Studies On Novel Detection Methods Of Point Mutation And On New-Type Biosensing-Interface Fabrication For Immunosensors

Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. Among these changes, the point mutation, i.e. single nucleotide polymorphisms (SNPs), are the most abundant form of genetic variation. Achieving early, accurate, simple and rapid identification to these single-base mutations is of particular importance for the pathogeny and early therapy of corresponding diseases. Up to now, many techniques have been developed for SNP detection. However, these conventional procedures are generally time-consuming, lowly quantitative, complicated, and necessary for expensive instrumentation and technical skills, which are applied to date in very few laboratories. Accordingly, exploring some simple, cost-effective, accurate and easy to be clinically popularized detection methods for SNPs is still of considerable interest. In additional, the electrochemical biosensors have been widely used for the assay of biological analyte due to the advantages of this approach including their simple-design, high-sensitivity and low-cost. However, the method of immobilization, the reproducibility and the reusability still remain to be solved in the design and applications of these sensors. Among these problems presented, the key step for the fabrication of biosensors with excellent property is the immobilization of bio-species on the surface of transducers.Focused on these said topics in the detection techniques for SNPs and the formation of the biosensing interface, several new detection methods for SNPs and several new procedures for immobilizing bio-species to construct biosensor have been developed in the presented paper and described as follows:(1) Using the distance-dependent optical properties of aggregated Au nanoparticles functionalized with oligonucleotides, we first reported a novel colorimetric detection system for SNPs through the gold nanoparticle assembly and the ligase reaction (in Chapter 2). By incorporating the high fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold...