Detection Of Nucleic Acids And Proteins Based On Magnet Beads,Signal Amplification And Chemiluminescence

Due to the importance of nucleic acids and proteins in our life,the high sensitive detections of them are of great importance.Chemiluminescence(CL)is used for the detection of nucleic acid and protein as a result of its high sensitivity and wide linear range.However,it is limited for ultra-trace analysis.And signal amplification would be effective to solve the problem.Some classical signal amplifications have many shortages,for example,requires annealing so many times and adding a plenty of enzymes.In addition,the homogeneous detection is easily influenced by the impurities which would lead high background,even cause false positive.To overcome these problems,we have combined CL and magnetic separation with isothermal signal amplification,such as catalytic hairpin assembly(CHA),isothermal strand polymerase reaction(ISDPS)and hybridization chain reaction(HCR)which not need annealing.And a series of CL methods have been developed for the detections of nucleic acids and proteins by combing isothermal signal amplification and magnetic beads.The detail contents are as follows:1.Two kinds of DNA walker sensors with different legs based on CHA have been constructed in this study.A toehold on MMPs surface-bound H1 could interact with target DNA and the hairpin be opened via toehold-mediated strand exchange.And then a newly exposed DNA segment within H1 would hybridize to a toehold on biotin-labeled H2.As strand displacement proceeds,the target DNA releases from the complex can participate in subsequent reaction cycles to achieve the target recycling,which is like a walker moves on the surface of MMPs.The cause of the phenomenon that their limits of detection were different was explored.Besides,it's expanded to the detection of HIV sequence.What's more,this DNA walker method has been applied for the detection of T4 polynucleotide kinase activity successfully.2.In order to apply DNA walker for protein detection,multi-pedal DNA walker biosensors based on CHA for the chemiluminescent detection of streptavidin are constructed in this study.In the presence of SA,multi-pedal DNA walker has been constructed by biotin-modified catalyst as a result of the terminal protection for avoiding the digestion by exonuclease I.Then a 'leg' of multi-pedal DNA walker could interact with a toehold of CHA-H1 conjugated with MMPs and opens the hairpin via toehold-mediated strand exchange.A toehold of biotin-labeled H2 could be hybridized with the newly exposed DNA segment in CHA-H1.Via the strand displacement process,one 'leg' of multi-pedal DNA walker has been displaced,and the other 'leg' could still hybridize with neighboring H1 to initiate the next cycle and move on the surface of MMPs.In order to solve the high background of CHA DNA walker,the other model based on ISDPR has been designed.Unlike CHA,ISDPR relies on the extension of a short primer,and successfully reduce the background signal.3.A novel DNA walker sensor was designed which is based on ISDPR,and the behaviors of it walking on obstructive surface of magnetic beads have been studied.The DNA strands which could prevent the movement of DNA walker,is called block.While the track is full of plenty of blocks,stereo hindrance would affect the movement and speed of DNA walker.The phenomena have been studied by coupling with different length or different concentrations of blocks on the surface of MMPs,and the speed of DNA walker could be controlled.Besides,under proper length,the signal-to-noise ratio even has been improved and this consequence is of great significance for improving the sensitivity of DNA detection.Furthermore,the DNA walker sensor also has been applied for DNA detection.4.In order to improve the sensitivity of protein detection,an ultrasensitive CL biosensor for the detection of thrombin is developed in this study based on HCR.First,the MMPs bound thrombin aptamer could hybridize with the primer.Then part of the primer triggered HCR which is a kind of signal amplification.In the presence of thrombin,the HCR products assembled on the surface of the MMPs were released after the aptamer recognized and bound to its target molecule.After the addition of H2O2-luminol,a significant CL imaging signal could be produced by the released HCR products in the supernatant.The detection limit of thrombin has been calculated be as low as 9.7 fM.The proposed method couldn't be applied in complex environment,so the sensing strategy was modified by changing the primer and the adding order of reagents.In the presence of thrombin,a sandwich structure was formed through the binding between thrombin and two aptamers.Then the remaining segment of primer triggered the HCR.As the HCR products were existed on the surface of MMPs instead of the supernatant,so the interferences from the supernatant could be avoided,this was favor to detect thrombin in complex biological environment.