Novel Surface Plasmon Resonance Technology For Nucleic Acids Detection

Nucleic acids, as a group of basic life substance, play vital roles in the processes of genetic information processing, including storage, transmission and expression of genetic information. Thus, nucleic acids have become one of hotspots in life sciences. As biomarkers, nucleic acids might provide useful information for the occurrence and development of some severe diseases such as cancers. In particular, the concentrations of relevant biomarkers are often present at a very low level in the early stage of disease. Therefore, the detection of specific DNA sequences with extremely low abundance is important for gene therapy, mutation analysis and clinical diagnosis. Surface plasmon resonance (SPR) was introduced in the early 1990s as the underlying technology in affinity biosensors for biomolecular interaction analysis. More recently, due to the advantages of label-free, real-time, high sensitivity and less sample consumption, SPR biosensor has greatly attracted the researchers in related field and has been widely applied to analyze biomarkers. This research has been performed based on SPR biosensor by integrating analytical chemistry and molecular biology for simple and sensitive detection of nucleic acids. This dissertation mainly contains the following two parts:1. Determination of the invA gene of Salmonella using surface plasmon resonance along with streptavidin aptamer amplificationWe have developed a sensitive method for the determination of Salmonella by integrating a streptavidinylated aptamer (SA-aptamer) as a signal amplification unit along with a modified asymmetric polymerase chain reaction (PCR) technique into the surface of an SPR sensor chip. The gold film of the sensor was first modified with a thiolated probe, and the target sequence and SA-aptamer were then induced to form a sandwich structure. If SA is added, the SA-aptamer forms a complex with SA which will amplify the response signal. Under optimal conditions, this sensing scheme had a linear response in the 50 pM to 200 nM range, and the lower detection limit was 20 pM (for a synthetic target sequence). This strategy was successfully applied to the determination of Salmonella bacteria at levels as low as 60 CFU mL-1. This biosensor is sensitive, selective and highly stable. These features make this strategy a promising and powerful screening tool to detect pathogens in food, and in clinical and environmental samples.2. A simple and novel DNA-nanotechnology strategy for detecting microRNA based on surface plasmon resonanceMicroRNAs (miRNAs) play significant regulatory roles in a variety of diseases and have been emerging as a group of promising biomarkers in cancer cells. Here, a novel and simple surface plasmon resonance (SPR) biosensor was developed for specific and highly sensitive detection of target miRNA employing the mismatched catalytic hairpin assembly (CHA) amplification coupling with streptavidin aptamer (SA-aptamer). The presence of target miRNA triggered the CHA amplification reaction, which brought about the recycling of the target miRNA and large amounts of CHA products. Meanwhile, the plentiful CHA products could hybridize with the capture probe on the sensor chip and combine with streptavidin to achieve enhancement and output of the detection signal. Benefiting from the outstanding performance of the enzyme-free CHA amplification and non-label SPR biosensor, the established biosensor exhibited simplified process, highly sensitivity and selectivity. Under the optimal conditions, the designed strategy could detect target miRNA down to 1 pM with a dynamic range from 5 pM to 100 nM, and successfully applied to the determination of target miRNA spiked into human total RNA samples. Thus, this SPR-based biosensor might become a potential alternative tool for miRNA detection in medical research and early clinical diagnosis.