Establishment And Application Of Novel Signal Amplification Technology

The main idea of this research is to explore novel signal amplification technology formolecules that can not be detected because of it’s low concention. In this parper we willcombine electrochemical techniques, chemiluminescence analysis, nucleic acid aptamerrecognition technology, signal amplification technology to construct a chemical andbiological sensors with high sensitivity and selectivity for the detection of dopamine,DNAand other substances.. The text includes the following chapters:Chapter1, Introduction. This chapter introduced several major signal amplificationtechnologies and its principle,application and development. We also introduced two kindsof detection methods including electrochemical analysis and chemiluminescence analysis.Chapter2, Signal amplified strategy based on gold nanoparticles(AuNPs) couplingwith aptamer for the electrochemical determination of dopamine. First, thionine(Th) wasadsorbed to the surface of AuNPs as the electrochemical marker and then the probe singlestrand DNA was loaded at AuNPs, the electrochemical probe(EC probe) was prepared.Then the aptamer was added into the system, the probe and ampater would form doublestrand DNA(dsDNA). When DA was added, the ampater would binding with DNA andreleased from the EC probe. The EC probe was absorbed to the gold electrode modifiedwith carbon nanoparticles (CNP/GE) for electrochemical detection. It provides a detectionlimit of1.0×10-8M (3σ) and the linear range from3.0×10-8to6.0×10-6M for DA.Chapter3, Signal amplification technology based on entropy-driven molecularswitches for ultrasensitive electrochemical determination of DNA. First, the goldelectrode was modified with AuNPs and the hairpin DNA was fixed on the electrodesurface by self-assemble, the target DNA(DNA1) was added to open the harpinDNA(DNA2). When the other harpin DNA (DNA3)was added, DNA1was releasedbecause DNA3has more complementary base with DNA2. Then the released DNA1 would open the harpin DNA again. As a result,low concentration of DNA1could open alarge amount of harpin DNA on electrode surface. Thionine(Th) was also used aselectrochemical marker in this chapter, Th and the probe single strand DNA(DNA4) wasloaded on Au@PtNPs respectively as EC probe. At last, the EC probe was coating on theelectrode for electrochemical detection.The chemical and biological sensors proved to bea sensitive and selective method for DNA determination. It provides a detection limit of1.0×10-14M (3σ) and the linear range from3.0×10-14to2.0×10-12M for DA.Chapter4, In this work, a new signal amplified strategy based on target-inducedstrand release coupling cleavage of nicking endonuclease for the ultrasensitive detectionof ochratoxin A (OTA) is reported. OTA aptamer (DNA1) and OTA aptamercomplementary (DNA2) were immobilized onto a magnetic bead (MB). In the presenceof OTA, DNA2was dissociated and released from the MB. The released DNA2thenhybridized with DNA3, which was linked at the5’terminus of the amplification templateand can extend along the template in the presence of Phi29DNA polymerase. Theformed double-stranded DNA was cleaved by nicking endonuclease Nb.BbvCI andproduced a short single-stranded DNA. The cleaved DNA strand generated a new site byPhi29DNA polymerase and the process of extension and cleavage was cyclical. Thus, aamount of the short single-stranded DNA were produced. Using DNA and ABEI labeledcarboxylic silica nanoparticles chemiluminescence (CL) probe, the short single-strandedDNA could be sensitively detected. The CL intensity (DI) versus the concentration ofOTA was linear in the range from1.0×10-12to5.0×10-8g mL-1. The detection limit was3.0×10-13g mL-1, and the RSD was3.4%at1.0×10-10g mL-1(n=7). The developedmethod has been applied to detect OTAin natural contaminated wheat samples. Due to itssimplicity, sensitivity and no need of specific recognition of aptamer for cleavage, this CLbioassay offers a promising approach for the detection of OTAand other biomolecules.Chapter5, A novel electrochemical (EC) method based on gold nanoparticles(AuNPs) modified carbon paste electrode (CPE) was developed for detection of malachitegreen (MG). The experimental parameters, such as dose of AuNPs, scan rate and pH,were optimized. The electrochemical response of MG at the AuNPs modified CPE wasinvestigated in details. The method exhibited good linearity for MG over the range from2.7×10-8to2.7×10-5mol L-1with the detection limit of3.0×10-9mol L-1by differentialpulse voltammetry (DPV) under optimum condition. The developed method was applied to analyze the content of MG in fishery water samples with recoveries ranging from95.4–100.9%, showing a satisfactory result.Chapter6, A sample and highly sensitive electrochemical method has beendeveloped for the detection of hydroquinone (HQ) at gold nanoparticles modified goldelectrode (AuNPs/GE). The AuNPs/GE showed excellent sensitivity on the redox of HQ.The electrochemical response of HQ at the AuNPs/GE was investigated in details. Themethod exhibited good linearity for HQ over the range from5.0×10-7mol L-1to2.0×10-4mol L-1mol L-1with the detection limit of1.0×10-7mol L-1by differential pulsevoltammetry (DPV) under optimum conditions. The developed method was applied toanalyze the content of HQ in water samples with recoveries ranging from95.4–100.9%,showing a satisfactory result.