Construction Of Unlabeled Glucose Oxidase Based Nucleic Acid Selective Signal Amplifiers And Their Applications For Biosensing

The demand of precise assay of nucleic acids and related bioanalytes has been increasing enormously in various areas including medical and health,military,food safety control,environmental monitoring and so on.The electrochemical nucleic acid sensors with the advantages of easy operation,rapid response,sensitive and low-cost have been widely used in biochemical analysis.In the actual detection,it is often encountered that the analyte concentration is low and mixed with more and complicated other interferences.Therefore,the selective amplification of the analyte signal is one of the important methods to improve the performance of electrochemical nucleic acid sensors.In the past decades,the design and development of high-efficiency nucleic acid selective signal amplifiers are of great significance for the construction of ultra-sensitive electrochemical nucleic acid sensors.In homogeneous solution,the mediated electron transfer behaviors between redox enzyme and different redox mediators are different due to the electrostatic interaction,steric hindrance,and hydrophilic/hydrophobic factors.Inspired by this,we reported the concept of unlabeled glucose oxidase(GOD)-based nucleic acid selective signal amplifier,and this nucleic acid selective signal amplifier had a unique selective signal amplification effect on the ferrocene-labeled DNAs(Fc-DNAs)with different specific structures.By means of electrochemical experiments and digital simulations,the selective signal amplification was systematically studied from the aspects of concept verification,quantitative evaluation,principle analysis,significance exploration and application investigation.Briefly,this selective signal amplification effect caused by the specific interactions between GOD and Fc-ss DNAs with different lengths can be quantitatively evaluated using the reaction constant(ks)of the homogeneous reaction between them.And this selective signal amplification effect is beneficial to improve the discrimination of these probes signals.A series of novel,simple and sensitive self-powered nucleic acid sensors had been developed by using this nucleic acid selective signal amplifier.In view of the fact that modern studies of the selectivity of most nucleic acid signal amplification strategies largely depends on the specificity of nucleic acid hybridization reaction,the development of unlabeled GOD-based nucleic acid selective signal amplifier provides a new idea for the construction of nucleic acid sensors.The main research contents of this dissertation are as follows:(1)The specific interactions between GOD and Fc-labeled single-stranded DNAs(Fc-ss DNAs)with different lengths may cause different mediated electron transfer behaviors in homogeneous solution.Based on this principle,we developed an unlabeled GOD-based nucleic acid selective signal amplifier,which could selectively amplify the signals of Fc-ss DNAs with different lengths.By means of electrochemical experiments and digital simulations,the selective signal amplification was systematically studied.In addition,this selective signal amplification effect has the advantages of simplicity and generality in nucleic acid sensing.As practical examples,a sensitive and universal self-powered nucleic acid sensing platform had been developed based on this nucleic acid selective signal amplifier,which realized the highly sensitive self-powered detection of different kinds of analytes,including infectious viral nucleic acid fragment,common energy small molecule and highly toxic heavy metal ion.In view of its easy implementation and potential popularization,this nucleic acid selective signal amplifier is expected to be a widely used nucleic acid selective signal amplifier for electrochemical nucleic acid sensing.(2)Based on the reasonable conjecture that the specific interactions between GOD and Fc-labeled double-stranded DNAs(Fc-dsDNAs)with different lengths may cause different mediated electron transfer behaviors in homogeneous solution,we developed an unlabeled GOD-based nucleic acid selective signal amplifier with selective signal amplification effect on Fc-dsDNAs containing different lengths and explored its application in the detection of the disease-related nucleic acid fragments and small molecules.Through electrochemical experiments,digital simulations and spectral characterizations,this selective signal amplification effect were discussed.By combining this nucleic acid selective signal amplifier with different nucleic acid reaction strategies to construct self-powered nucleic acid sensors,which realized highly sensitive self-powered detection of the B-type Raf kinase(BRAF)gene fragment and adenosine triphosphate(ATP),respectively.And these assays could also be applied to detect target analytes in human serum samples.Considering the flexibility and simplicity of this nucleic acid selective signal amplifier applied to different nucleic acid reaction strategies,it has a broad application prospect in the design of electrochemical nucleic acid sensing platforms.(3)Based on the study of selective signal amplification effect between GOD and Fc-labeled DNAs(Fc-DNAs)with different structures,we constructed an unlabeled GOD-based nucleic acid selective signal amplifier and explored its application in the detection of single nucleotide polymorphisms(SNPs).Taking four Fc-DNAs with different structures as research models to systematically study this selective signal amplification phenomenon.After that,different novel and simple self-powered nucleic acid sensors were constructed by combining this nucleic acid selective signal amplifier with direct hybridization strategy and strand displacement reaction(SDR),respectively,and their abilities to detect mutant genes were compared.Among them,the GOD-SDR system can better realize the highly sensitive and selective self-powered detection of the common melanoma associated SNP allele(BRAF gene V600 E mutation,BRAF V600E),and the detection limit was 38 f M.And this assay could also be applied to detect the amplifiers of loop-mediated isothermal amplification(LAMP)reaction(in buffer and human serum).In view of the role of the nucleic acid selective signal amplifier in improving signal differentiation,this work provides a promising scheme for disease diagnosis.(4)Based on the above works,the electrochemical experiments,digital simulations and spectral characterizations were used to further investigate the selective signal amplification effect caused by the specific interactions GOD and Fc-labeled double-stranded DNAs(Fc-dsDNAs)with different single base mismatched alleles in homogeneous solution.From this,we constructed an unlabeled GOD-based nucleic acid selective signal amplifier and verified its potential application in self powered genotyping.This will open up a new direction for designing novel and simple genotyping sensors.