Highly Sensitive Protein Detection Based On Local DNA Cascade Reaction

Objective: Base on local DNA chain reaction,DNA nanostructure and proximity hybridization,a simple and fast homogeneous method is presented for highly sensitive and selective detection of protein biomarkers in complex sample,which provide a new design for biomolecules detection.Methods: The circular DNA template was synthesized through enzymatic linkage reaction,and the DNA scaffold was prepared through rolling circular amplication.DNA nanolight was constructed by assembling a pair of DNA hairpins(H1 and H2)with DNA scaffold,and was used as the signal probe.A pairs of aptamer were used as proximity probes.In prescence of target protein,two probes simultaneously bind with the target,which induced the proximity of the linked DNA and formed a new single-strand DNA.The single-strand DNA can open the hairpin H1 by toehold mediated DNA displacement reaction,which triggered the cascade reaction between H1 and H2 to produce the fluorescent signal.The stability of DNA nanolight was demonstrated with serum stability experiment.The reaction rate and efficiency of DNA local cascade reaction were showed by dynamic assay.The target protein was quantified by fluorescent detection.Using three kinds of proteins as non-target model,the specifity of the method was demonstrated.The accuracy of the proposed method was demonstrated by detection of blood samples.Results: Compared with hairpin probe,DNA nanolight showed higher serum stability.Using thrombin as model target protein,the proposed methold can detect target with 30 min.Compared with homogeneous DNA cascade reaction,the proximity-triggered local DNA cascade reaction was more efficient and fast.In addition,it shows high sensitivity and the detection range is 0.1 Nm to 20 n M with a detection limit(the signal mean of blank measures + 3?)of 0.091 n M.The recoveries for the spiked thrombin are in the range of 97.1% to 103.0% with relative standard deviations from 2.28% to 6.78%,indicating the good repeatability and selectivity of the proposed strategy for thrombin detection in serum samples.The relative deviation was less than 6.5% in all real blood samples,indicating the accuracy and feasibility of the proposed method for the detection of clinical samplesConclusion: This work developed a highly selective and sensitive method for protein detection.It is easy operation,without separation and can be finished in one-step.The method can be used in complexed biological samples.By changing the recognition molecules,it can be extended to detect other proteins,demonstrating good versatility and application prospect in biological analysis and clinical detection.