An Ultrasensitive Fluorescence Method Based On Grapheme Oxide Suitable For Thermodynamic Analysis Of Nuclease Enzyme And Drugs Screening

Rapid detection of biological molecules is an important research topic in the field of biomedical and chemical analysis in modern society. Compared to some of the high cost, the experiment jumbled traditional biotechnology, fluorescent probe technique is a simple research methods and means with low cost, high sensitivity and specificity. It can be used to detect the biomolecule complex information into an easily detectable fluorescent signal, which is widely used in a variety of biological molecules such as proteins, nucleic acids, enzymes rapid detection. However, those methods have many disadvanteges such as the poor biocompatibility of the materials, and varied reactive conditions and so on. Thus, further improving and developing new fluorescent molecular probe technology is important and valuable.In recent years, scientists have found that Graphene oxide (GO) can absorb single-stranded nucleic acid and protect it against nuclease degradation. In addition, the nucleic acid molecules absorbed on the GO surface have a higher cell transfection efficiency, low toxicity and strong biological stability. Thus, GO binding fluorescent probe can reduce the background signals, improve biocompatibility, enhance sensitivity, which are widely used in the cell-specific genes and metabolites detection and imaging.According to the characteristics of GO, we developed a series of quick, simple and highly specific graphene oxide fluorescent probe technology for mung bean nuclease, DNase I and RNase H thermal dynamics analysis and inhibitors in vivo screen.The main contents are as follows:1. Thermodynamic analysis and inhibitors screening test of Mung Bean Nuclease (MBN) based on graphene oxide technologyThis chapter is based on a different mung bean nuclease-induced single-strand DNA breaks generated 5'terminal phosphate of a single nucleotide or oligonucleotide characteristics of graphene oxide and the number of different bases for different length s of single-stranded DNA affinity based on a design the quantitative fluorescence probe technique mung bean nuclease qualitative analysis experiments. Because of mung bean nuclease can be used as a marker to monitor the growth and development of the green bean sprouts, so we started the optimization of mung bean nuclease enzymatic reaction conditions, we found that:this method can be detected by measurin the concentration of such a nucleic acid in the range 2×10-4 to 4×10-2 unit/mL, the detection limit is 1×10-4unit/mL, GO optimum concentration of 15?g/ml, the optimal concentration of enzyme reaction system was 8×10-2unit/mL, at 37? for 30min is most appropriate. In this way you can filter out a variety of chemical and antibiotic effect of metal ions on the enzyme activity that, in addition, the inhibitor function is confirm ed by in vitro experiments. Comprehensive results showed:This measurement method is sensitive for mung bean nuclease in vivo quantitative and qualitative analysis. Interestingly, we found that:in vivo inhibition of mung bean nuclease activity can lead to growth inhibition of green bean sprouts, in addition, we also used agarose gel-electrophoresis to verify our assay results.2. Quantitative and qualitative analysis for DNase I deoxy nucleic acid analysis based on the graphene oxide fluorescence techniquesDNase I is an enzyme which can digest single-stranded or double-stranded molecule fluorescent probes. This chapter aims to carry out thermodynamic analysis and inhibitor screening in vitro and in vivo of DNase I according to graphene oxide fluorescent molecular probe. GO as a universal quencher has different affinities for different lengths of DNA. This method has advantages of high selectivity, high sensitivity, easily operation.After screening the most appropriate probe, this probe was used for quantitative analysis of DNase I. The results showed that the linear detection range of DNase I was from 1×10-4 to 6×10-2unit/mL, the detection limit is 0.5×10-4unit/ml. The method was further used to screen DNase I inhibitors in vitro. Results showed that gentamicin, kanamycin are inhibitors of DNase I, erythromycin is an activator; AS3+, Pb 2+,Hg2+,Cu2+,Cd2+ are inhibitors while contrary for Mg2 and Mn2+. Further application for quantitative detection of DNase I in tumor and serum show that the method can be used to accurately detect the levels of DNase I inl ?g of total protein.3. The establishment and application of RNase H quantitative analysis method based on graphene oxide qeunched fluorescence probeThis chapter designed and synthesized DNA-RNA heteroduplex as enzymatic substrates and a signal reporter molecule. Fluorescence method for RNase H analysis was established by using graphene oxide quenched fluorescence substrate. The method was furtherapplied for kinetic study and inhibitor sceening of RNase H. The results showed that the linear detection range of RNase H was form 1.0×10-4 to 4.0×10-2unit/ mL, the detection limit can reach 0.5×10-4 unit/mL. The result of inhibitor screen indic ated that gentamicin and metal ions have a significant impact on this enzyme. Finally, this method is used to detect the expression level of RNase H in different tumor cells and serum samples.