Two Kinds Of DNA Electrochemical Biosensors

DNA electrochemical biosensor is a kind of sensor with DNA as the recognition element for its highly specific base-paring interaction between complementary sequences. The DNA-based electrochemical sensors take advantages of high sensitivity, low cost and good selectivity as well as easy microminiaturization. It holds great promise in research on life science, genetic screening and clinical diagnosis. In this dissertation, the author reports two kinds of DNA electrochemical biosensors to monitor the activity of CpG Methyltransferase and Oxytetracycline (OTC) respectively.1. A biosensor to assay the activity of CpG MethyltransferaseDNA methylation, catalyzed by methyltransferase, is known to play an essential role in many biological events. Different from adenine methylation of the sequence5’-GATC-3’catalyzed by Dam methyltransferase in prokaryote, DNA methylation in mammalian cells occurs mainly at the cytosine in CpG dinucleotides, and its deregulation is often associated with carcinogenesis and some other diseases. In this thesis, we have proposed an electrochemical biosensor to monitor the activity of CpG Methyltransferase based on the enzymatic cleavage protection of methyl-CpG binding protein1(MBD1) which can bind to the single methylated CpG. When DNA molecules rich in CpG is methylated by CpG Methyltransferase, MBD1can then bind to the methylated site which may hinder the exonuclease activity and protect the downstream nucleotides, thus the unmethylated CpG DNA is digested. Since the DNA molecules have been modified onto the electrode surface, which may absorb hexaammineruthenium(III) chloride, the electrochemical species, the removal of unmethylated DNA strand from the electrode surface will result in the decrease of the detected electrochemical signal. Therefore, an electrochemical biosensor to assay the activity of CpG methyltransferase is proposed since the cleavage of the electrochemical signal is initially activated by CpG methyltransferase. The sensor may be a good candidate for monitoring DNA methylation in this future.2. A biosensor for the detection of OTCOTC is a broad-spectrum antibiotic with the ability to interfere with the production pathways of essential proteins for bacteria. It may also have efficacy in respiratory and gastrointestinal therapies, and positive effects on livestock’s growth, thus OTC has been widely used as food additive for animals. OTC, however, may cause gastrointestinal and photosensitive allergic reactions especially when we humans are extensively exposed to OTC, so it is essential to detect the content of OTC in food. In this thesis, we have proposed an electrochemical aptamer sensor to quantify OTC. Firstly, DNA1strand modified with ferrocene(Fc), an electrochemical reporter, was immobilized on a gold electrode surface, followed by the hybridization with DNA2, the aptamer of OTC. When OTC is added in the test system, the aptamer will be disassociated from the double strands for a higher affinity between OTC and its aptamer. As a result the DNA1strand modified on the electrode surface return to be single and form a hairpin structure with the help of Mg2+, which result in the proximity of Fc to the surface of the electrode. Consequently, the signal of Fc can be obtained. This method may have a linear detection range from10to600ng/mL with a detection limit of9.8ng/mL. Detection of OTC in mouse blood serum or urine can also be realized.