Research On Nucleic Acid Probe And Novel Biosensing Technology

Recently, the novel methods in biosensor and biochemical analysis have promoted the development of analytical chemistry and life science. They play more important roles in interdisciplinary field of analytical chemistry and life science both for scientific research and practical applications. Nucleic acid probes have got more and more attention in the field of biosensor and biochemical analysis due to good stability, ease of modification, sequence diversity and target diversification. Here, we have developed many new methods by coupling nucleic acid probe, novel nanomaterials with optical detection methods for microRNA profiling, UDG and TDG activity analysis, researching small molecule-protein interactions and lipopolysaccharide quantification. Our analytical methods developed in the paper are simple, sensitive and selective, the detailed contents are described as follows:MicroRNAs (miRNAs) are a group of short (approximately23nucleotides) and endogenous non-protein-coding RNA molecules. These mature miRNAs can bind to the complementary sites on target messenger RNA, promoting their degradation and/or translational inhibition by incorporation into an active RNA-induced silencing complex (RISC). Recent studies have shown that miRNAs have been implicated in various diseases. Several researches have established links between altered expression levels of miRNAs and cancer type, tumor stage and response treatments. In chapter2, we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase, and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease Ⅳ. The ERA reaction can be configured as a single-step, close-tube and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of0.1fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification, and may hold the potential for miRNA expression profiling and related theranostic applications.Uracil DNA glycosylase (UDG) is one of the most important base excision repair enzymes. It plays an important role in protecting the genome from damage and sustaining the genome integrity. Quantitative activity assay of UDG is a challenge and of fundamental importance in biosensor. In chapter3, we demonstrated a new analytical method by coupling a fluorophore-labeled hairpin probe with graphene oxide (GO) as a homogeneous analysis technology for sensitive UDG activity quantification. In the presence of UDG, the uracil base could hydrolyze from the hairpin probe, and further excision of the leaving abasic site released high fluorescence. Thus, the strategy provided a simple approach for UDG activity assay. As the outstanding fluorescence quenching ability of GO, a quite low background fluorescence signal can be reached for the efficient fluorescence resonant energy transfer from the fluorophore modified in the hairpin probe to the GO sheet. A quite wide dynamic range was achieved for UDG assay (from0.0017U/mL to0.8U/mL) and the detection limit was0.0008U/mL. From the results, we can know that this strategy offers a cost-effective, simple, highly selective and sensitive homogeneous detection platform for UDG activity analysis and related biochemical studies.The interaction between small molecules and protein receptors is a quite important topic in molecular diagnostics, drug developments, and fundamental research. In chapter4, we reported a novel transcription nanomachine built from biotin-labeled DNA heteroduplex, T7RNA polymerase and other functional factors for detection of protein binding. The small molecule-labeled dsDNA containing T7promoter sequence can perform a transcriptional reaction catalyzed by T7RNA polymerase. However, the reaction will be blocked after the binding of target protein receptor streptavidin. Conjugating transcripts with aptamer of the dye Malachite Green a simple, rapid and sensitive homogeneous fluorescence assay with a detection limit of0.2nM is obtained. And the protein and small molecule interaction assay shows dynamic responses in the concentration range from0.5to64nM. The transcriptional inhibition assay offer a general platform by using the strategy for three kinds of small molecule-protein interactions researches. Moreover, the developed small molecule protein interaction strategy might create a new methodology for developing intrinsically robust platform due to its label-free, homogeneous, and fluorescence-based detection format.Thymine DNA glycosylase (TDG), as a base-specific glycosylase, plays an important role in the epigenetic regulation and sustaining genome integrity. Recent advances in understanding the mechanisms of active DNA demethylation in mammal is the discovery that besides its glycosylase activity, TDG is involved in epigenetic regulation through an active5-methylcytosine demethylation pathway. In chapter5, we reported a sensitive and selective homogeneous fluorescence assay for TDG activity quantification with exonuclease-mediated amplification. TDG excises thymine base from G/T mispair which initiates two consecutive enzyme digestion reactions catalyzed by Endonuclease IV and T7exonuclease respectively. The results indicated that the enzyme activity analysis shows dynamic response to the concentration of TDG ranging from0U/μL to0.17U/μL with a detection limit of0.00018U/μL. The developed method offers high selectivity, desirable sensitivity, excellent reproducibility, simplified experiment sets and low cost, indicating that this method might hold considerable potential for TDG activity assay and associated biochemical function researches.In chapter6, we proposed a new analytical method for lipopolysaccharide (LPS) detection. This strategy relies on SERS-based detection of formaldehyde which is produced from the oxidation reactions of LPS and NalO4. Firstly, under room temperature, the LPS is oxidized by NalO4with the production of formaldehyde. A thiolated reactive molecule, Purpald, is used for the special reaction with formaldehyde, and the production can self-assemble on the gold nanoparticles to deliver the SERS signal. But as a control experiment, it provided a quite weak SERS signal in the absence of LPS. Therefore the quantification of lipopolysaccharide can be finished using the strategy. Under the optimized conditions, we observed the SERS signal increased with the increasing concentrations of the LPS and showed a linear correlation to the LPS concentration in the range from33to667μg/mL. The limit of detection is21μg/mL. This strategy was sensitive, without costly labeled reagents and sophisticate experiment procedures, fast and easy to realize. This developed strategy was potential to offer a convenient and sensitive homogeneous detection format for LPS assay.