Construction And Application Of Polya-DNA Probe Electrochemical DNA Biosensor

The electrochemical DNA biosensor has the advantages of high sensitivity,good specificity,convenient portability,and low energy consumption.In combination with various signal amplification strategies and renewable strategies,it can achieve trace analysis of detection targets.Their extensive attention has now become a cutting-edge topic in the fields of biology and medicine.However,developing high-sensitivity electrochemical DNA sensors still faces many challenges.At the interface between the electrode and the solution,the decrease in mass transfer rate and the increase in crowding effect make DNA recognition and hybridization greatly affected.The advent of DNA nanotechnology has brought new solutions to the problems in the field of electrochemical DNA biosensor.We design different two-dimensional DNA nanoprobe structures to improve chemical stability and biocompatibility at the nanosensing interface.In this thesis,two-dimensional DNA nanostructures are used to construct a multi-dimensional and cooperative nanosensing interface,and the nano-interfaces are regulated to achieve highly specific capture and recognition of target DNA molecules.Three electrochemical DNA biosensors were constructed in this paper.The specific research content of nanostructures is as follows:1.Traditional microbiology analysis is usually hindered by the long time-cost and lack of portability in many urgent situations.In this work,we developed a novel electrochemical DNA biosensor(E-biosensor)for sensitive analysis of the 16 S rRNA gene of five bacteria,using a consecutive adenine(polyA)probe.The polyA probe consists of a polyA tail and a recognition part.The polyA tail can combine onto the gold surface with improved controllability of the surface density,by conveniently changing the length of polyA.The recognition part of the capture probe together with two biotin-labeled reporter probes hybridize with the target DNA and form a stable DNA-tetramer sandwich structure,and then avidin-HRP enzyme was added to produce a redox current signal for the following electrochemical detection.Finally,we realized sensitive quantification of artificial target DNA with a limit of detection(LOD)of 10 fM,and excellent selectivity and reusability were also demonstrated.Importantly,the detection capability was equally good when facing bacterial genomic DNA,due to the base-stacking force of our multireporter-probe system,which can help to break the second structure and stabilize the probe-target complexes.Our biosensor was constructed on a 16-channel electrode chip without any polymerase chain reaction(PCR)process needed,which took a significant step toward a portable bacteria biosensor.2.Multiblock DNA probe attracted a large amount of scientific attention,for the development of multitarget biosensor and improved specificity/sensitivity.However,the development of multiblock DNA probes highly relied on the chemical synthesis of organic linkers or nanomaterials,which limited their practicability and biological compatibility.In this work,we developed a label-free assembling strategy using a triblock DNA capture probe,which connects two DNA probes with its intrinsic polyA fragment(probe-PolyA-probe,PAP).The middle polyA segment has a high affinity to the gold electrode surface,leading to excellent reproducibility,stability,and regeneration of our biosensor.Two flanking capture probes were tandemly coassembled on the electrode surface with consistent spatial relationship and exactly the same amount.When combined with the target DNA,the hybridization stability was improved,because of the strong base stacking effect of two capture probes.The sensitivity of our biosensor was proved to be 10 fM,with a wide analysis range between 10 fM to 1 nM.Our PAP-based biosensor showed excellent specificity when facing mismatched DNA sequences.Even single nucleotide polymorphisms can be distinguished by each probe.The excellent practicability of our biosensor was demonstrated by analyzing genomic DNA both with and without PCR amplification.3.A preliminary investigation was made of the charge transfer in the space of the "triangular" polyA-DNA constructed on the surface of the gold electrode.It was initially found that when no target exists,there is no current signal value.When the target exists,the stem loop of the ferrocene-modified capture probe is opened This makes the ferrocene closer to the electrode surface and can detect the current signal,which opens up some new ideas for studying the configuration of the interface assembly.