of target.After magnetic separation,the released mDNAs hybridized with the hairpin DNA immobilized on the electrode to trigger HCR and form linear duplex-DNA structure.It was used as an excellent template to in-situ grown silver nanoparticles(AgNPs)to achieve label-free detection.Under optimized experimental conditions,this developed biosensing strategy achieved a low detection limit of 0.56 fM and a wide linear range from 10 fM to 10 nM for sensitive detection of Type b3a2 with good selectivity and stability.A novel electrochemical biosensing strategy was proposed to detect cytokeratin fragment antigen 21-1(CYFRA 21-1)DNA based on Exo Ⅲ-assisted digestion of dsDNA polymer(EADDP)from hybridization chain reaction(HCR).Primarily,the presence of target driven a catalytic hairpin assembly(CHA)reaction in homogeneous solution,which was aim to achieve target recognition and circulation.Then the HCR was triggered to generate long dsDNA polymer with signal tags.Subsequently,the introduction of Exo Ⅲ digested the long dsDNA polymer to produce large amounts of DNA fragments with signal tags at both ends(DSF).The released DSF were captured onto the electrode directly by capture probe(CP)and a remarkably amplified electrochemical signal can be detected.The target recognition and a series of amplification were all in homogeneous solution,which circumvented complex interface reaction and tedious operation steps on electrode.Besides,under the synergistic effect of CHA,HCR and Exo Ⅲ,the sensitivity was greatly improved.This biosensing strategy achieved ultrasensitive detection of CYFRA 21-1 DNA at 0.0348 fM with a linear range from 5 fM to 50 nM.A novel strategy for preparation of bipedal DNA walker(BDW)based on hybridization chain reaction(HCR)with the assistance of Exo Ⅲ was proposed.Based on this strategy,an electrochemical biosensor was constructed and achieved ultrasensitive detection of CYFRA 21-1 DNA.Firstly,a one-step catalytic hairpin assembly(CHA)reaction was utilized to achieve target recycling,and then hybridization chain reaction(HCR)was initiated to form duplex-stranded DNA(dsDNA)polymer in homogeneous solution.In particular,the elongated single strand of hairpin DNA for HCR was designed as Mg2+ DNAzyme sequence.With the assistance of Exo Ⅲ,dsDNA polymer were digested and transformed into large amounts of BDW.These BDW cleaved the signal probes driven by Mg2+,which were modified on the electrode surface and thus achieved "signal-off" detection of target.This preparation method of BDW based on HCR with the digestion of Exo Ⅲ greatly increased the conversion amount of single target to BDW.Coupled with the walking cleavage of BDW and a series of cascade amplification techniques,the sensitivity of this biosensor was improved significantly and realized ultrasensitive detection of target DNA with the detection limit as low as 3.01 aM.