Study On New Methods For The Detection Of Acid Modifying Enzymes Based On Surface-enhanced Raman Scattering And Fluorescence Sensing Technology

Optical biosensing technique has the advantages of short analysis time,good selectivity,simple operation and high sensitivity.It has been widely used in the detection of protein,nucleic acid,bioactive small molecule and cell,and became an important technique in the filed of chemical and biological analysis.The gene modification process is closely related to cancer,and the gene modification process is regulated by various nucleic acid modifying enzymes.The nucleic acid modifying enzymes hold the potentials to be the biomarkers and therapy target of cancer.Therefore,developing new methods for the detection of nucleic acid modifying enzymes is of great importance in cancer diagnosis and therapy.Nucleic acid signal amplification technology provides technical support for achieving highly sensitive detection and has been widely used to build a variety of biosensors.In this thesis,combining with optical biosensing technique and nucleic acid signal amplification,we developed three optical biosening methods for the detection of uracil-deoxyribonucleic acid glycosylase(UDG),apurinic/apyrimidinic endonuclease 1(APE1),Dam methyltransferase(Dam).The details are as follows:Part one:This work developed a surface-enhanced Raman scattering(SERS)method for the simple,sensitive and homogeneous detection of UDG activity based on silver nanorods(AgNRs).It utilizes the different adsorption properties of single-strand DNA(ssDNA)and double-strand DNA(dsDNA)on the metal surface.UDG-catalyzed uracil removal from the dsDNA-structured substrate will cause the separation of the substrate into two ssDNA probes,which were free to be adsorbed on the AgNR surface.The FAM dye labelled on the substrate would be close to the metal surface,achieving a strong SERS signal.This method is sensitive with a 0.003 U mL/L detection limit due to the high enhancement factor of AgNRs.The proposed approach also could be used for evaluating the inhibition of UDG activity.This method may have potential applications in UDG-related biological research.Part two:A label-free fluorescent assay for the detection of apurinic/apyrimidinic endonuclease 1(APE1)activity was developed based on the polymerase and nicking endonucleases co-assisted isothermal amplification and G-quadruplex-liagand interaction.APE 1-catalyzed the cleavage of DNA substrate will trigger the isothermal amplification,liberating numerous of G-quadruplex.N-Methyl Mesoporphyrin ?(NMM)dye will interact with G-quadruplex to produce strong fluorescence signal.This method displays a high sensitive for APE1 activity with a detection limit of 0.006 U/mL.This method has a potential application in cancer diagnosis and other APE1-related biological researches.Part three:In this work,we constructed a label-free and dual-amplified fluorescence method for sensitive analysis of DNA methyltransferase based on exonuclease ?(Exo?)-assisted DNA cycling and hybridization chain reaction(HCR).After the catalysis reactions of DNA methyltransferase and methylation dependent restriction enzyme,DNA fragements for inducing Exo ?-assisted DNA cycling were released.Another DNA fragment was generated and then triggered the HCR amplification.After that,multiple G-quadruplex-contained HCR products were obtained.Finally,the G-quadruplex structures bonded with the NMM which yielded an enhanced fluorescence signal,realizing the label-free detection.This method has a high sensitivity to the activity of DNA methyltransferase with a limit of 0.1 U/mL.It also can be used for studing analysis of the inhibitor of DNA methyltransferase.This work may provide usful technical reference for DNA methyltransferase-related disease diagnoise and medical research.