Fluorescence Analysis Based On Small Molecule Mediated DNA Signal Amplification

Signal amplification is an effective way to achieve sensitive analysis.DNA has become a good material for signal amplification because of its unique base-complementary pairing characteristics and the ease of chemical synthesis.DNA signal amplification has been widely used for sensitive analysis of biological molecules.In this paper,small molecule mediated DNA hybridization reaction?HCR?and DNA Walker?DNA Walker?were realized through the formation of amide bonds,click chemistry,and hydrazine chemistry,and two kinds of fluorescence methods have been constructed for the analysis of glucosidase and their inhibitors and the detection of lipopolysaccharides and furfural,respectively.The main research contents are as follows:1.New method for in situ assay of?-glucosidase activity and the inhibitor screening through enzyme substrate mediated DNA hybridization chain reactionIn this section of this paper,a new method for in situ analysis of?-glucosidase??-Glu?and its inhibitors based on small molecule mediated DNA hybridization chain reaction?HCR?has been proposed.As an important digestive enzyme,?-Glu catalyzes the hydrolysis of the substrate p-aminophenyl-?-D-glucopyranosine?pAPG?to p-aminobenzene?pAP?and?-D-glucopyranose.pAPG is used to be covalently linked to a magnetic nanoscale?AMNSs?,which contains a cis-glycol group that can form a phosphodiester bond with a boronic acid-labeled starting chain,causing the starting chain to be fixed to AMNSs,which in turn drives the occurrence of HCR around AMNSs.However,the catalytic reaction product pAP does not have the ability to capture the boric acid-labeled starting chain,so HCR does not occur.Therefore,?-Glu activity is related to the degree of HCR reaction around AMNSs.In the concentration range from 0.4 U/mL to 1.7 U/mL,the fluorescence intensity is linearly related to the enzyme activity,and the minimum detection limit is 0.023U/mL.At the same time,the established method can also be used for screening and analysis of enzyme inhibitors such as gallic acid and quercetin.In addition,the method can be applied to in situ detection of?-Glu on cell membrane surface and screening of inhibitors at cell level.2.Construction of DNA Walker mediated by split walking chain and its application in double target analysisThis section proposes and constructs a DNA Walker mediated by split aptamer with the addition of azido-acetylene and hydrazone chemistry and it has been further applied for the analysis of lipopolysaccharide?LPS?and furfural.The method is accomplished by two reaction systems.In the first reaction system,the magnetic beads?MB?with streptavidin on its surface can bind to the lipopolysaccharide aptamer chain modified by biotin at the end,and the recognition chain DNA can subsequently bind to the aptamer chain by base complementary pairing.In the second reaction system,MB can be modified by the long chain DNA modified with hydrazine and dibenzocyclic octyne at the end,respectively,through specific recognition between streptavidin and biotin with high affinity,and by the two short chain DNA modified with different fluorescence groups.With the addition of lipopolysaccharides at different concentrations in the first reaction system,the recognition chain modified with azide group can be dissociated into the solution which can subsequently be transferred into the second reaction system.Meanwhile,the aldehyde group modified recognition chain and furfural was added into the second reaction system.The two recognition chains are combined with long DNA on the MB by clicking chemistry and hydrazone chemistry respectively,resulting in the occurence of DNA hybridization.In the presence of the Nicking enzyme,the short chain DNA is continuously cut,resulting in the happening of continuously amplified fluorescence signal in the solution.For the DNA Walker system for single-target detection,there was a linear correlation between fluorescence signal intensity and LPS in the concentration range of 10^-44 ng/mL to 10⁷ ng/mL,with the minimum detection limit of 91 fM.This system shows high sensitivity and could be applied to the actual sample analysis of LPS.For the double-target DNA Walker system,the quantitative analysis of LPS and furfural can be performed,where the linear detection range of LPS is 10^-2 ng/ml-10⁷ ng/mL and the linear detection range of furfural is 1mM-50 mM.This method can be used to detect LPS and furfural in food with an important application prospect.