Hydrogen Peroxide Signal Amplification Labeled Dna Fluorescence Detection Method

DNA carries the genetic information of humans, therefore the detection of DNA hybridization are very important. At present, numerous DNA detection methods have been developed, the traditional method for DNA detection generally uses gel electrophoresis. During the past decade, the fluorescence method has been extensively used because of its high sensitivity. Here, we present a fluorescence method for label-free detection of DNA.This paper is consisted of the following three chapters.Chapter1:IntroductionThe structure and principle of molecular beacons, the key feature of the molecular beacon and the structure of G-quadruplex and its application were introduced. We also introduced the concept and theoretic basis of mimetic enzyme, elaborated introduction and application of mimetic peroxidase. We described the research background, significance, purpose and content.Chapter2:Label-free fluorescence detection of DNA hybridization based on switch of molecular beacon to mimetic peroxidase enzymeIn this chapter, we developed a new method, which based on a new molecular beacon and a exogenous fluorescent dye (10-Acetyl-3,7-dihydroxypenoxazin), for enhanced fluorescence detection of target DNA. Compared to the traditional MB, the stem of the new MB is compose of G-riched DNA sequence. In the presence of the target DNA, G-quadruplex DNA can be free from the MB. G-quadruplex are foure-stranded DNA structure stabilized by coordination cation, e.g., K+. K+-stabilized G-quadruplex (with hemin as cofactor) exhibit superior peroxidase-like activity and effectively catalyze the H2O2mediated oxidation of10-Acetyl-3,7-dihydroxypenoxazin (ADHP). ADHP has not fluoresence, while oxidation product of ADHP showed strong fluorescence in the580nm. Therefore, the detection of the fluorescence signal can indirectly detect the target DNA. Due to the activity of G-quadruplex-Hemin complex and accumulation of oxidation production, the sensitivity can be improved significantly. After hybridization of mis-matched target DNA, the ADHP oxidation products showed a weak fluorescence, which clearly showed that the new MB probe can distinguished between complementary target from single-base pair mismatched and non-complementary ssDNA. The results of experiment prove that the change of the fluorescence intensity is liner with the concenstration of target DNA in the range from1×10-11-1×10-8mol/L. The detection limit for target DNA was3.0×10-12mol/L. The liner regression equation was I=2.0c (n mol/L)+33.6, the relative standard deviation R2=0.994.Chapter3:Colorimetric Detection of glucose based on Mimetic peroxidase enzymeG-quadruplex is a foure-stranded DNA structure stabilized by coordination cation, e.g., K+. K+-stabilized G-quadruplex (with hemin as co-factor) exhibit superior peroxidase-like activity. Glucose oxidase catalyze glucose into gluconic acid and H2O2. The G-quadruplex-Hemin complex effectively catalyze the H2O2-mediated oxidation of10-Acetyl-3,7-dihydroxypenoxazin(ADHP), reflected by a strong absorbance. Upon the addition of an increasing amount of gluose, there is a gradual increase in adsorption was observed, the detection of limit for glucose analysis with colorimetry is3.0×10-6mol/L. In this paper, the dual-enzyme system effectively amplifies the signal, improves the sensitivity.