Studies On Fluorescence Detection Small Biomolecules And DNA Based On Enzyme Assisted Cycle Amplification

A biosensor is a device that converts target identification into a physically detectable signal,such as optical,electronic,or magnetic.Typically,the biosensor contains two substantially functional components: target recognition and signal transduction.In theory,identifying components is the key to sensor performance.Ideal recognition components should have the characteristics of high sensitivity,enviable selectivity,fast response,powerful performance and versatility of various goals.With the continuous development of biochemical analysis,a single biosensor has been unable to meet our needs.As the small molecules can not be directly amplified,the concentration of the test object is too low and other limitations,so vigorously develop the signal amplification technology has become the field of biochemical analysis of the main points of work.In this paper,we introduce the method of DNA enzyme cycle amplification and DNA self-assembly and amplification to realize the high sensitivity,high specificity,high selectivity and rapid direct detection of DNA and biological small molecules.Mainly includes the following three aspects:1.Analysis of DNA methylation and MTase activity is important in the early clinical diagnosis of cancer in order to provide insight into the mechanism of gene arrest and to develop new drugs for the treatment of methylation-related diseases.A new fluorescence method for the detection of DNA methyltransferase was constructed by the combination of dendrimer roll amplification and DNasespecificity.In the presence of DNA methyltransferase(MTase),the double-stranded DNA probe was methylated to generate a specific recognition site for the restriction endonuclease DpnI.The cleaved hybrid DNA probe then acts as a signal primer to initiate the dendritic amplification reaction.Subsequently,the roll loop amplification produces a complementary sequence with Mg2+-dependent DNase,releasing the fluorescent signal in the presence of Mg2+.The detection limit of DNA methylation was 0.36 U/mL and the linear range was 1.0~10 U/mL.In addition,the use of this method can assess and screen for MTase inhibitors,which may help to detect anticancer drugs.2.Based on the three-way cross-structure and self-assembly cycle amplification technique,the DNA was detected by the target DNA triggering three-way(3-WJ)structure and the polymerase cleavage of DNase,and the DNA self-assembly technique(CHA)Secondary amplification,the design of a highly specific,sensitive,rapid DNA detection methods.CHA,as an enzyme signal amplification technique,has been used several times in a protocol for catalytic expansion.The target DNA binds closely to the 3-WJ primer and the 3-WJ template to form a stable 3-WJ structure,which forms a primer chain under the action of DNA polymerase and chimeric enzymes.These primer chains contain a multi-stage catalytic probe self-,Can be complementary hybrid way to open hairpin probes H1 and H2,triggering self-assembly reaction,the signal secondary amplification.After the reaction,the fluorescent probe was released,and the high sensitivity and high selectivity of the target DNA were realized by two-step amplification technique.3.Based on the tyramine signal amplification new immunosensor for highly sensitive detection of thrombin.The system uses the specific binding of thrombin and its two aptamers to connect the magnetic microspheres with AuNPs to form a composite structure of magnetic microspheres-thrombin-AuNPs.At the same time,tyramine and horseradish peroxidase(HRP)binding,and the previous complex connected to form a signal amplification.HRP catalyzes the reaction substrate hydrogen peroxide and p-hydroxyphenylacetic acid.Through the detection of fluorescent signals,to achieve the sensitivity of tumor markers thrombin detection.