Research On Nucleic Acid Probe And Novel Optical Biosensor Method

As a tool for rapid and sensitive detection of biomolecules, biosensors have always been the research interest in the field of bioscience and analytical chemistry. Optical biosensing technology has been widely used in the detection of many biomolecules, such as enzyme activity, protein and nucleic acids, due to its advantages of high sensitivity, simple, no separation and real-time. Furthermore, nucleic acid probes have attracted great attention in the development of new method of biosensor and biochemical analysis, attributing to their advantages of sequence diversity, various recognition, good stablity and so on. Based on the current literature reports, in this thesis, we have established several new strategies throuth the combination of diverse nucleic acid probes with optical analysis methods for the detection of polynucleotide kinase, mi RNA and disease-related gene sequence. Compared with the traditional methods, the proposed detection approaches are simple, cost-effective, real-time and sensitive. The detailed contents are listed as follows:In chapter two, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5’-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by d CTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5’-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real-time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.In chapter three, we develop a new homogeneous method for sensitive detection of mi RNA on the basis of bioluminescence monitoring the released AMP from the target-triggered hybridization chain reaction(HCR)-mediated ligation. The presence of target mi RNA can efficiently initiate HCR between two species of hairpin probes,generating the ds DNA polymers with a large number of ligatable nicks, which can be covalently ligated by the ligase, and the released AMP from the ligation can be used for quantitative analysis of micro RNA. The introduction of HCR not only improves the sensitivity of mi RNA assay, but also provides a universally amplified platform for the detection of different biomolecules by designing specific nucleic acid probes. Importantly, this bioluminescence assay employs the target-dependent ATP from the ligation byproduct of AMP as the reporter without the requirement for the sophisticated luciferase manipulation, complicated immobilization, and separation steps. The proposed method has significant advantages of simplicity, low cost, high throughput, and high sensitivity, and holds a great promise for practical point-of-care applications.In chapter four, we described a novel and simple fluorescent sensors based on the combination of the gravity of gold nanoparticles and Taq DNA polymerase reaction for the detection of nucleic acids using a disease-related sequence of p53 gene as the model target. Firstly, the primer is immobilized on the Au NP surface via strong self-assembly of thiol-gold bond. In the presence of target DNA, it hybridized with the primer then the formation of a partial duplex can be extended by Taq DNA polymerase with the present of d NTPs, generating a stable ds DNA duplex with a blunt terminal. Finally, the elongation product is ligated with the fluorophore labled ds FAM by T4 ligase. After centrifugation, a significant fluorescence can be achieved in the precipitation while the signal in supernatant can be neglected. By contraries, a single base mismatch in the target DNA or random DNA sequence causes failure of hybridization, extension, ligation, resulting no obvious fluorescence signal in the precipitation. The proposed method not only have the advantages of simple, convenient, high selectivity, but also can be successfully applied for the detection of related enzyme activity.