CRISPR Electrochemical Biosensor Based On Signal Enhancement Strategy For Detection Of Disease Biomarkers

The development of rapid,sensitive and accurate biosensors for nucleic acid detection has become an urgent need in clinical practice.Polymerase chain reaction(PCR)is usually used as the gold standard of nucleic acid detection,which is widely used in the analysis of disease biomarkers in clinical practice.However,the PCR method still faces some problems in the application of point-of-care in the field because it requires complicated instruments and equipment,standardized laboratories and professional technicians.Electrochemical detection,as a simple and fast detection method,has the advantages of sensitive detection,easy to miniaturize instrument,lowcost,and has been widely concerned in the field of point-of-care.Especially in recent years,the development of CRISPR-based electrochemical biosensor provides new ideas for nucleic acid detection,and broadens the development of biosensor.However,the sensitivity of CRISPR-based electrochemical biosensor is still a problem.In this work,two kinds of interfacial signal amplification strategies based on nucleic acid amplification are discussed,the feasibility of the two strategies is investigated,and the detection performance is also analyzed.The details are as follows:1.LAMP-CRISPR electrochemical sensor based on temperature-controlled electrode amplifying interface signal for biosensing of BRAF V600 E mutationHerein,we report a simple and sensitive temperature controllable loop-mediated isothermal amplification(LAMP)-CRISPR based electrochemical sensors(namely TCLCES).The target can be amplified and recognized on the sensor surface in situ at required temperatures with the temperature controllable device.The electrode surface heating generating the natural convection of the solution to the electrode surface leading to the damping of the signal with no target.In addition,the signal with target showed no damping,which showed enhanced signal gain by heating the electrode surface.The enhanced mass transfer and optimization of detection parameters and condition of reaction are revealed by numerical simulations(COMSOL).The effect of temperature on electrochemical interface signal enhancement was verified experimentally,and the signal difference would be further amplified after temperature control of the reaction system by temperature control device.In addition,the performance of TCLCES was also investigated and it was found that the detection limit reached 1 f M when the signal gain was 10%,which maintained good detection performance when stored in the buffer for five days.It is expectable that the sensor can be used to significantly help the development of CRISPR-based biosensor for diagnosing applications.2.CRISPR-powered electrochemical biosensor for target amplification-free mi RNA detection using primer exchange reaction to enhance interface signalHere,we report a new CRISPR-based electrochemical biosensor using primer exchange reaction(PER)to enhance electric current signal(which we term PER-ECRISPR)for target amplification-free mi RNA detection.In this detection system,Cas13a/cr RNA complex was used to specifically identify target mi R-21 and activate trans-cleavage activity(non-specific cleavage of nontargeted RNAs)to cleave HP1,competitively opening HP2 modified on electrode.As a result,the PER bridge sequence binding site at 3’-end in HP2 was exposed to capture the pre-synthesized PER concatemers which are composed of a bridge and multiple repeating units.Multiple signal probes were then hybridized to PER concatemers to achieve signal enhancement.Under the optimal reaction conditions,compared with the unextended sequence,the detection signal of PER series as a signal amplification strategy increased by 6.17 times.Comparing with previous detection methods,the developed method does not require pre-amplification of target RNA,but improve sensitivity by introduction of PER concatemers as signal enhancer.Besides,by designing double hairpin probes(HP1 and HP2),we can achieve a “turn-on” electrochemical signal.The experimental results demonstrated this biosensor have excellent analytical performance with a LOD of 30.2f M without target amplification and can accurately distinguish the breast cancer patients from healthy donor by analyzing the plasma samples,showing great promise in the molecular diagnosis.