Fluorescent Biosensor Based On Signal Amplification Of 3-D DNA Walker Technology

The early diagnosis of cancer is of great significance to improve the cure rate of tumor.Especially,detection of tumor markers is very important and practical for early tumor screening,diagnosis,prognosis,etc.Fluorescent biosensor as a mature detection means has been widely used for tumor detection due to its excellent characteristics,including quick detection,high stability,etc.DNA walker can move on the predetermined orbit through dynamic interaction and thereby directionally transmit and amplify the detection signals.This thesis focuses on fluorescent biosensor systems based on DNA walker technology to identify the tumor markers.The main research contents of this manuscript are as following:1.DNA Walker on Live Cell Membrane for Evaluation of Cell Surfaces(Chapter 2)Membrane proteins play an important role in receptor recognition,signal transduction,etc,so in situ imaging analysis of membrane proteins is very important to understand specific proteins and identify the physiological state of cells.In this work,DNA walker based on the catalytic hairpin assembly(CHA)on live cell membrane was developed for high sensitivly imaging membrane proteins.the hydrophobic cholesterol modified DNA hairpin H1 was fixed on the cell membrane by hydrophilic or hydrophobic interaction,and the fluorescence group FAM and quenching group BHQ1 modified DNA hairpin H2 was dispersed in the solution;the double catalyst was brought to the surface of cell membrane by the recognition between aptamer sgc8 and membrane protein;The DNA catalyst can bind to a toehold on H1 and open the hairpin H1 via branch migration.A newly exposed single-stranded region within the H1 will bind to H2 dispersed in solution,forming an unstable tripartite complex.Ultimately,the catalyst is dissociated form the tripartite complex,resulting in the thermodynamically stable construction,the H1: H duplex,and then participate the next reaction cycles.The open of hairpin H2 would separate FAM and quencher,so the targeting cell would lighted by FAM.The design can retain the activity of receptor protein during reaction and will be applied to study on other proteins by simple replacement of aptamer,so it has good versatility and is applied to different biological specimens,such as living cells,tissues,zebrafish,etc.2.Highly sensitive detection of telomerase via Toehold mediated DNA walker on Au nanoparticles(Au NPs)surface(Chapter 3)Here,a DNA walker on surface of Au NPs for detection of telomerase activity in live cells was designed.Priming strand and DNA1 were assembled onto the surface of Au NPs through Au-S bonds.FAM fluorescent labeled at 5’ end of DNA was used as a reporter probe.DNA1 and DNA2 were hybridized with the 5’ end and middle moiety of the reporter probe respectively to fix the reporter probe on the Au NPs surface.The fluorescence group was close proximity to the Au NPs surface,resulting in fluorescence quenching of FAM.In the presence of the target telomerase,the telomerase priming strand can be extended.The extended DNA sequence was hybridized with the exposed part of the report probe.Driven by Fuel strand,the reporter probe was displaced into the solution through Toehold mediated strand replacement reaction(TSDR),resulting in fluorescence recovery.This method can analyze and detect various substances by designing different DNA recognition sequences.