Preliminary Establishment And Research Of Biosensors And Droplet Microfluidic System For Nucleic Acids Detection

Vitro amplification and detection of nucleic acid take an important part in pathogen analysis,molecular diagnosis,tumor early screening,environmental monitoring,forensic identification and many other fields.There is an increasing demand in developing micro-convenient,simple,sensitive,efficient,low energy and reagent consumption,low-cost analysis means.Isothermal amplification acquired development unceasingly and is applied more in nucleic acid detection,because it can achieve more specificity,high sensitivity and low cost nucleic acid analysis without using complicated device for temperature control.Nucleic acid detection technologies that combined with electrochemical biosensors and droplet microfluidic chip have the potential of being greater detection systems,because they have the advantages of both electrochemical biosensors,which include simple to operate,responsive and droplet microfluidic chip,which include low regent consumption,high droplet capacity,avoidance of contamination among droplets.In this paper,we have studied the performance of electrochemical DNA sensor based on gold electrode on polystyrene sheet in nucleic acid detection,and developed a nucleic acid detection system based on droplet microfluidic technology,combined with isothermal amplification technology to detect DNA effectively.This paper is divided into four chapters;the main contents are as follows:In Chapter One,we reviewed the current methods of nucleic acid detection and their main applications,and summarized the principle of electrochemical biosensor and droplet microfluidic technology and their applications in nucleic acid detection field.In Chapter Two,we used polystyrene plate instead of the traditional material-glass plate as the substrate,the gold electrode was fabricated by chemical plating technology.The electrochemical characteristics and the ability to immobilize the SH-DNA probe of this gold electrode were compared with gold electrode on glass substrate made by the sputtered photolithography.We explained the principle of making polystyrene substrate gold electrode and possible factors which account for its better ability to immobilize the SH-DNA probe.The polystyrene substrate gold electrode was applied in DNA immobilization and hybridization,the detection limit was 7.2×10-11 M(S/N=3).After all we provide a good electrodes fabrication way that is worthy to be popularized.In Chapter Three,we made the PDMS-PDMS microfluidic chip to form the micro-droplets,we designed the three-hot-block heating device with the groove and the snake-like capillary channel to carry out the HRCA reaction,and we set up the detection platform based on the fluorescence detection technology to establish a system of nucleic acid amplification and detection.This system was used to amplify and detect DNA,the detection limit was 7.5 X 10-10 M(S/N =3).The heating device and the heating channel material of the system could be optimized to be capable of being used in PCR,and has a greater range of applications;droplet preparation microfluidic chip and detection system could be improved for their potential to be applied in digital PCR or digital RCA detection system.In Chapter Four,the summary and prospect were stated.We will combine electrochemical DNA sensor and droplet microfluidic technology for nucleic acid detection in the next work,with the hope to make advantages of both to detect nucleic acid more effectively and conveniently.