DNA Strand Displacement Reaction Based Electrochemical Biosensor In Application Of Nucleic Acid Detection

Nanotechnology refers to a technology that explores materials and objects at a scale less than 100 nanometers.Nadrian Seeman first proposed the concept of DNA nanotechnology in the early 1980 s.The key in nucleic acid is its precise Watson-Crick pairing principle that makes the operation controllable.DNA nanotechnology includes two fields: structural DNA nanotechnology and dynamic DNA nanotechnology.The latter uses a toehold-mediated strand displacement mechanism to dynamically recombine the double and single chains to form a more stable new double strand and release a single strand.A series of nanodevices,including logic circuits,nanometer robots and cascade amplifier,are based on the toehold-mediated strand displacement.The powerful dynamic and predictable base pairing make dynamic DNA nanotechnology suitable for molecular electronics,medical and other fields.DNA molecule is generally used as a biometric element to identify target in a DNA biosensor.During the molecular recognition,small changes that occur can be converted into measurable signals as well as visually observable graphics by signal conversion elements,and then trace analysis is achieved.Common signal conversion components,such as electrochemical,fluorescent,quartz crystal microbalance and field effect transistor,render DNA biosensor prosperous.Among them,electrochemical DNA biosensor with its unique advantages such aslow cost,easy miniaturization,high sensitivity,and strong anti-interference ability will have a good prospect in point-of-care test and molecular diagnosis.In this work,electrochemical DNA biosensor integrates with DNA nanotechnology to develop a series of structure-switchable platform,focusing on how to improve the signal to noise ratio,achieve rapid sensor-preparation,detection in one step and other key issues.Two kinds of strand displacement based biosensors are operated: one is “Signal off” mode,in which the signal changes from high to low before and after the target are detected;and the other is the “Signal on” mode,in which signal changes conversely from low to high before and after the target are detected.Experiments carried out in this paper are the methods explorations of gene detection.The specific contents are as follows:1.Toehold inspired semiduplex probe to construct a “Signal off”electrochemical biosensor for nucleic acid detectionIn the “Signal off” mode,the sensitivity of structure-switchable electrochemical DNA sensors is generally limited by the irremovable redox labels which often mask the signal generated by the target.To address this issue,a semi-duplex probe composed of a longer mercapto DNA and a shorter signal DNA is designed,analogous to a “nicked loop”.Semi-duplex can serve a toehold to mediate a target-responsive strand-displacement reaction.When the target comes in,it hybridizes with the mercapto DNA through the toehold to form a newly and more stable duplex,while the signal strand is displaced away from the surface of the electrode.Such a reaction can fundamentally eliminate the postresponsive background current that arises from the irremovable probe,and thus improve the sensitivity.This novel toehold E-DNA sensor is able to achieve a detection limit as low as 0.2 p M,which is lower than that of the classic stemloop structured sensor by two orders of magnitude.Moreover,the toeholddomain endows the sensor an excellent selectivity against a single-base mismatched sequence and high binding kinetics.In addition,the sensor is reproducible,reusable and can detect low abundance of target even in complex human serum.These advantages make the sensor more suitable for clinical detection than traditional electrochemical DNA sensor.2.Toehold mediated one-step conformation-switchable “signal-on”electrochemical DNA sensing enhanced with homogeneous enzymatic amplification“Signal off” mode of the electrochemical sensor is limited by the maximal signal suppression of 100%,so another mode of “Signal on” electrochemical sensors is designed in this chapter.It is still a double-stranded probe that is first fixed on the electrode surface,but at this time the mercapto chain is modified with a methylene blue at its other end.The other chain is an auxiliary chain without any modification,but only to provide a toehold.When the target exists,the auxiliary chain hybridizes with the target through the toehold,forming a new duplex released into the solution.Another limitation of the former design is the1:1 response,meaning that each target sequence could only induce a single signaling event by hybridizing with one copy of capture probe.This may thus limit its sensitivity and application in real world.So a signal amplification strategy by exonuclease III is introduced,since the new duplex has a blunt end,which is specifically cleaved from 3' blunt end by the exonuclease in the solution.The auxiliary chain is completely digested and the target is released for the next round of strand displacement reaction.After 60 min of reaction,most mercapto chains fixed on the electrode surface return to its primitive stem-loop structure in the solutions with salt ions.The electroactive molecules are pulled onto the electrode surface for rapid electron transport,resulting in a strongelectrical signal.This 1: N target cycle strategy achieves a detection limit of 42 f M,which is one order of magnitude lower than that of the previous enzyme-free electrochemical DNA sensor.In addition,this strategy shows high selectivity for single base mismatches and enables detection of low aboundance of target DNA directly in human serum and cell culture medium with minimal interference.The high selectivity of toehold,the enzyme-catalyzed target cycle amplification strategy,and the high efficiency of the one-step conversion of the readout signal are integrated into a single sensor,which is believed to provide a new idea for the detection of gene.