RAFT Polymerization Based Electrochemical Biosensing Assay For Nucleic Acid Detection

Highly selective detection and recognition of DNA has always been an important research of biochemical analysis and precision medicine.In recent years,DNA biosensors have attracted more and more attention due to their advantages of low cost,high sensitivity,high detection rate,ability of strong process control and high portability.Among them,electrochemical DNA biosensors stand out among DNA biosensors because of their advantages of environmental friendliness,high sensitivity,multi-selectivity and short detection time.Low concentrations of target DNA in samples remains an challenge for DNA detection.To improve the sensitivity and specificity of DNA detection,researchers have proposed the strategy of using natural enzymes,biomimetic enzymes and nanomaterials to amplify the target signals,however,these traditional signal amplification strategies still have problems such as high cost,heavy metal pollution and long operation time.The electrochemical DNA sensor based on RAFT signal amplification strategy can not only avoid these problems,but also improve the detection efficiency and accuracy of electrochemical DNA sensor.In this paper,two RAFT polymerization signal amplification strategies with different initiation mechanisms were combined with electrochemical biosensors to achieve ultra-sensitive detection and performance analysis of target DNA at low concentrations.The main research contents are as follows:1.Stable nitronyl nitroxide monoradical TEMPO as monomer of RAFT polymerization for lung cancer DNA detectionIn this work,it was come up a hydrophilic and stable nitronyl nitroxide monoradical4-methacryloyloxy-TEMPO as unused monomer of polymerization for DNA detection,the detection limit was more than 1,000,000 times lower than TEMPO derivatives as electrochemical labels alone.Within this approach,a single biomolecule can be converted into numerous electrochemical signals,therefore lung cancer DNA can be detected at low concentration.Firstly,the HS-PNA probe was fixed on the Au eletrode.After hybridization with target DNA,4-cyano-4-（phenylcarbonothioylthio）pentanoic acid（CPAD）was bonded to DNA as chain transfer agent of RAFT polymerization reaction via coordination bond of Zr⁴⁺.Electroactive polymers had grown by means of surface-induced thermally initiation RAFT polymerization with 4-methacryloyloxy-TEMPO as monomer.TEMPO-contained polymer improves significantly the electrochemical signal.Under optimal conditions,the proposed method can detect DNA from 0.01 f M to 10 p M,and detection limit is 1.51 a M.The sensitivity of this method is greater than that of most other reported signal amplification strategy of DNA biosensor,which shows that it is appropriate for single nucleotide polymorpism analysis and will open broad prospects for biological molecules detection application.2.Coenzyme-catalyzed electroinitiated RAFT polymerization for ultrasensitive electrochemical DNA detection.In this study,β-nicotinamide adenine dinucleotide（NAD⁺）was used as the initiator of electro-RAFT polymerization for the first time.The electro-RAFT polymerization technique was combined with DNA electrochemical sensor,which ensured the high sensitivity of DNA electrochemical sensor.The problem of CTA decomposition due to high temperature of RAFT polymerization is also avoided.In this coenzyme-catalyzed electro-RAFT polymerization,numerous ferrocenylmethyl methacrylate（FCMMA）as monomer with electrochemistry signal were linked to the biomarker on Au electrode.Afterwards,a strong oxidation peak appears at the potential of about 0.3 V that represents a typical oxidation potential of FCMMA.The sensitivity of this methodology was presented by detecting DNA from 10^-1 to 10⁴ f M concentration and limit of detection（LOD）being down to 4.39 a M in 10μL samples.This is lower by factors than detection limits of most other ultra-sensitive electrochemical DNA assays.