Label-free Detection Of Nucleic Acids, Enzymes And Small Biomolecules Based On Nucleic Acids Probes

The developments in biology over the past 20 years have fully proved that nucleic acids are not only hereditary materials for the storage of genetic information but also could be used in gene analysis, disease diagnosis. Nucleic acids are recognized by complementary strands through specific base pairing. Since aptamers were first reported in 1990, they have been widely used in analytical detection, separation and imaging. Expand the target of nucleic acids probe from nucleic acids to small molecules, proteins and ions. In order to improve the sensitivity, many techniques have been introduced in nucleic acds probe strategy including the use of nanoparticles, enzymes and some signal amplification techniques.In this thesis, a series of label-free methods based on nucleic acids probes have been developed.We have proposed a simple and sensitive label-free fluorescence strategy by employing graphene (GO) and the tediously familiar dye Hoechst 33258 to detect DNA and enzymes. We chose a dye, Hoechst 33258, one of the most popular for DNA stain. To our knowledge, Hoechst 33258 is nearly a planer structure which should be attracted by GO and its fluorescence will be quenched even in present of ssDNA. However, in present of dsDNA, Hoechst 33258 may release from GO and exhibit fluorescence with dsDNA. This is based on the different affinity of Hoechst 33258 to ssDNA and dsDNA. The different fluorescent responses of Hoechst can be applied to detect DNA sequence, and extended to detect several enzymes, including Exonuclease ? (Exo ?) and Dam methyltransferase (Dam MTase).DNA methylation has received the most attention due to its close relationship to a wide range of biological phenomena, such as gene activation, gene imprinting, chromatin stability, X chromosome inactivation in females and tumor. In this part, we have reported a simple and reliable fluorescence turn-off assay for Dam MTase detection combining site recognition and the fluorescence enhancement of DNA-AgNCs by sequence with 5'-C-rich/G-rich-3' tails with a detection limit of 1 U/mL. In the presence of Dam MTase, the methylation-sensitive restriction endonuclease Dpn ? which has the same recognition site with the Dam MTase can split the probe, making G-rich sequence detach from C-rich sequence, thus quenching the fluorescence of DNA-AgNCs. The fluorescence of Ag nanocluster was easily obtained by the in-situ modification on the oligonucleotide probe, which eliminated the cost of fluorescence labeling. Through fluorescence detection, gel electrophoresis and inhibitor research, it demonstrated that the DNA-AgNCs can be used as a substitute to traditional fluorescence molecular beacon and accomplish simple and low-cost detection of Dam MTase.As the ubiquitous energy currency of all living organisms, adenosine 5'-triphosphate (ATP) plays an important role in many biological processes such as synthesis and degradation of biological molecules, active transport, and muscle contraction. In addition, it has been suggested that ATP acts as an indicator for cell viability, cell injury, cell motility and other cellular process. In this part, we designed a simple and highly sensitive method for analysing the ATP levels in vitro with a detection limit of 1 nM on the basis of branched rolling circle amplification (BRCA) and achieve the visualization of ATP distribution in cells by using fluorescence in situ hybridization.