Novel Bioanalysis Methods Based On Aptamer And G-quadruplex Probe With Signal Amplification

Biosensors have wide application in biochemical analysis, environmental monitoring, clinical diagnostics and drug screening, because of their high sensitivity, easy operation, excellent selectivity, short analysis time and low-cost. Meanwhile, with the advantages of easy to synthesize, good stability, simple design, good biocompatibility, flexibility and signaling mechanisms, nucleic acid has important significance in the construction of biosensors. In addition, biosensors can realize the detection of targets rapidly, efficiently and high sensitively depending on a variety of signal amplification methods. In this thesis, using excellent features of aptamers, molecular beacons, G-quadruplex, gold nanoparticles and deoxyribozymes, new design ideas have been deconstructed for detection of different analysis objects. Compared with the traditional detection methods, the proposed detection methods are highly sensitive, easy to operate, low cost, and high speed of analysis.Immunoglobulin E (IgE) detection of allergic rhinitis, allergic asthma and other related diseases is important in clinical diagnosis. In chapter2, an electronic channel switching-based (ECS) aptasensor was developed for ultra-sensitive protein detection. In the detection mechanism of sensor, the hairpin structure of aptamer was designed to pull electroactive species towards electrode surface and use the surface-immobilized IgE to serve as a barrier that separated enzyme from its substrate. As a result, the IgE binding to the aptamer has been shown capable of causing the decrease in peak current intensity. In the presence of target IgE, the aptamer could specifically "capture" its taget ligand that served as separator between ALP and1-NP, inhibiting the enzymatic reaction. Moreover, the formation of dielectric layer of IgE could impede the subsequent oxidation of naphthol. For this biosensor, the achievement of electrochemical signal did not depend on the conformational change of aptamer probe, and no other oligonucleotide probes were involved, these features can overcome the difficulties encountered by the conventional electrochemical aptasensors. The method had high sensitivity, the detection limit reached4.44×10-6μg·mL-1(22.7fM). It also exhibited good recoveries in diluted serum samples.Ochratoxin A (OTA) is a hazard element for human and animal health, including nephrotoxicity, teratogenicity, carcinogenicity, cytotoxicity, genotoxicity and so on. In chapter3, combination of the high specificity of aptamer, using OTA as a model analyte, an electrochemical biological detection method was designed based on the reconstructive aptamer platform. Two parts of split aptamer can specifically recognize adenosine together, alkaline phosphatase (ALP) could then stained in the electrode by affinity of biotin and avidin. ALP plays enzymatic role which enzymatic conversion of1-Naphthyl phosphate (1-NP) into an electroactive naphthol, leading to the electrochemical signal generation. This method possesses high sensitivity, good selectivity and simplicity in operation. It also provides an efficient way for the detection of other biomolecules using the methods of cleft aptamers.Adenosine triphosphate (ATP) is the direct source of energy with which all biological cells in vivo participate in the life activities, including the synthesis of adipose, sugar, protein and nucleotide synthesis. Detecting ATP rapidly and accurately has very important significance in researching metabolic processes and clinical diagnosis. In chapter4, using ATP as the model analyte, aptamer is split into two parts. Simultaneously, hybridization chain reaction (HCR) is combined with the above platform to accomplish the goal of signal amplification technology. It can be said that the procedure is easy. Aptamer is split two fragments, with one part to mark with mercapto and other part to extend with bases. Lots of ALP attached the electrode for HCR which contribute to high sensitivity for ATP detection, and the detection limit of0.2nM.Literature reports demonstrate that there is a "signal misreading" behavior in existing machines where the target recognition process and signal transduction is separated from each other. In chapter5, we established an integrated signal transduction-based autonomous machine, in which the recognition element and signal reporter are integrated into the same DNA strand. This new biosensing machine can execute the amplification of target-induced signal. Using exonuclease III to execute signal amplification method, which generated a large number of G-quadruplex-heme as catalyzer in the system of H2O2and ABTS. The machine was employed to detect the p53gene in a more ascendant fashion, and improved assay characteristics are achieved, including dynamic response range and sensitivity. The proposed strategy is also selective and sensitive with a detection limit of1pM. However, we hope that the proposed platform of the p53gene detection is more sensitive. Thus, in chapter6, strand displacement amplification (SDA) was executed signal amplification based on the integrated signal readout mode. The present strategy is highly selective, possessing wide dynamic range and sensitive for p53gene detection with a detection limit of25fM. Moreover, the evaluation of p53gene using this colorimetric method was also successfully demonstrated.In chapter7, multidimensional devices of G-rich oligonucleotides were designed and applied in gold nanoparticle aggregation-based colorimetric sensor for cancer diagnosis. When p53genes hybridize with molecular beacons embedded G-rich strand, multidimensional devices of G-quadruplex form for particular DNA. Simultaneously, when multidimensional devices were added in solution, it happened aggregation of gold nanoparticles (AuNPs) modified with capture probe. And the colorimetric system exhibited an obvious red-to-purple color change within10-min hybridization. The colorimetric sensor can not only provide nanomolar level of detection capability but also visualize the mutant p53gene. The method provides potential application for the detection of tumor clinical diagnosis, drug screening and DNA nanodevice design.