Studies On Amplification Methods Based On Nucleic Acid Design And Enzyme In Fluorescence Detection

Nucleic acid is an important hereditary material, which has been widely used in the biological analysis. It is important to detect DNA sequence or some molecules for disease diagnosis and clinical applications. Compared with the traditional modification and amplification, the design of DNA nanomachine exhibits remarkably important advantages such as simple operation and high sensitivity. Especially, the combination of DNA nanomachine and different enzymes is becoming an increasingly promising method for biosensors. In this material dissertation constitutes by four parts besides the preface:(1) A novel DNA nanomachine system was designed to study the possibility of signal amplification based on hybridization chain reaction (HCR) with modified hairpin oligonucleotides as fuels and magnetic beads (MBs) separation. Gel electrophoresis was explored to reveal the growth of the double-strand DNA beginning with the target. The system was found a good sensitivity and selection. The Quantitative analysis of target ssDNA was thus carried out by measuring the reduction of the fluorescence intensity in the system and obtained a detection limit of 1.58×10^-13M and a linear range from 0.5 pM to 100 pM.(2) A signal amplification strategy based on hybridization chain reaction combined with enzyme-enhanced fluorescence detection is proposed for improving the sensitivity and detection limit of the detection of DNA sequences. The target DNA added as catalyst triggered the HCR system to allow fuels——biotin modified hairpin oligonucleotide with different sequences combine into one long double-strand DNA which is immobilized and separated by magnetic beads (MBs). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacetic acid (DBDA) generated from the substrate 4-hydroxyphenylaceticacid (p-HPA) under the catalysis of HRP and obtained detection limit of 2.7×10^-16M and a linear range from 2×10^-15 M to 4×10^-14 M. The system was found a good sensitivity and selection.(3) A fluorescent DNAzyme probe for detecting Pb²⁺ in aqueous solution has shown high sensitivity and selectivity. Due to the intermolecular hybridization between 17E DNAzyme and the substrate strand, in the presence of Pb²⁺, the substrate strand was cleaved under the catalysis of enzyme strand, and the fluorophore-modified fragment was released, which resulted in the increase of fluorescence. The interaction mechanism between Pb²⁺ and DNAzyme was studied by fluorescence spectroscopy. The experiments indicated that, under optimal conditions, a linear correlation existed between the fluorescence intensity and the concentration of Pb²⁺ in a linear range from 5.0×10^-8 to 5.0×10^-7 M, the detection limit is 3.15×10^-8 M(4) A singly FAM-labeled DNA-hairpin smart probe (SP) in which the GGG-triplet quenched the fluorescence and the cocaine aptamer were applied in the detection for cocaine. The exonucleasⅢdigestion has a special reaction site for the double strand DNA. The fluorescence intensity would rise after the cocaine reacting with the aptamer and smart probe because FAM was far away from the GGG-triplet in the new the configuration on which the exonucleasⅢdigestion would occur. After the digestion, the cocaine would be released in the solution, which completes a reaction cycle. In the system, the cocaine could be quantified in the range of 4.0×10^-9 to 8.0×10^-8 M and the detection limit is 1.76×10^-9 M...