Signal Amplification Strategies And Graphene-type Nanomaterials Used In Optical Biosensing Technology

The rapid development of modern society imposes higher requirements for analytical chemistry, especially for biochemical analysis. Developing new analytical methods and techniques for solving more and more analytical problems and social crisis has proposed a great challenge for analysts. Recently, biosensing technology have provided novel strategies for the research in biomedicine a nd analytical chemistry, and greatly promoted the development of biomedicine, clinical diagnosis and drug screening, because of their high sensitivity, good selectivity, short analysis time and low-cost. Moreover, optical detection technology has attracted a great attention of many biochemical researchers, due to many advantages such as easy operation, short analysis time and ease of implementation. This doctoral thesis developed a series of new methods by coupling nucleic acid probe, nanomaterials with opt ical detection technology for detection of base excision repair enzyme and their inhibitors, nucleic acid, small molecule, p H and micro RNA profiling. Compared with the traditional methods, the proposed analytical methods are simple, sensitive and selective. We also verified the feasibility and practicability of these developed methods. The detailed contents are described as follows:Repair of DNA damage plays vital roles in maintaining the integrity of the genomes, base excision repair(BER) is the major pathway of DNA damage repair process, and the activity of BER enzyme is connected to several diseases. In chapter 2, using uracil-DNA glycosylase(UDG) as a model analyte, we developed an enzyme-free amplification for highly sensitive detection of UDG activi ty by combining HCR amplification and GO-based fluorescence quenching platform. The helper hairpin probe HP contains four uracil bases in its stem which can be excised by UDG, significantly decreasing melting temperature and the self-folding stability of the probe and inducing a single-stranded conformation for the probe. This conformation change activates the initiator, which, in turn, triggers the HCR, and generates a chain-like double-stranded DNA(ds DNA) assembly. By combining of fluorescence quenching ability of GO with its different adsorption for ss DNA and ds DNA, a strong fluorescence signal is obtained as a quantitative indicator for the activity of UDG. The proposed strategy is highly selective and sensitive for UDG assay with a wide dynamic range from 0.0001 to 100 U/m L and a detection limit of 0.00006 U/m L. Moreover, this assay held the potential for quantitative scr eening UDG inhibitors and capability of screening UDG activity sensitively in real complex samples. The proposed approach is simple, highly sensitive and selective, which might offer a cost-effective and highly sensitive homogeneous detection platform for UDG activity analysis and related biochemical studies.Intracellular p H is one of significant regulation factor in cell physiological activity, strategies for detecting p H plays an important role in the physiological and pathological process. In chapter 3, using GO as a fluorescence quencher, different adsorption properties on GO for nucleic acids associated with p H induced conformation changes were ultilized to design a simple, rapid assay for p H. In this assay, we designed a p H-sensitive DNA probe, in acidic condition, the probe form s an intramolecular triplex DNA structure, however, when p H is greater than, the probe form a hairpin structure. The triplex DNA possesses stronger rigidity, which would not be absorbed by GO, leading to a strong fluorescence signal. In contrast, the hairpin DNA can be adsorbed on the GO surface via its sticky end, thereby displaying a substantially quenched fluorescence signal, and then, realizes rapid analysis of p H.The assay of nucleic acid and small molecule is very important in biomedicine and clinical diagnosis. In chapter 4, we synthesized graphene-hemin hybrid nanosheets(GHs) by a simple wet-chemical method. The GHs possessed excellent properties of both graphene and hemin. Moreover, it can distinguish the ss DNA and ds DNA. On the basis of special property of GHs, a novel label-free colorimetric assay has been developed for detection of nucleic acid an d small molecule. The proposed strategy is simple and cost-effective, which might provide a promising method of choice for convenient nucleic acid and small molecule detection.Micro RNAs(mi RNAs) are a group of short(approximately 19-25 nucleotides) and endogenous noncoding RNA molecules. These mature mi RNAs can bind to the complementary sites on target messenger RNA, promoting their degradation and translational inhibition by incorporation into an active RNA-induced silencing complex. Recent studies have found that the dysregulation of mi RNA expression is closely associated with various diseases. In addition, mi RNAs have been regarded as biomarker candidates in clinical diagnosis and therapy. In chapter 5, using SYBR Green I as a fluorescence indicator, a label-free fluorescent assay was designed based on the specific binding of coralyne and polyadenosine. Coralyne can binds specifically to polyadenosine with a stoichiometry of one coralyne per four adenine bases, and promoting the formation of adenosine2-coralyne-adenosine2(A2-coralyne-A2) complexes, leading to the conformational folding of polyadenosine from a random-coil to a folded structure. Mi RNA can extend poly(A)-tailing at 3′ end with the assistance of poly(A) polymerase, coralyne can bind the poly(A)-tailing and drives the conformational folding of poly(A)-tailing from a random-coil to a folded structure, and exhibit greatly enhanced fluorescence induced by SYBR Green I through intercalating into the grooves of the A2-coralyne-A2 complex, thus realized the detection of target mi RNA. In chapter 6, a new strategy for sensitive detection of mi RNA was developed based on combining super fluorescence quenching ability of WS 2 nanosheet and duplex-specific nuclease signal amplification. We report for the fi rst time that WS2 nanosheets can exhibit differential affinity toward short oligonucleotide fragment versus ss DNA probe, and can act as an efficient quencher for the adsorbed fluorescence DNA probes. Target mi RNA can hybridize to the ss DNA probe to form DNA/RNA heteroduplex. The heteroduplex will become the substrate for duplex-specific nuclease(DSN) cleavage, since DSN only cleaves the ss DNA probe in the DNA/RNA duplex, which initiates DSNSA and generates a great amplification of fluorescence signal. After the product from the DSNSA reaction incubated with WS 2 nanosheets, the cleaved short oligonucleotide fragments will not adsorb on the nanosheets because of their weak affinity and thus retain a strong fluorescence signal. The proposed method shows high sensitivity with a low detection limit of 300 f M, with an improved single-base mismatch discrimination. In virtue of these advantages, the proposed strategy implies the potential of combining the WS 2 nanosheets based fluorescence quenching with nucleic acid amplification techniques as a high-performance sensing platform for biomedical research and clinical diagnostics.