Electrochemical Biosensing Strategy Based On DNA Molecular Machine

Inspired by the natural protein molecular machines,many artificial molecular machines have been designed to achieve precise nano scale movement.As an excellent biomaterial,the molecular machines constructed by DNA have excellent performance.One kind of DNA molecular machines that continuously move on the interface is called DNA walkers.In the field of electrochemical biosensors,DNA walkers have attracted more and more attention due to their interface motion.Through the design and synthesis of DNA sequences,a series of electrochemical biosensors based on DNA walkers are proposed,which are used to detect cell,protein,nucleic acid and other targets.In this study,we use the neighborhood chain substitution reaction as the recognition switch to start the monopod DNA walkers after the specific recognition of the target,and realize the signal amplification by constantly walking and opening the hairpin.By adjusting the length of the anchor structure sequence of the monopedal walker,the sensor has excellent detection sensitivity and specificity.The model of protein and target DNA as the target proves that the sensor has excellent scalability.Objective: to construct a universal DNA molecular machine electrochemical biosensor by using the highly programmable DNA molecular machine and the excellent interface reaction amplification efficiency.In order to verify the detection performance and expandability of the sensor,we take DNA and protein as the detection target concept verification at the same time,aiming to provide a new idea for the realization of sensitive,fast and portable electrochemical biosensor.Methods:(1)the repeatability of the electrode array was confirmed by cyclic voltammetry(CV).(2)the feasibility of the reaction was verified by Polyacrylamide gel electrophoresis(PAGE)and the moderating effect of the anchorage sequence on the residence time of the walking strand W on H1(Hairpin 1).(3)Electrochemical impedance spectroscopy(EIS)and differential pulse voltammetry(DPV)were used to characterize the preparation process and detection feasibility of the sensor.(4)Thrombin and target DNA were used as targets for protein and nucleic acid detection to verify the sensitivity and specificity of the method.The detection performance of the method was improved by optimizing the H1 concentration,the ratio of P1-W to H1,the length of anchor structure sequence,reaction time and H2 concentration.Results: The high coincidence of the curves of 8 gold working electrodes and the difference between the oxidation peak and the reduction peak less than 80 mV in the CV spectrum show that the gold printing electrode array has good repeatability and the results between the working electrodes are comparable,and the electrode can be directly used in the modification and subsequent preparation process.(2)It is proved by non-denatured page that w chain can open H1 and generate cycle after being replaced by H2(hairpin 2).In this process,w can be stably combined with H1 by introducing "anchor" structure sequence,and then it is proved theoretically that W can open adjacent H1 by direct contact collision with the help of "anchor" structure sequence.(3)The EIS Nyquist diagrams of the gold electrode,p1-w and H1 modified on the electrode,6-mercapto-1-hexanol(MCH)sealed on the electrode and Tris HCl added with thrombin,P2' and H2-Fc showed that the step-by-step modification of DNA or the process of making DNA more vertical to increase the electrochemical impedance effect.By scanning,the biosensor was placed on the DPV curve of(a)Tris HCl,(b)(a)adding H2-Fc,(c)(b)adding P2',and(d)(c)adding thrombin.It is concluded that the strategy has the ability to develop into a quantitative method.(4)The proposed biosensor has a linear range of 0.2 pM ~ 2 nM and 1 pM ~ 10 nM for thrombin and target DNA at room temperature,and a low detection limit(LOD)of 0.11 pM and 0.61 pM respectively.By detecting DPV peak currents of AFP,CEA,thrombin standard and their mixtures,it is concluded that this method has high detection specificity.The results show that the concentration of H1,the ratio of P1-W to H1 and the length of "anchor" structure sequence have the best value for biosensor.Conclusions: In this paper,we propose an anchor based DNA walkers with neighborhood chain instead of switch activation,which improves the efficiency of walking amplification by adjusting the dwell time of DNA walkers based on the "anchor" structure sequence.(2)The electrochemical biosensor strategy proposed by DNA walkers has the characteristics of one-step,enzyme-free,and can be directly detected at room temperature,which makes the strategy have better scalability in protein or nucleic acid detection.