Development And Application Of DNA Biosensors And Logic Gates

DNA biosensor is a new field which is developed well in recent years. DNA biosensor is applied widely to the environment and medicine areas with some merits such as high sensitivity, good selectivity, simple procedures and economy. The coalescent of DNA biosensor and the fluorescence analysis method makes the line range wider, the amount of sample used smaller and the repeatability better. Logic gate is the linchpin to the design of digital logic circuit and the development of computer. DNA logic gate has been applied widely to the biochemistry and medicine fields including the treatment of digital information, the development of chemistry sensor and the diagnosis of disease. However, the research on the construction of logic sensor is just beginning from logic to sensor. Therefore, it is significant to realize the practicality of molecule logic through connecting the sensor and logic. This work aims to develop label-free DNA biosensor and DNA logic molecule logic gate and detect small molecule. The main context was shown below :Part 1: A label-free, highly sensitive and selective detection of hemin based on the competition between hemin and protoporphyrin IX binding to G-quadruplexes. Hemin has a lot of biological functions in our human body and an assay of hemin is of great significance, but researches on the detection of hemin are relatively scarce. In this work, a simple and label-free fluorescent detection method for hemin is reported using protoporphyrin IX(PPIX) as a fluorescent signal repo rter. PPIX emits weak fluorescence in aqueous solution. When PPIX binds to G-quadruplexes, the fluorescence intensity of PPIX is greatly increased. While in the presence of target hemin, PPIX will be replaced by hemin in G-quadruplexes as its affinity to G-quadruplexes is weaker than that of hemin. With the formation of hemin–G-quadruplex complex, PPIX is released to the solution from the G-quadruplex accompanied by the quenching of fluorescence of the system. This fluorescence change of the system can be used to monitor hemin with a low detection limit of 36 n M. In addition, the possible binding sites for PPIX binding to the G-quadruplex are discussed based on the competition between hemin and PPIX.Part 2: Thiazole orange as a fluorescent probe: label-free and selective detection of silver ions based on the structural change of i-motif DNA at neutral p H. Silver ions have been widely applied to many fields and harmful effects on environments and human health. Herein, a label-free optical sensor for Ag+ detection is constructed based on thiazole orange(TO) as a fluorescent probe for the recognition of i-motif DNA structure change at neutral p H. Ag+ could be employed to fold a C-rich single stranded DNA sequence into i-motif DNA structure at neutral p H and that folding is reversible by chelation with Cys. We discovered that this DNA folding process can be indicated by TO, which is non-fluorescent in free molecule state and emits strong fluorescence after the incorporation with i-motif DNA. Thus, a rapid, sensitive, and selective method for the detection of Ag+ and Cys is developed based on thiazole orange(TO) as a fluorescent probe for the recognition of i-motif DNA structure change at neutral p H with a detection limit of 17 and 280 n M, respectively. It is worth noting that the mechanism underlying the increase of the fluorescence of thiazole orange in the presence of i-motif structure was explained and a fluorescent DNA logic gate is successfully designed based on the Ag+/Cys-mediated reversible fluorescence c hange in this system. In addition, this system shows a great promise for i-motif/TO complex to analyze Ag+ in the real samples.Part 3: Application of the target DNA fragment from the transgene cauliflower mosaic virus 35 S promoter to DNA Logic gates using DNA junctions based on graphene oxide and SYBR Green I. A DNA fragment with twenty-nine bases(DNA-T) from the transgene Ca MV 35 S promoter was applied to build logic gates(YES, AND and OR) based on graphene oxide(GO) and SYBR Green I(SG) to test DNA-T. A four-way DNA junction is mainly designed through the hybridization of DNA-T and other three strands. The different functional domains of the four-way DNA junction structure are separated on different strands. Only when corresponding strands are inputted and the completely complementary double-stranded DNA(ds DNA) or the four-way DNA junction is formed, the fluorescence intensity of SG is remarkably increased due to the weak GO/ds DNA binding and the combination between ds DNA and SG. In addition, the sensitive detection of DNA-T is also developed GMOs can be identif ied and quantified by this method as the enhancement of fluorescence signal shows the existence of Ca MV 35 S promoter and indicates wide linear ranges from 5 to 600 n M with a detection limit of 2 n M. What is more, the label-free method is economical, simple and rapid, and might pave the way for applying logic gates to more meaningful test.