Studies Of Label-free DNA Biosensors Based On Enzymatic Catalysis

The study related with nucleic acid was developed rapidly because of its special physical and chemical properties.It has wild application in the medical prevention,diagnosis of disease and gene therapy.With the development of the study,nucleic acid has competitive properties in environmental monitoring and food safety detection.It draws much attention to develop fast,sensitive and convenient biosensors.Our research,based on DNA,constructed biosensors for the detection of NAD+,SA and Pb2+ using the detection instruments fluorescence spectrophotometer and Raman spectroscopy.This research works-mainly include:In chapter 1,the introduction of the research progress of DNA in analytical chemistry especially in fluorescence and Raman field,and the introduction of signal amplification strategy that DNA biosensors usually used.Finally,the research work of this dissertation is summarized.In chapter 2,we developed a novel label-free fluorescence sensing approach for the detection of nicotinamide adenine dinucleotide(NAD+).According to the characters of Exonuclease Ⅰ(Exo Ⅰ)and exonuclease Ⅲ(Exo Ⅲ),SYBR Green Ⅰ(SGⅠ)and the fact that E.coli DNA ligase works only with the presence of NAD+,a novel DNA probe was designed to detect NAD+.A single-stranded DNA probe that formed a self-complementary structure at both ends with two stem-loop structures,like a dumbbell,and a nick was designed in the middle of the probe.The 5’-end was modified with a phosphate group and the 3’-end was exposed.In the absence of NAD+,the ligation reaction did not occur;the probe was digested to mononucleotides after the addition of Exo Ⅰ and Exo Ⅲ,resulting in weak fluorescence intensity due to the weak interaction between SG Ⅰ and mononucleotides.In the presence of NAD+,the DNA probe was ligated by E.coli DNA ligase,blocking the digestion by Exo Ⅰand Exo Ⅲ.As a result,SG Ⅰ was intercalated into the stem part of the DNA dumbbell probe and fluorescence enhancement was achieved.According to the relationship between the fluorescence intensity and concentration of NAD+,the quantitative detection of NAD+ was achieved.The fluorescence intensity showed good linear correlation to the concentration of NAD+ within the range 0.5-1500 nM,and had a wide response detection range of four orders of magnitude.Detection limit of the method is 0.1 nM.This method was simple in design,fast to operate,with good sensitivity and selectivity which could discriminate NAD+ from its analogs.The capacity of resistant to interference was strong,and had a good performance in the complex biological environment like human blood serum samples.It was also successfully applied to detect NAD+ in human cells.In chapter 3,we developed a novel label-free turn-on fluorescence biosensor for the determination of streptavidin(SA)based on terminal protection of small molecule-linked DNA(TPSMLD).According to the theory of TPSMLD,we designed DNA probe that can form a self-complementary structure at both ends with two stem-loop structures,and the 3’-end was modified with biotin to resist digestion by exonucleases in the presence of target SA.Strong enhancement of the fluorescence signals was obtained after the addition of SG Ⅰ.In the absence of SA,DNA probe could be completely digested to mononucleotides after the addition of exonucleases,and can get low background signal.This method showed a good linearity for SA from 0 to 200 nM and low detection limit of 0.4 nM,had good ability to distinguish its target with other nonspecific proteins.It also had good reproducibility that the average relative standard deviations for the series of repetitive measurements with different concentrations of SA was 4.48%,and it had a strong capacity to resistant to interference in the complex biological environment and the recovery range from 96%to 118%.It holds great promise for practical applications with good specificity and sensitivity.In chapter 4,an ultrasensitive DNA biosensor was developed for the detection of Pb2+ in water based on DNA amplification strategy.A DNA probe was designed with GR-5 DNAzyme and its complementary substrate.GR-5 DNAzyme worked as cleavage enzyme in the presence of Pb2+ and it can recognize and cut the specific site of the substrate DNA.The released substrate DNA can partly complement to the biotin-DNA bounded to the magnetic bead,and the free part sequence can hybridize with p1 and p2 probe inducing HCR-like reaction with the result of long length DNA.Then the following addition of 4-ATP-AuNPs can connect to p1 and p2 probes.By washing magnetic bead,the unreacted DNA and 4-ATP-AuNPs can be removed.Through Raman measurement,we can get enhanced SERS Raman signal.In the absence of Pb2+,the DNA probe keep its intact structure,no released DNA can react with biotin-DNA so that the reaction of extending DNA length can’t occur.So we can get low background Raman signal through SERS measurement.The concentration of Pb2+ had relation to the Raman intensity of the choosed Raman characteristic peak,so the quantitative determination can be achieved.This method showed a good linearity in 10 pM-100 nM,detection limit 4 pM and can well distinguish Pb2+from other metal ions.Combining the sensitivity of this method with the characteristic of Handheld Raman Spectrometer of rapid and simple,this method is promising in situ test of water environment.