Fluorescence Analysis Method To Detect The Activity Of DNase Ⅰ And The Study Of A549 Cells And E. Coli O157:H7

Deoxyribonuclease I is an important part of biological structure and life activities.Its research has extensive and important application prospects in the clinical diagnosis of medical diseases,drug development,industrial production and so on.Through the detection and analysis of the activity of hydrolases and their inhibitor,access to important biological information,which can effectively play an enzyme in various fields of application value and significance.In addition,cancer cells and bacteria,as one of the factors that cause serious harm and loss to human lives and property,are in urgent need of research to reduce their perniciousness.At present,the analysis methods for detecting hydrolases,cells and bacteria are various and have their own characteristics.The fluorescence analysis method is one of those methods,which has become a kind of very simple detection method because of its advantages of small background interference,high sensitivity,etc.There is great potential for development of analytical methods like this.In this paper,the activity of DNase I,A549 cells and Escherichia coli 0157:H7 were detected by fluorescence spectroscopy and fluorescence polarization,respectively.Two simple,rapid,sensitive and specific methods for detecting DNase I activity,cells and bacteria were constructed,especially,and enzyme activity inhibitors and real samples were also studied.This paper was composed of four parts.The chapter 1 is an introduction.It is composed of three parts.The first part is an overview of DNase I,which mainly introduces nuclease and DNase I and their detection methods.In addition,the preparation and application of DNA hydrogel are summarized.The second part is the overview of cancer cells and pathogens,mainly introduces the biological characteristics of A549 cells and E.coli 0157:H7,besides pathogenicity,clinical manifestations and research progress of the detection methods;the third part mainly illustrates the background,research purposes and research content of the thesis.The chapter 2 studied the DNase I activity,by preparing DNA hydrogels embedded with quantum dots and nanoparticles as the detection system and fluorescence quenching for the detection method.Firstly,the hydrogel was self-assembled by hybridization of complementary DNA strands,and quantum dots and nano-particles were simultaneously added into the gel at the formation process to prepare DNA hydrogels with biological characteristics.Then DNase I was added to DNA hydrogel system containing fluorescent quantum dots.After the hydrolysis of DNase I,fluorescence quenching would occur and the fluorescence intensity of the reaction system would be changed because of the relative distance change between the quantum dots and the nanoparticles,the degree of attenuation of the fluorescence intensity in the process was used to detect the activity of DNase I.The linear range of fluorescence intensity to DNase I concentration was 20 U/L～4000 U/L,and the limit of detection was 13 U/L(S/N=3).The method was used to detect DNase I activity with high sensitivity,and it was easy to operate.In addition,we investigated not only the activity of DNase I,but also the inhibitors affecting the catalytic activity of DNase I.The chapter 3 used fluorescence polarization to detect A549 cells.In this chapter,we constructed a fluorescent labeled DNA strand as aptamer probe to detect cells.The fluorescent probe was an aptamer DNA strand formed of a 45-base single strand DNA labeled with a fluorescent group FAM.Before the targets were added,the FAM-labeled single strand DNA was polarized due to its relatively small molecular weight.When the light passed through the detection system,the corresponding fluorescence polarization was also relatively small.When the target A549 cells were added to the system,the probes with the FAM group captured the cells and bound themselves to the surface due to the specific recognition of aptamers and cells.At this time,the molecular weight of the aptamer probe increased.The corresponding fluorescence polarization value was accordingly increased,and A549 cells were detected based on the change in fluorescence polarization in the process.The linear range of the fluorescence polarization and the concentration of A549 cells in this method was 102 CFU/mL to 106 CFU/mL,and the detection limit was 83 CFU/mL(S/N=3).The method had the advantages of high sensitivity,good selectivity,etc.It could not only efficiently detect A549 cells,but also had a good effect in the analysis of actual samples.The chapter 4 detected E.coli 0157:H7 by fluorescence polarization.This chapter also constructed a method for detecting bacteria using FAM-labeled DNA as fluorescent probe.The probe was also aptamer formed by FAM-labeled 52-base single strand DNA,the E.coli 0157:H7 was detected based on changes in fluorescence polarization before and after addition of bacteria.The linear range of fluorescence polarization change and E.coli 0157:H7 concentration in the method was 103 CFU/mL～107 CFU/mL,and the detection limit was 622 CFU/mL(S/N=3).As such,the method exhibited a higher selectivity in the rapid and highly sensitive detection of E.coli 0157:H7 and actual sample studies.