Application Of Dnazyme-based Colorimetric Methods For Detection Of Food Safety Factors

Food safety incidents caused by food safety factors contamination in food have been repeatedly reported in recent years, so the detection of food safety factors in food have become more and more important. However, although the traditional detection methods including high performance liquid chromatography, enzyme-linked immunosorbent assay and gas chromatography possess high accuracy and sensitivity, they need complicated operation, sophisticated instruments, and high cost. Therefore, a simple, rapid, sensitive and economical detection method is urgently needed. DNAzyme-based colorimetric methods have attracted substantial concern in recent years because they have high specificity, wide range of targets, easy synthesis and modification, and no necessary for any expensive and sophisticated instruments. In this thesis, we explored and developed the DNAzyme-based colorimetric methods for detection of food safety factors in food samples.1. We developed a new kind of DNAzyme-based colorimetric bisoensing method for visual detection of Alicyclobacillus acidoterrestris. In the presence of H2O2, DNAzyme facilitated the redox reaction between H2O2 and guaiacol that was produced by the metabolic activity of Alicyclobacillus acidoterrestris, accompanying with an obvious color change from colorless to amber. By analyzing the color signal, Alicyclobacillus acidoterrestris could be visual detected and the limit of detection was 85 cfu mL–1.2. A Pb2+ detection kit with simplicity, good selectivity and sensitivity was developed by using Pb2+-promoted DNAzyme. Addition of Pb2+ enabled the G-rich DNA to bind hemin to fabricate the Pb2+-promoted DNAzyme, which could catalyze the H2O2-mediated oxidation of colorless guaiacol to produce amber tetraguaiacol, causing a visible color change. On the basis of such color change, a detection kit was developed for detection of Pb2+. Under optimum conditions, the detection limit of the kit for Pb2+ was 1 nM.3. Herein, we for the first time report a DNAzyme-based colorimetric method for the detection of chlorpyrifos(CP) in fruit juice. In the absence of CP, ABTS2- is directly oxidized by H2O2 with DNAzyme as a biocatalyst to produce the blue-green-colored free-radical anion(ABTS?-). However, the amount of H2O2 used in H2O2-DNAzyme-ABTS2- system will be decreased if CP is firstly reacted with H2O2, leading to the decrease of the amount of ABTS?- and a color change. Under optimal conditions, the limit of detection of the method was 0.01 μg/mL.4. We constructed a DNAzyme-based colorimetric and fluorescent detection method for the detection of cholesterol. In the absence of cholesterol, there were no color production and fluorescence intensity change because of no H2O2 production in reaction system. In the presence of cholesterol, using the generated H2O2, DNAzyme could effectively catalyze the H2O2-mediated oxidation of guaiacol, resulting in the change of color and the decreasing of fluorescence intensity. By analyzing the color and fluorescence signal, the limit of detection of the colorimetric method was 0.16 μM and the limit of detection of the fluorescent method was 25 nM.