RAFT Polymerization-based Signal Amplification Strategy For Highly Sensitive Electrochemical Sensing

Conventional electrochemical biosensors are unable to achieve the requirements of highly sensitive and selective detection of biomolecules with extremely low content.To address this problem,we fabricate an novel electrochemical biosensor by using recognition peptides,peptide nucleic acids(PNA),and aptamers as bio-recognition elements,along with reversible addition-fragmentation chain transfer(RAFT)polymerization as a signal amplification strategy.The proposed electrochemical biosensor achieves highly sensitive and selective detection of thrombin,target DNA,and protein kinase.Experimental results demonstrate that the proposed electrochemical biosensor is highly sensitive and selective.This paper can be divided into three main parts:(1)Sensitive and sequence-specific detection of trace amounts of nucleic acids is of great importance to early diagnosis of diseases,infectious diseases control,pathogen screening,etc.Herein,an ultrasensitive electrochemical DNA biosensor is reported by using peptide nucleic acid(PNA)as the bio-recognition elements and the coenzyme-mediated nicotinamide adenine dinucleotide(NAD~+)electro-RAFT(NAD~+-eRAFT)polymerization as a novel amplification strategy.As the electro-grafting of polymers involves simply i)the tethering of RAFT agents and ii)the NAD~+-eRAFT polymerization,it is highly efficient,easy to use,and low-cost.Under optimal conditions,the voltammetric signal correlates linearly with the logarithm of DNA concentration over the range from 0.1 fM to 0.1 nM(R~2=0.996),with a detection limit of 0.067 fM(S/N=3).The PNA-based electrochemical DNA biosensor can also differentiate even single base mismatch and is applicable to DNA detection in the presence of complex serum matrices,thus showing great promise in the sequence-specific detection of DNA targets at ultralow concentrations.(2)The detection of kinase activity with high sensitivity is important to medical diagnostics and drug discovery.Herein,we report the biologically mediated RAFT polymerization(BMRP)and its potential use as an efficient amplification strategy in the ultrasensitive electrochemical sensing of kinase activity.The BMRP-based strategy is biologically friendly,highly efficient,uncomplicated,and quite low-cost.Under optimal conditions,the voltammetric signal correlates linearly with the c AMP-dependent protein kinase(PKA)activity over the range from 25 mU/mL to 175 mU/mL(R~2=0.998),with a detection limit of 1.85 mU/mL(S/N=3).Moreover,the proposed kinase sensor is applicable to inhibitor screening and kinase activity sensing in serum samples.By virtue of its low cost,high sensitivity and selectivity,and uncomplicated operation,the proposed kinase sensor holds great potential in medical diagnostics and drug discovery.(3)As the abnormal expression of thrombin is closely associated with various pathological conditions,the clinical assay of thrombin is of great importance to disease diagnosis.On the basis of the signal amplification by target-synergized biologically mediated reversible addition–fragmentation chain transfer(RAFT)polymerization(tsBMRP),we describe herein a simple and cost-effective method for the electrochemical aptasensing of thrombin at the femtomolar levels.This method involves the immobilization of the thrombin-binding aptamers,the labeling of the oligosaccharide chains of thrombin with RAFT agents via the BA crosslinking,and the recruitment of ferrocene(Fc)tags via the BMRP of ferrocenylmethyl methacrylate(FcMMA).As the BMRP of FcMMA can result in the grafting of long polymer chains,each cis-diol site can be decorated with hundreds of Fc tags.The tsBMRP-based strategy is biologically friendly,cost-effective,easy-to-use,and enables the dual amplification of the detection signal.Under optimal conditions,the voltammetric signal correlates linearly with the logarithm of thrombin concentration over the range from 0.05 fM to 100 fM(R~2=0.998),with a detection limit of 35.3 fM(S/N=3),and is applicable to thrombin detection in serum samples.The merits of high sensitivity and selectivity,low cost,good anti-interference capability,and simple operation make the tsBMRP-based electrochemical thrombin aptasensor greatpromise in biomedical and clinical applications...