| Objective: Dengue fever is one of the most important vector-borne viral diseases,transmitted by Dengue virus(DENV)infected mosquitoes.Due to the fact that mosquitoes exist widely,Dengue fever is easy to be prevailed.As a result,numerous people died for Dengue epidemic,and large numbers of countries paid a heavy economic price.However,the trend of Dengue prevalence continues to spread,and the Dengue epidemic areas in China have gradually expanded from the southeast coastal areas to the southwest,central,northeast and northwest regions.This disease poses a big risk to all human beings.In order to cope with the spread of Dengue fever,it is important to develop a high-efficency detection method,and virus’ nucleic acid detection is a potent potential method.Nevertheless,the biggest challenge of nucleic acid testing is that the concentration of patients’ DENV nucleic acid is very low,which requires a sensitive method.At present,the most commonly used DENV nucleic acid detection methods are PCR and gene sequencing.These approaches demand expensive laboratory equipment and professional technicians,which limits the rapid diagnosis of DENV.Method: A universal and highly sensitive electrochemical biosensing strategy was developed with a triplet nanostructure-mediated dendritic hybridization chain reaction for the analysis of DENV nucleic acid.A locked ds DNA was firstly recognized by the target DENV nucleic acid fragment to release the initiator,which was then captured by a capture DNA modified gold electrode to initiate the HCR in the presence of biotin labeled substrates A and B,and two helpers.After the recognition reaction of the captured initiator with substrate A and then helper 1,one DNA structure was formed to react with substrate B,which produces a triplet nanostructure to trigger the dendritic HCR by the toehold of the hybridization structure.After the dendritic product was bound with avidin labeled horseradish peroxidase(avidin-HRP),an amperometric signal could be obtained to achieve ultrasensitive electrochemical detection of DENV.The non-enzymatic isothermal amplification was driven by only entropy.The stable three-dimensional dendritic nanostructure with rigid double helix could assure the excellent performance of the designed electrochemical biosensing strategy.Results:(1)Through polyacrylamide gel electrophoresis(PAGE)experiment,we found that adding initiator into the reaction system could consume substrates and produce a large number of byproducts.This proves that the initiator could trigger the amplification reaction,which verifies the feasibility of the strategy.(2)EIS was used to characterize the stepwise modification of gold electrode.Each step of the modification was accompanied by an increase in resistance,indicating that each substrate was successfully modified on the electrode;(3)We verified the optimal number of cycles for our reaction.Half cycle,one cycle,two cycles and infinite cycles of dendritic HCR were tested,and we demonstrated that the signal values of the two cycles were equivalent to that of infinite cycles,indicating that the amplification efficiency reached a plateau when it reached to the second cycles.(4)We also compared the efficiency of dendritic HCR amplification and without amplification.The formation of dendritic nanostructure significantly improved the sensitivity of DENV detection.(5)Under the optimal experimental conditions,we analyzed the performance of the proposed detection strategy.The DENV nucleic acid concentration ranges from 1.6 p M to 1000 p M,and the linearity of the target concentration and chronoamperometry signal was obtained.(6)The extendibility of this strategy was verified by the application of two different DENV nucleic acids,indicating that our biosensor can be easily applied to different nucleic acid detection;(7)The specificity of the sensor was verified by testing the signal of the single base,double base,triple base,and five base mismatched DNA and the perfectly matched target DENV nucleic acid fragment.It was found that all the mismatched base signals were weaker than those of the perfectly matched target,indicating that the strategy can be used to detect single base mismatches;(8)The recoveries were performed in diluted serum samples and the recovery rates ranged from95.69% to 105.26% with relative standard Deviations ranging from 1.29% to 7.04%.It shows that this method has good accuracy and precision in the analysis of target DNA in clinical samples.Conclusions: The proposed method showed a detection range of 1.6 ～ 1000 p M with a detection limit of 188 f M and an ability to distinguish single-base mutation.By changing the recognition sequence of initiator,the detection of different DENV nucleic acid fragments could be achieved with the same performance.Thus,this method had good extendability for other nucleic acids,providing a promising candidate for nucleic acid detection in early clinical diagnosis. |
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