Studies On New Methods Of Biochemical Analysis Based On Functional Nucleic Acid And Isothermal Amplification Technologies

In recent years, the development of biological sensing technology has greatlypromoted the application of analytical chemistry in life science field. Due to manyadvantages such as high sensitivity, good selectivity, low cost, fast analysis speed,continuous on-line monitoring in complex system and so on, the biological sensingtechnology has a high application value in the chemistry, biology, food, environment,medicine and other fields. In order to further improve the performance of thebiological sensing technology, this doctotal thesis uses the specific combining abilityof functional nucleic acid with cells, proteins, small molecules and ions, based onisothermal signal amplification technology of nucleic acid to develop several bioassaysystems for enhancing the detection sensitivity, broadering the assay concentrationrange of biosensors, simplifying experimental operation, and reducing the cost andusing in actual samples. The detailed contents are described as follows.(1) In chapter2，a novel electrochemical aptasensor based on hybridization chainreaction (HCR) with enzyme-signal amplification was constructed for the detection ofinterferon-gamma (IFN-γ). In this aptasensor, the recognition probes which containedthe sequence of IFN-γ aptamer were initially binded to IFN-γ, and the unboundrecognition probes were captured on the electrode as an initiator to trigger the HCR.The two DNA hairpins bio-H1and bio-H2were opened by the recognition probe, andbound one by one on the electrode. The biotin was used as a tracer in the hairpins andstreptavidin-alkaline phosphatase (SA-ALP) as a reporter molecule. Then, SA-ALPconverted its electro-inactive substrate1-naphthyl phosphate into an electroactivederivative1-naphthol generating amplified electrochemical signal by differential pulsevoltammetry (DPV). The activity of the immobilized enzyme was voltammetricallydetermined by measuring the amount of1-naphthol generated for enzymaticdephosphorylation of1-naphthyl phosphate. The electrochemical signal observed wasinversely related to the concentration of IFN-γ. The proposed approach showed a highsensitivity for IFN-γ in a concentration range of0.5-300nM with a detection limit of0.3nM. The sensing system also provided satisfactory results for the detection ofIFN-γ in the cell media.(2) In chapter3, based on exonuclease III (Exo III) aided amplification andgraphene oxide (GO) platform for fluorescence quenching, a novel, turn-on fluorescent aptasensor for lysozyme (Lys) protein was constructed. The system contains a hairpinprobe (HP) and a signal probe (SP) labeled with carboxylfluorescein (FAM) at its5’end. HP, which consists of the aptamer sequence of Lys, is partially complementary toSP. Lys can bind with the aptamer region of the HP and facilitated the opening of thehairpin structure of HP, exposing a single-stranded sequence to hybridize with SP. Thistriggered the Exo III aided amplification and caused the degradation of SP, whichliberated the free fluorophore labels. Upon the addition of GO, the releasedfluorophore could not be adsorbed and no fluorescence quenching occured, while theintact SPs could be adsorbed on GO surface with the fluorescence substantiallyquenched. The results revealed that the proposed method displayed fluorescenceresponses in a linear correlation to the concentrations of Lys within the range from0.125μg/ml to1μg/ml and the detection limit is0.08μg/ml. Besides such sensitivity,the proposed strategy is also low-cost and simple due to its homogeneous andfluorescence-based detection format.(3) Cancer is one of the most serious and lethal diseases around the world. Itsearly detection has become a challenging goal. To address this challenge, in chapter4,we developed a novel sensing platform using aptamer and RNA polymerase-basedamplification for the detection of cancer cells. The assay uses the aptamer as a captureprobe to recognise and bind the tumor marker on the surface of the cancer cells, andform an aptamer-based sandwich structure for collection of the cells in the microplatewells, then it uses SYBR Green II dye as a tracer to produce strong fluorescence signal.The tumor marker interacts first with the recognition probes which were composed ofthe aptamer and single-stranded T7RNA polymerase promoter. Then, the recognitionprobe hybridized with template probes to form a double-stranded T7RNA polymerasepromoter. This dsDNA region is extensively transcribed by T7RNA polymerase toproduce large amounts of RNAs, which are easily monitored using the SYBR Green IIdye and a fluorometer, resulting in the amplification of the fluorescence signal. UsingMCF-7breast cancer cell as the model cell, the present sensing platform showed alinear range from5.0×102to5.0×106cells mL-1with a detection limit of5.0×102cellsmL-1. This work suggested a strategy to use RNA signal amplification combiningaptamer recognition to develop a highly sensitive and selective method for cancer cellsdetection.(4) In chapter5, we developed a novel fluorescence assay for DNA phosphataseactivity based on the strand displacement amplification of nicking enzyme. Thismethod took T4polynucleotide kinase phosphatase (PNKP) as the model analyte and aimed at the detection of DNA3’-phosphatase activity. The designed3’-phosphoryl ofhairpin probe could be dephosphorylated by T4PNKP into a3’-hydroxyl end, leadingto the initiation of DNA polymerase extension and triggering the strand displacementamplification of nicking enzyme. The process resulted in many“polymerization-nicking” cycles and displaced plenty of single strand DNA. Afterhybridizing with molecular beacons, the single strand DNA opened the loop struactureof molecular beacons and yielded significant fluorescence signal. The detection ofDNA phosphatase activity can be accomplished by monitoring the fluorescenceintensity change. This proposed assay is convenient, fast and highly sensitive with thelimit of detection of0.17U/mL.(5) In chapter6, a simple lable-free fluorescent sensing scheme for sensitive andselective detection of nicotinamide adenine dinucleotide (NAD+) has been developedbased on DNA ligation reaction with ligand-responsive quadruplex formation. Wenoticed that the E. coli ligase specifically employed NAD+as cofactor and its catalyticactivity is cofactor-dependent. We employed Klenow fragment (exo-) which couldsuppress the blank signal and N-methyl mesoporphyrin IX (NMM) as the signalreporter. The DNA probe was designed as a single-strand molecule that formedself-complementary structure at both ends and a quadruplex-forming sequence wasdesigned as the loop close to5’-end. The5’-end was modified with a phosphate groupand the3’-end was exposed. In the presence of E. coli DNA ligase, together with thecofactor NAD+, the ends of DNA probe can be ligated to block the extension reactionfrom the3’-end and ensure the quadruplex-forming sequence reserved. This G-richoligomer fold into a quadruplex structure with monovalent ions. The strong interactionbetween the “activated” quadruplex and NMM, brings about a great fluorescenceenhancement. This approach can detect0.5nM NAD+with high selectivity againstother NAD+analogs.(6) In chapter7, we designed a novel lable-free fluorescent strategy for detectionof glutathione (GSH) and cysteine (Cys). The system consisted of two single strandDNA (ssDNA) containing thymine-thymine (T-T) mismatches and used Hg2+as amediator, N-Methyl mesoporphyrin IX (NMM) as the signal reporter. The assay isbased on a competitive reaction of Hg2+by GSH/Cys and by T-Hg2+-T double strandDNA (dsDNA) complexes. In the abcence of target, two ssDNA containing T-Tmismatches react with Hg2+to form a T-Hg2+-T dsDNA structure in the solution, whichhampers the formation of G-quadruplex structure. However, in the presence of target,GSH/Cys react with Hg2+to keep DNA probes in a free single state. This process results in the effective formation of G-quadruplex structure of DNA probe (GP).Subsequently, because of the strong interaction between the “activated” G-quadruplexand NMM, a great fluorescence enhancement was obtained. The concentration rangesof the strategy are10-400nM for GSH detection and10-500nM for Cys detection withthe limit of detection (LOD) of9.6nM for GSH and10nM for Cys.