Study On Highly Sensitive Electrochemical Biosensors Based On Multiple Bioamplification Technologies

With the rapid development of scientific research,the construction of simple,rapid and sensitive assay methods in disease diagnosis,environmental monitoring,and food analysis fileds,has become challenging research topics.Recently,electrochemical biosensor has the advantages of simple operation,low detection cost,high specificity,and short time consumption,which has been widely applied to various fields.The employment of newly-developingnanomaterials,mimic enzyme and self-assembly DNA that are easy to synthesize,high stability,and good biocompatibility is favorable for improving the analytical performance of biomolecule detection for construction of biosensing.In this paper,we mainly focused on the application of nanomaterials,DNA self-assembly methods,DNA amplification technology and mimic enzyme,which constructed three kinds of DNA biosensors with simple operation and high sensitivity for the determination of metal ion and Nosema bombycis gene PTP1.This main article contents are as follows:1.Toehold-mediated strand displacement reaction triggered by nicked DNAzymes substrate for amplified electrochemical detection of lead ionLead ion(Pb²⁺),a toxic heavy metal ion,is a nondegradable and easily accumulated pollutant and does seriously harm to environment and human health.So it is important to search a novel electrochemical assay method with high sensitivity and specificity.Herein,based on the integration of toehold-mediated strand displacement reaction?TSDR?-fuelled DNA recycling and the electrocatalysis of magnetic Fe₃O₄ for signal amplification,a sensitive electrochemical biosensor for Pb²⁺was developed.Firstly,probe hybrids containing three stands DNA were assembled in the electrode surface modified with Au nanoparticles?AuNPs?-loaded Fe₃O₄ nanocomposites?AuNPs@Fe₃O₄?.With the addition of Pb²⁺,the Pb²⁺-DNAzymes was specific cleaved,and the cleaved substrate fragments?rSS?was bound to toehold in the tail of probe hybrids,displacing the short DNA strands of probe hybrids and exposing the intermediate toehold region for hybridization with beacon probe modified with methylene blue?MB-SP?,which futher replaced both rSS and aother short DNA to activate TSDR.As such,the released rSS continuously activated TSDR,leading to the in-situ assembly of a large number of MB-SP on the resultant electrode surface and generation of increasing electrochemical signal.The assembly of MB-SP was resulted in the spatial proximity of MB close to Fe₃O₄ in the modified electrode surface.In this case,the amplified electrochemical output signal was further enhanced due to the robust electrocatalysis of Fe₃O₄ toward MB reduction.Herein,based on rSS recycling induced by TSDR and the catalysis of AuNPs@Fe₃O₄ to MB reduction for signal amplification,the proposed biosensor for assay Pb²⁺exhibited a reliable linear range of 1 pmol?L^-1 to 500 nmol?L^-1 with the limit of detection of 0.32pmol?L^-1.Importantly,owing to low cost,simply,the proposed free-enzymes assay platform biosensor would be easily applied for environmental monitoring.2.Dendritic structure DNA for lead ion biosensor based on catalytic hairpin assembly and a sensitive synergistic amplification strategyAll the time,cost-effective detection of heavy metal ions at trace or ultra-trace levelat trace or ultra-trace level plays an important roles in the field of environmental monitoring and food analysis.To further improve the sensitivity for metal ion determination,herein,a sensitive electrochemical Pb²⁺biosensing was proposed based on the high specificity of Pb²⁺-DNAzymes.The response signal was efficiently amplified by the catalytic hairpin assembly?CHA?-assisted DNA recycling,and synergistic catalysis of Pt@Pd nanocages?Pt@PdNCS?and manganese porphyrin manganese?MnTMPyP?for H₂O₂ reduction.Firstly,the substrate strand?rS1?of the Pb²⁺-specific DNAzymes was specifically cleaved by Pb²⁺,releasing one of the two fragments?rS1?to induce CHA in electrode surface.With the recycling of rS1,the electrochemical signal was amplified.Next,Tb-S3-Pt@PdNCs and Tb-S4-Pt@PdNCs bioconjugates consisting of Pt@PdNCS,electroactive toluidine blue?Tb?,DNA S3 and S4,respectively,were captured onto the resultant electrode surface,resulting in the formation of DSDNA triggered by hybridization reaction between S3 and S4.Owing to the formation of this DSDNA,an amount of Tb was immobilized in electrode surface,obtaining enhancing electrochemical response signal.In addition,it also could provide abundant binding sites for immobilizing mimic enzymes MnTMPyP and Pt@PdNCs with peroxidase activity.With the presence of H₂O₂,Pt@PdNCs and MnTMPyP synergistic catalyzed the reduction of H₂O₂,promoting the electron transfer to further synergistic amplifying the electrochemical signal,which greatly improved the analytical performance of the sensor.As a result,the detection range of proposed biosensor was varied from 0.1 pmol?L^-1 to 200 nmol?L^-1,and LOD was 0.033 pmol?L^-1.The proposed method with strong specificity and high sensitivity would be potential and promising for Pb²⁺and other metal ions environment monitoring.3.Highly sensitive electrochemical assay for Nosema bombycis gene DNA PTP1 via conformational switch of DNA nanostructures regulated by H⁺from LAMPDetermination of DNA with long base sequences has shown more and more attention in agricultural analysis and disease diagnosis.However,they suffered from inherent drawback of low sensitivity,resulting in great limitations in practical applications.Herein,a novel electrochemical biosensor was constructed for highly sensitive detection of Nosema bombycis gene PTP1 through transducing chemical stimuli H⁺from PTP1-based LAMP to regulate the conformational transition of pH-dependent DNA nanostructures?DNA NSs?.The key of the method is that the special structure of DNA NSs are transformed under the stimulation H⁺from LAMP reaction,leading to the release of signal probe labeled with the ferroelectric Fc?Fc-SP?to electrode surface,producing amplified electrochemical signal releyed on PTP1.As a consequence,the developed biosensor for PTP1 determination exhibited a reliable limit of detection of 0.31 fg·?L^-1.Thus,by transition of LAMP-based H⁺into electrochemical signal readout,this novel and simple transduction schemes with the advantages of convenience,rapidity and high sensitivity,would be interesting and promising to open up a novel analytical route in the field of various biological reaction monitoring and disease diagnosis.