Studies On Electrochemical Biosensor Based On DNA Signal Amplification Strategies

Electrochemical biosensor is a novel biosensor,which translates the biological signals between target and molecular recognition elements into electrochemical signals.Due to its inherent advantages of high specificity,simple operation,fast analysis speed,less sample consumption,simple instrument,electrochemical biosensor has become one of the most attractive research areas in analytical chemistry.It is particularly valuable to develop a highly sensitive,selective and reliable electrochemical sensor for the detection of trace targets(such as disease marker proteins,DNA,micro RNA,heavy metal ions,etc.).In this paper,In this paper,combined with a variety of DNA signal amplification strategy,a series of simple and low cost electrochemical biosensors were constructed,which realized the highly sensitive and selective detection of total protein,Hg2+ and ATP.The main research contents are as follows : 1.An electrochemical immunosensor for the total protein of Nosema bombycis detection based on Fe3O4NPs-DNA dendrimer for signal amplificationMetal nanoparticle catalyzed signal amplification mostly involved in the addition of catalytic substrate into an electrolytic cell,which suffered from far interaction distance between catalyst and substance,limiting the catalytic efficiency in signal amplification.Therefore,a sensitive electrochemical immunosensor was constructed to analyze and detect the total protein of Nosema bombycis(TPN.b)on the basis of Fe3O4NPs-DNA dendrimer and coimmobilization Fe3O4 NPs and methylene blue(MB)in the same electrode surface for signal amplification.On the one hand,the Fe3O4NPs-DNA dendrimer formed by layer by layer self-assembly can greatly increase the immobilization amount of electron mediator MB,thus improving the electrochemical signal.On the other hand,Fe3O4 NPs in Fe3O4NPs-DNA dendrimer could be utilized as catalyst toward the electrochemical reduction of MB intercalated in the DNA backbone without the addition of other substances,making the signal further enlarged,which can avoid the use of protease with the disadvantages of high cost and easy deactivation.More importantly,the self-assembly between Fe3O4 NPs and MB in the same electrode surface could shorten the interaction distance between them and thus efficiently improved the catalytic efficiency.Under optimal conditions,the proposed immunosensor displayed a good linear response in the concentration ranged from 0.001 to 100 ng·mL-1 with a detection limit of 0.35 pg·mL-1.The method has the advantages of simple operation,fast response and low cost,and provides a new method for the detection of other proteins with high sensitivity and high selectivity.2.A novel electrochemical sensor for sensitive detection of Hg2+ based on endonuclease assisted cyclic and terminal deoxynucleotidyl transferase assisted DNA amplificationPolymerase chain reaction(PCR),rolling circle amplification(RCA)and loop-mediated isothermal amplification(LAMP)were widely utilized to amplify DNA,nevertheless,these strategies often require relatively long assay time,complicated preparation,specific equipment or the complex process of designing probes.In this work,terminal deoxynucleotidyl transferase(TdT)was applied to amplify DNA,thereby presenting a facile,direct,and cost-effective way for signal amplification owing to the fact TdT,a template-independent DNA polymerase,could effectively prolong the growth of single strand DNA at 3'-OH end of DNA in room temperature.In the presence of the target Hg2+,hairpin DNA(HP1)undergoes conformational changes to form output DNA and hybridized with the 3'-PO4 terminus labeled HP2 on the surface of electrode.The endonuclease Nt.BbvCI was able to cut the HP2 at the cleavage site,accompanying with releasing output DNA and participating in the next hybrid cycle,which realized the recycling of output DNA.Compared with other enzymes,the endonuclease enzyme possessed excellent specificity.And the HP2 that was left on the surface of the electrode will be exposed substantial 3?-OH group.Upon the addition of TdT,HP2 will be extended to long single stranded DNA to absorb a large number of positively charged Ag+ via electrostatic interaction.The silver nanoparticles could be in-situ deposited on electrode surface for electrochemical signal output,which avoided the addition of electroactive subtances,thus,simplifying the experimental operation.The introduction of TdT led to a substantial increase in the loading capacity of Ag+,which amplified the electrochemical signal.The electrochemical biosensor constructed by this method had high sensitivity and good selectivity,and is expected to be extended to detect other toxic heavy metal ions.3.Target-driven catalytic hairpin assembly and Mg2+-dependent DNAzymes recycling technique coupled enzyme-free,dual-amplified electrochemical biosensor for ATP detectionEnzyme-assisted signal amplification can improve the senstitivity of biosensor,however,the enzyme with high cost is unstable and susceptible to environment such as temperature,pH.In consequence,it is an urgent challenge to develop a simple,sensitive and nonenzymatic electrochemical biosensor.In this work,we constructed an enzyme-free and dual-amplified electrochemical biosensor for sensitive detection of adenosine triphosphate(ATP)with dual amplification by using target-driven CHA and Mg2+-dependent DNAzymes recycling techniques.The binding of two split aptamers(Apt1 and Apt2)toward ATP triggered DNA assembly and thus be converted to enzymatic sequence(termed as HP1-HP2)via CHA reaction,accompanying with target recycling for dramatically enhancing the conversion ratio.The subsequent hybridization of enzymatic sequences with substrate hairpins(HP3)on magnetic polystyrene microspheres(PSC)made the formation of ion-dependent DNAzymes.Therefore,the corresponding metal ions catalyzed the cleavage of substrate sequences to obtain output S1 fragment and to release the enzymatic sequences to trigger another cleavage cycle,which realized the indirect conversion of input ATP into output S1 as well as further enhancement of co nversion ratio.Based on the dual recycling amplification,one target ATP could be converted into massive DNA S1,providing a significantly amplified approach for sensitive detection of ATP up to femtomole level.As a result,the established electrochemical biosensor exhibited high sensitivity and excellent selectivity with a wide linear range from 0.001 nmol·L-1 to 50 nmol·L-1 and a low detection limit of 0.45 pmol·L-1.More importantly,this approach avoided the utilizing of a ny protein enzymes and could undergo multiple turnovers without losing binding ability or activity.This work demonstrated here can thus offer new opportunities for the development of convenient and enzyme-free signal amplification strategies for detecting various targets such as DNA,metalion and protein.