Studies On Novel Functional Nucleic Acid Probes And Isothermal Signal Amplification Strategies For Assay Application

Functional nucleic acid probes(FNAPs)including the molecular beacon,aptamer,DNAzyme and T-Hg2+-T structure,can provide flexible signal transduction mechanisms for developing new analytical tools.Especially,the combination of functional nucleic acid probes with isothermal signal amplification strategies significantly improves the sensitivity of many assay methods.In recent years,the FNAPs-based assay methods have been widely applied in different fields,such as environmental monitoring,food security analysis and clinical diagnosis.Their excellent assay performance has drawn great attention.Meanwhile,various isothermal signal amplification techniques are explored to enable the construction of ultrasensitive assay methods.Therefore,in this thesis,research was focused on designing new FNAPs and isothermal signal amplification strategies to construct several sensitive assay methods.The main contents of this thesis are listed as follows:In Chapter 1:The definition,classification and development process of FNAPs were briefly reviewed;the working principles and assay applications of several isothermal signal amplification techniques were introduced in detail;research purposes and signification of this thesis were summarized.In Chapter 2:A simple,sensitive and reusable electrochemical sensor was designed for determination of mercury(?)(Hgig)by coupling target-induced conformational switch of DNA hairpins with thymine-Hg2+-thymine(T-Hg2+-T)coordination chemistry.Each hairpin was labeled with ferrocene(Fc)redox tag in the middle of the loop and was immobilized on the electrode.Due to the rigidity of stem in hairpin probe,the Fc tag could not approach electrode to transfer electron.In the presence of target analyte,Hg2+-mediated base pairs induced the conformational change from the sticky end to open the hairpins,resulting in the ferrocene tags close to the electrode for the increasing redox current.By monitoring the increased current signal,the concentration of Hg2+ could be detected with high sensitivity.The dynamic concentration range spanned from 5.0 nM to 1.0 mMu Hg2+ with a detection limit(LOD)of 2.5 nM.In Chapter 3:A novel magneto-controlled electrochemical DNA biosensor was designed for the ultrasensitive detection of lead coupling a lead-specific DNAzyme with DNA-based hybridization chain reaction(HCR).To construct such a magnetic lead sensor,DNAzyme-based molecular beacons,selective to cleavage in the presence of Pb2+,were initially immobilized onto magnetic beads.Upon addition of target lead,catalytic cleavage of substrate DNA segments in the double-stranded DNAzymes resulted in the capture of the initiator strands via the conjugated catalytic strands on magnetic beads.The captured DNA initiator strands triggered the hybridization chain reaction between two alternating hairpin DNA structures labeled with ferrocene,resulting in the enrichment of Fc tag.By monitoring the electrochemical signal of Fc tag,the concentration of Pb2+ could be detected with high sensitivity.The dynamic concentration range spanned from 0.1 nM to 75 nM Pb2+ with a detection limit(LOD)of 37pM.In Chapter 4:A novel colorimetric sensor based on the linear rolling circle amplification strategy was designed for sensitive screening of microRNA(miRNA)at an ultralow concentration coupling catalytic hairpin assembly(CHA)with DNAzyme formation.In this method,target miRNA acted as the cyclization template for padlock probe,as well as the primer for rolling circle amplification(RCA).By this means,RCA reaction was triggered to synthesize a long-chain DNA,which consisted of many repeated catalytic sequences.These catalytic sequences then catalyzed the automatic assembly of hairpin H1 and hairpin H2 to produce numerous H1/H2 duplexes(i.e.CHA reaction),resulting in the release of HRP-mimicking-DNAzyme sequence from hairpin H1.Upon binding with hemin,the HRP-mimicking-DNAzyme acted as peroxidase mimics to catalyze the TMB-H2O2 system,thus resulting in the amplification of detectable signals.A linear dependence between the absorbance and the logarithm of miRNA concentration was obtained in the range from 1.0 fM to 1.0 pM with a detection limit(LOD)of 0.68 fM.In Chapter 5:An ultrasensitive electrochemical DNA sensor was developed by using an immobilization-free hairpin probe(HP)to recognize target DNA and mediate strand displacement amplification(SDA).The hybridization of target DNA on the target recognization region of HP could trigger the SDA reaction in the presence of KF exo-polymerase and N.BbvC IA endonuclease.The polymerization reaction step in SDA opened the hairpin structure of HP,releasing the streptavidin(SA)aptamer from HP.Meanwhile,the SDA reaction produced a lot of trigger DNA fragments,which could also open HP through competitive combination.Through the binding of SA aptamer with SA,the opened HP was captured on a SA-modified electrode.By monitoring the electrochemical signal of ferrocene(Fc)labeled in HP,the concentration of target DNA could be detected with high sensitivity.The electrochemical signal increased with the increasing target DNA concentration in the dynamic range from 5 fhM to 10 pM with a detection limit(LOD)of 2.56 fM.In Chapter 6:A novel split-type photoelectrochemical immunoassay(STPIA)for ultrasensitive detection of prostate specific antigen(PSA)was developed by coupling rolling circle amplification(RCA)with enzymatic signal amplification.Firstly,the sandwich immunoreaction induced the capture of a secondary antibody/primer-template DNA-labeled gold nanoparticle tag(pAb2-AuNPs-ptDNA)in microwell plate,which could then trigger RCA reaction.Because the reaction substrate for RCA contained biotin-dATP,the long-chain DNA product was embedded with a lot of biotin.Through the binding of biotin with avidin,numerous avidin-alkaline phosphatase complexes(avidin-ALP)were captured in microwell plate.The captured avidin-ALP then catalyzed the hydrolysis of ascorbic acid 2-phosphate(AAP)to generate ascorbic acid(AA),which acted as an efficient hole-trapping reagent to amplify the photocurrent of CdS quantum dot(QD)-modified TiO2 nanotube array(CdS/TiO2 NTA).By monitoring the increasing photocurrent,the concentration of PSA could be detected with high sensitivity.The photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL-1 with a low detection limit(LOD)of 0.32 pg mL-1.