Research On Signal Amplification Strategies Based On Radical Polymerization And Its Applications In Nucleic Acid Sensing

Biosensing technologies based on signal amplification strategies are essential in point-of-care diagnostics,a portable biosensor capable of detecting nanomolar to attomolar concentrations of biomarkers without the use of advanced laboratory equipment.Due to the inherent amplification in the chain growth process,free radical polymerization has been exploited as a high-performance biological detection method,which can effectively replace conventional enzyme-or nanoparticle-based signal amplifications.Polymerization-based bioassays benefit from different types of initiation reactions and multiple monomer choices,making them suitable for different sensing conditions and detection methods.This paper introduces a variety of free radical polymerization chemistry that have been implemented in the biological detection platform,and evaluates their utility.Firstly,we describe the principle of each polymer signal amplification-based biological detection,and discuss its advantages and current limitations in practical application in this field.Then,a series of nucleic acid sensors based on free radical polymerization signal amplification strategy are characterized and analyzed by multiple means.Finally,we look forward to the future direction and opportunities of developing signal amplification strategies using free radical polymerization in biosensors.The main work and results of this paper are as follows:1.Ultrasensitive Nucleic Acids Biosensor Based on Electrochemical Atom Transfer Radical PolymerizationHere we report a highly selective and ultrasensitive DNA biosensor based on electrochemical atom transfer radical polymerization(ATRP)signal amplification and “Click Chemistry”.The DNA biosensor was prepared by immobilizing thiol and azide modified hairpin DNAs on gold electrode surface.In the presence of target DNAs(TDNA),hairpin probes hybridized with T-DNAs to form a duplex DNA,and the ring of hairpin DNA was opened to make azide groups accessible at 3’ ends.“Click reactions” proceeded between the azide and propargyl-2-bromoisobutyrate(PBIB)to initiate theATRP reaction which brought a large number of ferrocenylmethyl methacrylate(FMMA)on the electrode surface.The amount of FMMA was proportional to the concentration of T-DNA and quantified by square wave voltammetry.Combining ATRP signal amplification with “Click Chemistry”,the optimized DNA biosensor was capable of detecting 0.2 a M T-DNA.The preliminary application of the developed DNA biosensor was demonstrated by detecting target DNA in spiked serum samples.The developed DNA biosensor shows great promise for the detection of gene biomarkers.2.Ultra-sensitive Nucleic Acid Detection Based on Target Cycling of Triple Helix Molecular Switch and ATRP Double Signal AmplificationWe propose a novel electrochemical biosensor platform with triple helix structure as switch,Exonuclease III(Exo III)-mediated target cycle at 2-D level and electrochemical-mediated atom transfer radical polymerization(ATRP)at 3-D level as double signal amplification strategy for nucleic acid detection with ultra-sensitivity and high-selectivity.In this strategy,triple-helical DNA modified with N3 was first constructed on the gold electrode(Au E)surface.Exo III mediated target cycle was then triggered in the presence of Target DNA.N3 was released and exposed on the Au E surface,owing to the triple helix structure being destroyed in this process.propargyl-2-bromoisobutyrate(PBIB),the initiator of ATRP,was then introduced to the biosensor surface via click chemistry.Finally,electrochemically mediated ATRP polymerizes a large amount of electroactive monomer,ferrocene methacrylate(FMMA),onto the Au E surface.The target circulates to make target DNA reused at the 2-D level,and ATRP polymerizes a large number of electrochemical signal molecules at the 3-D level,making the biosensor detection limit as low as 1.954 a M.Moreover,the linear range of target DNA detection reaches 7 orders of magnitude(10 a M-10 f M,R~2=0.993).In addition,the biosensor shows excellent anti-interference ability when analyzing DNA in serum samples.In short,the preparation method is rapid,simple,easy to operate,and has potential applications in biological analysis.3.Dual Signal Amplification by eATRP and DNA Templated Silver Nanoparticles for Ultrasensitive Electrochemical Detection of Nucleic AcidsHerein,a novel,highly selective and ultrasensitive DNA detection method was reported,which combines electrochemical atom transfer radical polymerization(eATRP)with DNA templated silver nanoparticle(Ag NPs)signal amplification.Peptide nucleic acid(PNA)functionalized with thiol was immobilized on the surface of the gold electrode as a probe.In the presence of target DNA(T-DNA),PNA hybridized with T-DNA to form double-stranded PNA/DNA,and Zr4+ can bind to phosphate on DNA.Then Zr4+ was combined with α-bromophenylacetic acid(BPAA)to trigger ATRP reaction.A large number of glyco-syloxyethyl methacrylates(GEMA)were captured on the formed PNA/DNA duplex via ATRP,and assembled on the surface of the electrode.Afterwards,the polysaccharides were oxidized to the polymerized aldehydes with sodium periodate(Na IO4).And Ag NPs were deposited on the electrode surface by silver mirror reaction.The results indicate that the amount of Ag NPs proportional to the T-DNA was quantified through differential pulse voltammetry(DPV).Furthermore,it proves that the modi-fied electrode has good performance in DNA detection,indicating that the DNA sensor has high selectivity,high sensitivity and stable repeatability.The linear range of 10 a M to 10 p M is obtained,and the detection limit is 6.725 a M(S/N=3).The sensor is successfully used to detect DNA in human serum with satisfactory results,which shows great promise for detecting gene biomarkers and clinical analysis.4.Nucleic Acid Ultrasensitive Detection via SI-eRAFT and In Situ Metallization Dual-Signal AmplificationIn this work,we report a new amplification strategy based on electrochemically mediated reversible addition-fragmentation chain transfer(eRAFT)and in situ metallization for DNA electrochemical detection.First,peptide nucleic acid(PNA)probes were immobilized on the surface of the gold electrode,and when they hybridized with the target DNA,the chain transfer agent(CTA),4-cyano-4-(phenylcarbonothioylthio)pentanoic acid(CPAD),of RAFT was connected to the PNA/DNA heteroduplex formed by the coordination bonding of Zr4+.Then glycosyloxyethyl methacrylates(GEMA)were assembled on the surface of electrode by electrochemically mediated surface-initiated reversible addition-fragmentation chain transfer(SI-eRAFT)to form a polymer containing sugar glucose.Next,the orthohydroxyl groups on polysaccharide molecular skeleton were oxidized to aldehyde groups by sodium periodate(Na IO4).The aldehyde groups generated then reduce silver ions to silver particles deposited on the electrode surface in situ,and it was subjected to differential pulse voltammetry(DPV).Under optimal conditions,the intensity of the stripping current and the logarithm of the target DNA(t DNA)concentration has a good linear relationship in the range of 10 a M-1 p M(R~2=0.996),and the detection limit can go down to 5.4 a M(S/N=3).Moreover,the method is suitable for single nucleotide polymorphisms(SNPs)analysis and has strong anti-interference ability for the analysis of target ss DNA in serum samples.5.Ultrasensitive Nucleic Acid Electrochemical Biosensor Based on Mn TBAP Biomimetic Catalyzed AGET ATRP Signal Amplification ReactionIn this work,an ultrasensitive,highly selective and green electrochemical biosensor for quantifying DNA sequences(a M DNA)based on Mn TBAP catalyst for AGET ATRP reaction is proposed.Firstly,PNA is covalently bonded to the gold electrode surface by Au-S bond as a capture site for DNA.Then,the interaction between the T-DNA and the PNA allows the PNA probe to capture DNA on the surface of the electrode with high selectivity through the principle of base pairing,introducing a large amount of phosphate groups.Since the phosphate group also exhibits a strong complex interaction with the zirconium dichloride oxide octahydrate(Zr4+)linkage,and the remaining vacancies of Zr4+ can be coordinated with the carboxyl group.Subsequently,the phosphate group in the T-DNA backbone reacts with Zr4+,and the resulting electrode-PNA-T-DNA-Zr4+ can bind to theATRP initiator α-bromophenylacetic acid(BPAA)and the catalyst Mn TBAP.Then,a large number of electroactive monomeric ferrocenylmethyl methacrylate(FMMA)were polymerized in situ by Mn TBAP catalyzed AGET ATRP onto BPAA to form gold electrode-PNA-T-DNA-Zr4+-FMMAn.Finally,the electroactive polymer(FMMAn)on the electrode surface was used to detect T-DNA quantitatively by enhanced electrochemical signal.Under the optimal conditions,the method can be used for rapid analysis of T-DNA in the range of 100 a M-100 p M(R~2=0.997),and the detection limit is 11.79 a M.Moreover,the method has universality and strong anti-interference ability in the analysis of complex biological samples.For the first time,a combination of biomimetic catalyzed free radical polymerization and DNA electrochemical biosensor was used as a signal amplification strategy.