Aassy Of DNA Methylation And A New Method For Detection Of Ag(Ⅰ)

DNA methylation is an important epigenetic event that plays a pivotal part of epigenetics, associated with cellular differentiation, regulation of gene transcription and repair and is the most extensively investigated epigenetic mark because of the direct relevance to human health and various diseases such as cancers. Gold nanorods attracted attention and developed rapidly owing to anisotropic configuration and unique optical properties, which can be widely exploited to recognize specific analytes such as amino acid, metal ion, antibody, DNA sequence and for caner cell imaging and photothermal therapy, and to make different self-assembled mode. Great concerns are focused on monitoring and evaluating heavy metal pollution in water due to its characteristics, such as persistent, harmful and difficult to control. Silver is a highly toxic and widespread pollutant in the environment, which can lead to a variety of adverse health effects, brain damage, and damage to the nervous and immune systems. Therefore, sensitive and selective silver detection is very important. Based on all above mentioned, the main points of this thesis are listed as follow:1. Electrochemical strategy for sensing DNA methylation and DNA methyltransferase activity.A label-free signal-off electrochemical method for the determination of DNA methylation and the assay of methyltransferase activity has been developed in this work. A DNA biosensor was firstly fabricated on the gold electrode and the assay exploited electroactive complex [Ru(NH3)e]3+(RuHex) as a signal transducer. After methylated, the DNA biosensor was cleavged by Dpn I, leading to the decrease in electrochemical signal. The concentration of methyltransferase and electrochemical signal are linearly independent, thus providing a method for detection of activity of methyltransferase. The proposed method yielded a linear response to concentration of Dam MTase ranging from0.25to10U/mL with a detection limit of0.18U/mL (S/N=3). Furthermore, we developed a fast, simple and efficient way to screen anticancer drugs related to DNA methylation, thus providing a new platform to evaluate anticancer drugs.2. Gold nanorods-based FRET assay for ultrasensitive detection of DNA methylation and DNA methyltransferase activity.Based on the fact that positively charged GNRs have higher affinity for dsDNA than ssDNA because the surface charge density of dsDNA is much larger than that of ssDNA, a fluorescence method was developed to detect DNA methylation and its inhibition.In the absence of methyltransferase, the fluorescence of FAM-labeled DNA1(FDNA1) was quenched due to the electrostatic attraction-based fluorescence resonance energy transfer (FRET) between GNRs and FDNA1. After the addition of complementary DNA1(cDNA1). The formation of FDNA1/cDNAl hybrid enhanced the electrostatic interaction between GNRs and DNA, leading to an increase in FRET efficiency and a significant decrease in fluorescence intensity. However, if FDNA1/cDNAl was treated with Dam MTase, methylate was generated. Fluorescence enhancement was observed when further treated with Dpn I, which cleaved FDNA1/cDNAl hybrid substrate into small fragments, leading to weakened electrostatic interaction between GNRs and small DNA fragments. So the fluorescence of the system was restored. By monitoring the change of fluorescence, Dam methyltransferase and inhibition has been systematically investigated, thus providing a practical tool for studying DNA methylation and assay of activity of methyltransferase and inhibition. This method is simple, fast and selective with a detection limit of0.5U/mL at a signal/noise ratio of3.3. A label-free colorimetric assay of Ag (I) ions using unmodified gold nanorods.Based on the structural change of cytosine (C)-rich silver-specific DNA and the interactions of unmodified GNRs, a novel colorimetric biosensor was developed for the selective detection of silver (I) ions utilizing positively charged gold nanorods (GNRs) as a recognition platform. The electrostatic interactions between single strand DNA probe and gold nanorods (GNRs) lead to GNRs spacing closely. However, the electrostatic interaction is not strong enough to alter the aggregation state of GNRs. In the presence of Ag+, the formation of C-Ag+-C base pairs strengthened the electrostatic interaction between the GNRs and DNA, resulting in the decrease in the longitudinal absorption of GNRs due to the stronger interaction inducing aggregation of GNRs. The sensitive determination of Ag+was achieved through changes in longitudinal absorption of GNRs. The detection limit of this sensor was15nM (S/N=3). In addition, this method has some other characters, such as short detecting time and excellent selectivity.