Studies Of Novel Fluorescence Biosensing Technology Based On DNA Probes And Nanomaterials

Sensitive and rapid detection of samll biomolecules and heavy metal ions havealways been the research hotspot in biomedical and analytical chemistry. However,with the development of the scientific research, developing new analytical methods fordetecting sensitively and accurately is still a great challenge for analysts. Fluorescentbiosensing technology, as a cutting-edge sensing technology in analytical chemistry,has been used in different areas broadly such as bioscience, agriculture science,biomedical and so forth, owning to the advantages of high sensitivity, excellentselectivity, low cost, simple operation, fast analysis, continuous monitoring, etc.Integrating with related reports, this thesis aims at simplifying the experimentaloperation, enhancing detection sensitivity, reducing the cost, etc. In this thesis, a seriesof novel biosensing strategies based on DNA probes and nanomaterials were developedfor heavy metal ions, proteins and enzyme activity detection, respectively. Theseresults primarily proved that the proposed technologies were feasible, reliable andaccurate. The detailed content was described as follows：(1) A simple, label-free fluorescent assay strategy for sensitive detection of lead(II) using a nitrocellulose membrane biosensor has been developed. The surface ofnitrocellulose membrane was modified by glutaraldehyde to conjugate streptavidin,followed by the immobilization of a DNA probe via the biotin modifier. Thebiotinylated DNA probe can fold into a G-quadruplex structure in the presence of K+that selectively binds to N-methyl mesoporphyrin IX and yield a strong fluorescencesignal. The presence of lead (II) can induce a conformational change of theG-quadruplex to a more compact structure, which results in the release of K+andN-methyl mesoporphyrin IX with a concomitant reduction of the fluorescence signal.The biosensor shows a detection limit as low as10nM with excellent selectivity forlead (II) over other heavy metal ions. The developed biosensor is further demonstratedfor the detection of lead (II) in spiked river water samples.(Chapter2)(2) A novel biosensor was designed for sensing of targets such as protein andsmall molecule based on the self assembled aptamer-MoS2nanosheets architecture.This DNA-MoS2nanosheet was constructed with aptamer labeled with fluorophoreonly at one end. The aptamer can self assembled onto the surface of MoS2nanosheet toform stable aptamer-MoS2nanosheet architecture, still keeping the binding affinity andspecificity to the aptamer. DNA-MoS2nanosheets can act as a low background signal platform used for the small molecule (Adenosine triphosphate) or protein (humanα-thrombin) detection based on long-range resonance energy transfer. In the absence oftarget, the adsorption of the aptamer labeled with fluorophore on MoS2nanosheetsmakes the dyes approaching closely toward the proximity to MoS2nanosheets surfaceresulting in high efficiency quenching of fluorescence of the dyes with very lowbackground. With the addition of target, binding of the aptamer probes to the targetcan release the aptamer away from the MoS2nanosheet, the quenched fluorescence isrecovered significantly. This biosensor has the advantages in its superb specificity,being rapid, and convenient. Morover, aptamer-MoS2aptasensor design can be easilyextended to develop a variety of probes for detection of a wide range of targets bysimply changing the fluorophores and altering aptamer sequences.(Chapter3)(3) A novel fluorescent nanosensor based on phosphorylation-specificexonuclease reaction and efficient fluorescence quenching of single-stranded DNA(ssDNA) by WS2nanosheet has been developed for monitoring the activity of PNKusing T4polynucleotide kinase (T4PNK) as a model target. The fluorescentdye-labeled double-stranded DNA (dsDNA) remains highly fluorescent when mixedwith WS2nanosheets because of the weak adsorption of dsDNA on WS2nanosheets.While dsDNA is phosphorylated by T4PNK, it can be specifically degraded by λexonuclease, producing ssDNA strongly adsorbed on WS2nanosheets with greatlyquenched fluorescence. Because of the high quenching efficiency of WS2nanosheets,the developed platform presents excellent performance with a wide linear range, lowdetection limit and high signal-to-background ratio, the detection limit of T4PNK was0.01U mL-1. Additionally, inhibition effects from adenosine diphosphate, ammoniumsulfate, and sodium chloride have been investigated. The method may provide auniversal platform for PNK activity monitoring and inhibitor screening in drugdiscovery and clinic diagnostics.(Chapter4)...