Research On New Strategy Of Fluorescence DNA Detection Using Enzyme And Nanomaterials

DNA is a biological molecular that carries genetic informations. The detection of DNA is of vital significance in medical diagnosis, monitoring of environmental pol-lutants, food safety and so on. Fluorescence detection method has been widely utilized in the detection of DNA due to its advantages including high sensitivity, simple pro-cudures, stability and the capability of homogeneous detection. DNA amplification techniques have been combined with DNA auto-assembly to develop the sensitive and specific DNA detection method. To reduce the labeling procedure and develop more effective signal transduction method, nanomaterials have been applied to the DNA detection. Considering that fluorescence DNA detection has been a promising analyt-ical method. This thesis focuses on the proposal to develop more effective and sensi-tive DNA detection method, utilizing nucleases and nanomaterials. This work in-cludes two parts as follows:1. A DNA machine for sensitive and homogeneous DNA detection via lambda exonuclease assisted amplificationThis work designs a DNA machine with three assistant DNAs and lambda exonuclease (Exo-λ) for sensitive and homogeneous fluorescent detection of DNA. The selective digestion of Exo-λ to blunt or recessed 5’-phosphorylated strand of Probe 1-Probe 2 duplex results in the release of target DNA and Probe 2 to produce the fluorescence restoring of fluorophore labeled to Probe 1. The released target DNA could hybridize with another Probe 1-Probe 2 duplex to trigger the target recycling for signal amplification, while the released Probe 2 hybridized with molecular beacon to restore its fluorescence for signal enhancement. This DNA machine showed a fast response to target DNA with a linear concentration range from 0.4 pM to 4 nM. The limit of detection was 68 fM at a signal-to-noise ratio of 3. The high selectivity of the method may result from the Exo-λ’s specific recognition-site of double-stranded DNA and the specific hybridization of target DNA with Probe 1-Probe 2 duplex. This DNA machine with the homogenous detection, rapid response as well as simplicity provides a new approach for sensitive detection of DNA.2. Label-free ratiometric fluorescence detection of DNA utilizing fluorescent DNA-regulated Ag nanoclustersTo realize the sensitive homogeneous detection of DNA, a probe has been designed as template for generating two kinds of Ag nanoclustes (Ag NCs). Through introducing target DNA to regulate the structure of the probe, the fluorescence properties of Ag nanoclusters could be influenced and then the ratio of these two intensities of Ag nanoclsuters could be used to quantify the concentration of target DNA. The principle of the detection was as followed:without target DNA, two fluorescence emissions were observed at 620 and 675 nm. When the target was introduced, the structure of probe was changed. Thus the fluorescence intensity at 620 nm was enhanced while the fluorescence intensity at 675 nm was decreased. The ratios of these two fluorescence intensities of Ag NCs were used to quantify the target concentration. The designed fluorescent strategy shows a linear concentration range from 10-1000 nM with a detection limit of 7.3 nM for the detection of target DNA. Nanomaterial-based ratiometric fluorescence method provides a proof-of-concept in homogenous detection of DNA.