Studies On New Biochemical Sensing Methods Based On Nanomaterials And Signal Amplification Technologies

In recent years, biosensors have greatly promoted the development of chemistryand clinical diagnosis, due to their’s many advantages such as short analysis time, lowcost, high sensitivity and good selectivity. Furthermore, the rapid development ofnanomaterials and signal amplification technologies provide more new plat forms andideas for the construction of biosensing methods. In this thesis, we proposed severalmethods for the detection of cocaine and diseased-related gene. In order to obtain thebetter performance of the sensing methods, nanomaterials and signal amplificationtechnologies are combined to decrease the detection limit and improve detectionsensitivity. The detailed contents are shown as follows:In chapter2, a label-free fluorescence aptamer sensor method for cocaine wasconstructed based on polymerase aided isothermal circular strand-displacementamplification (ICSDA) and graphene oxide (GO) absorption. Owing to theinstantaneous and overwhelming effects on the central nervous system and potentialillegal use of cocaine, the sensitive and selective determination of cocaine is ofsignificant importance in clinical diagnosis and law fields. In this assay, hairpin probe(HP) which consists of the aptamer sequence of cocaine, is partially complementary tothe primer. When cocaine bound with HP, the primer could anneal with the single-stranded sequence of the opened HP then triggered polymerase elongation, generatinga double-stranded DNA (dsDNA) and displaced the cocaine. In the absence of cocaine,the HP kept intact and ICSDA would not occur. Through combining the uniqueproperties of SYBR Green I (SG) and the preferential absorption of GO to single-stranded DNA over dsDNA, a significant fluorescence enhancement was achieved.Whereas, the SG-stained HP without polymerase elongation was absorbed andquenched by GO. The proposed method is simple, convenient, and has high sensitivityand selectivity. This method displayed a good linear correlation within the cocaineconcentrations range from0.2μM to100μM, and the detection limit was down to190nM. In addition, this aptamer-based sensor method was also successfully applied forcocaine quantification in human urine samples.In chapter3, we developed a multiple-amplification based electrochemicalsensor method for ultrasensitive detection of nucleic acids using a disease-relatedsequence of the p53gene as the model target. A capture probe (CP) with a hairpin structure is immobilized on the electrode surface via thiol-gold bonding, while itsstem is designed to contain a restriction site for EcoR I. In the absence of target DNA,the probe keeps a closed conformation and forms a cleavable region. After treatmentwith EcoR I, the target binding portion (loop) plus the biotin tag can be peeled off,suppressing the background current. In contrast, the CP is opened by the targethybridization, deforming the restriction site and forcing the biotin tag away from theelectrode surface. On the basis of the biotin-streptavidin complexation, goldnanoparticles (GNPs) modified with a large number of ferrocene-signaling probes (Fc-SPs) are captured by the resulting interface, producing an amplified electrochemicalsignal due to the GNP-based enrichment of redox-active moieties. Furthermore, Fctags can be dragged in close proximity to the electrode surface via hybridizationbetween the signaling probes and the CP residues after EcoRI treatment, facilitatinginterfacial electron transfer and further enhancing the signal. With combination ofthese factors, the present system is demonstrated to achieve an ultrahigh sensitivity ofzeptomole level and a wide dynamic response range of over7orders of magnitude.High-sensitivity and high-selectivity bioassay for single nucleotidepolymorphism (SNP) genotyping is of vital importance for the early diagnosis andeffective therapy of many diseases, especially cancer. In chapter4, we developed anelectrochemical SNP genotyping sensor for the analysis of cancer-related genesequence. To improve the detection sensitivity, an effective signal amplificationstrategy was developed via combining gold nanoparticle (GNP)-based enrichmenteffect with the surface hybridization-based dragging strategy. With the large numberof ferrocene (Fc) probes enriched by GNP and dragged in close proximity to theelectrode surface through DNA hybridization, a detection limit of femtomolar level (4fM,40zmol in10μL sample) can be achieved with a wide linear dynamic range (from10fM to1nM). By using the allele-specific nucleotide ligation, high selectivity forsingle point mutation was verified. Moreover, PCR amplicons from both patientsamples and standard cell lines have been successfully tested, demonstrating the greatpotential of this sensing scheme in medical diagnosis of genetic diseases.