DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide ...DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme.Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes,the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores.Thus,it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection.Herein,we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1(FEN1),human alkyladenine DNA glycosylase(hAAG)and uracil DNA glycosylase(UDG)based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence(CE-LIF)detection.The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation.The method demonstrated a detection limit of 0.07 U/mL(0.08-160 U/mL)for FEN1,2.40 U/mL(2.5-250U/mL)for hAAG and 2.1×10^(-4)U/mL(0.0004-2.5 U/mL)for UDG,the relative standard deviations(RSDs)of peak time and peak area for different analytes were as follows:2.50%-4,37%and 3.24%-7.18%(inter-day);1.37%-2.71%and 1.43%-3.02%(intra-day),4.28%-6.08%and 4.16%-7.57%(column to column),respectively.And it can identify the inhibitor-like drugs,evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts,providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.展开更多
The design and manufacturing of microchannels are crucial aspects of modern micro/nanomanufacturing processes,offering a versatile platform for manipulating and driving micro/nanoparticles or cells.In this study,we pr...The design and manufacturing of microchannels are crucial aspects of modern micro/nanomanufacturing processes,offering a versatile platform for manipulating and driving micro/nanoparticles or cells.In this study,we propose a method for manufacturing microchannels using optically induced dielectrophoresis technology to induce the polymerization of polyethylene glycol diacrylate solution.To overcome limitations related to the light intensity energy and the size of intact microchannels,we design and manufacture microstructures of various shapes with a height of 4µm.Additionally,we simulate and analyze the movement of and forces acting on polystyrene(PS)microspheres at different spatial positions within the microchannels.Finally,we successfully demonstrate applications involving the transport of PS microspheres in custom-fabricated microchannels.This novel biocompatible microchannel manufacturing method is simple and non-biotoxic.It provides a new approach for simulating physiological environments in vitro and cultivating and manipulating cells.展开更多
基金supported by the National Natural Science Foundation of China(Nos.21874060 and 22174058,U21A20282)the Science and Technology program of Gansu Province(No.22JR5RA476)。
文摘DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme.Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes,the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores.Thus,it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection.Herein,we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1(FEN1),human alkyladenine DNA glycosylase(hAAG)and uracil DNA glycosylase(UDG)based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence(CE-LIF)detection.The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation.The method demonstrated a detection limit of 0.07 U/mL(0.08-160 U/mL)for FEN1,2.40 U/mL(2.5-250U/mL)for hAAG and 2.1×10^(-4)U/mL(0.0004-2.5 U/mL)for UDG,the relative standard deviations(RSDs)of peak time and peak area for different analytes were as follows:2.50%-4,37%and 3.24%-7.18%(inter-day);1.37%-2.71%and 1.43%-3.02%(intra-day),4.28%-6.08%and 4.16%-7.57%(column to column),respectively.And it can identify the inhibitor-like drugs,evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts,providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.
基金funded by the National Natural Science Foundation of China(Project No.62273289)The Youth Innovation Science and Technology Support Program of Shandong Province(Project No.2022KJ274)+1 种基金Natural Science Foundation of Shandong Province(Grant No.ZR2024MF007)Graduate Innovation Foundation of Yantai University,GIFYTU.
文摘The design and manufacturing of microchannels are crucial aspects of modern micro/nanomanufacturing processes,offering a versatile platform for manipulating and driving micro/nanoparticles or cells.In this study,we propose a method for manufacturing microchannels using optically induced dielectrophoresis technology to induce the polymerization of polyethylene glycol diacrylate solution.To overcome limitations related to the light intensity energy and the size of intact microchannels,we design and manufacture microstructures of various shapes with a height of 4µm.Additionally,we simulate and analyze the movement of and forces acting on polystyrene(PS)microspheres at different spatial positions within the microchannels.Finally,we successfully demonstrate applications involving the transport of PS microspheres in custom-fabricated microchannels.This novel biocompatible microchannel manufacturing method is simple and non-biotoxic.It provides a new approach for simulating physiological environments in vitro and cultivating and manipulating cells.
