Novel Electrochemical Biosensing Based On DNA Tetrahedron And Signal Amplification Technology

In recent years,cancer and infectious diseases have become two major killers,which threatening life and health of human beings.The concentrations of their biomarkers are extremely low,especially in the early stage.The rapid and sensitive detection of their biomarkers is of great significance to achieve early diagnosis and treatment of them.Electrochemical biosensors have attracted widespread attention for its simple operation,high sensitivity,low cost and easy miniaturization.The highly ordered surface and effective signal conversion and amplification go hand in hand with the sensitivity and accuracy of the biosensor.DNA tetrahedrons are relatively simple nanostructures,which can provide rigid support for identification elements,control the density and orientation between identification elements,and improve their utilization efficiency.In this thesis,by using of DNA tetrahedron and signal amplification strategies,new type of electrochemical biosensors are constructed,which are employed to reduce the background signal and increase the detection signal simultaneously,to achieve high sensitive and effective detection of ultra-low abundance biomarkers.The main researches in this thesis are as follows:1.Herein,a label-free and double recognition-amplification(LDRA)strategy for carcinoembryonic antigen(CEA)detection was developed,based on a new designed dual-function messenger probe(DMP)coalescing with DNA tetrahedron probes(DTPs)and hybridization chain reaction(HCR).The DMP possesses dual-function to replace CEA for specific interface hybridization and initiate hybridization chain reaction.The interfacial hybridization event was quantitatively converted to an electrochemical signal by using hemin/G-quadruplex(h-Gx)formed after the hybridization chain reaction.Self-assembled DNA tetrahedron probes,which were readily decorated on an electrode surface as a scaffold with rigid support and ordered orientation,enabled the highly efficient strands hybridization and greatly increased target accessibility as well as significantly decreased noise.The proposed assay integrated dual recognition processes and HCR signal amplification processes,achieving the identification of low concentration of CEA as detection limit of 18.2 fg/mL(S/N=3)and wider linearity range of 0.0001 ng/mL-50 ng/mL.This new electrochemical sensing method was proposed for CEA detection and used in real clinical samples.The obtained results were good consistency with those of clinical diagnosis.2.In experiments,we found that new DNA tetrahedrons need to be redesigned and synthesized for different targets,which is likely to cause a decrease in their purity and yield.In response to this problem,we designed and synthesized graft-type DNA tetrahedrons.The recognition sequence is transferred to a shorter bridge probe.Herein,a robust and highly ordered three-dimensional electrochemical DNA biosensor was proposed,and its orientation was controlled from top down by poly adenine oligonucleotides(polyA-ODNs)mediated rolling motor(PRM)and graftable tetrahedron DNA(GTD).The GTD with a grafting domain was immobilized on the electrode surface to construct a well-organized sensing interface and controlled the orientation and distribution of the whole system at the "bottom" of this biosensor.The polyA-ODNs regulated the direction and density of the leg DNA attached on PRM at the "top" of the biosensor.The motion was achieved through the target induced cyclic cleaving,which triggered the motor rolling rather than walk.Impressively,the duplex strand DNA formed after grafting,as a girder,provided a stable support to the soft long single strand,which facilitated the formation of the catalytic center,elevated the efficiency of the rolling cleavage.Under the optimal conditions,this method exhibited a lower detection limit of 0.17 nM and wide linear range from 0.5 nM to 1.5μM for adenosine rapid detection.Unique dual orientation regulated characteristics of this system increased the probability hybridization enormously and improved the motion efficiency significantly,which offered new avenue of DNA nanomachines development in biosensor platform.3.In order to improve the detection efficiency of the sensor,shorten the detection time,and achieve high-sensitivity detection of different targets at the same time,a multi-task processing system is constructed.As the amount of tasks increases,the analysis space will become more limited,and the background signal will increase accordingly.How to carry out effective signal conversion to achieve high-sensitivity detection of different targets is a problem.In this work,a grafting homogenous electrochemical biosensing strategy is proposed by integrating of GTD,reverse proximity ligation and exonuclease Ⅲ(Exo Ⅲ)assisted target circulation to analyze hepatitis B(HBV)and human immunodeficiency(HIV).The linear range of this method is 1 fM to 100 pM,and the detection limits are 0.32 fM and 0.18 fM,respectively.Compared with the conventional strategies,the grafting homogenous electrochemical biosensing strategy not only achieved convenient sensitive detection of multiple communicable diseases DNA simultaneously,but also performed well in the detection of sole target.This strategy effectively decreases the background,homogenizes the distribution of probes,and avoids the complex and time-consuming modification process of the working electrode,which holds great potential application in early diagnosis for communicable disease in the future.4.DNA tetrahedrons can not only be used as base materials,but also directly used as DNA nanomachines.The motion mode of DNA machines goes hand in hand with its moving efficiency.Since both semifixed and completely free modes show unsatisfactory efficiency,there is an urgent need for a machine that maintains free movement while being immobilized on the electrode surface.In this work,a free lateral motion multi-pedal DNA tetrahedron machine(MTM)was designed to improve the mentioned shortages and applied to construct a biomimetic electrochemical ratiometric strategy for ultra-sensitive target DNA analysis.The biomimetic interface was constructed by decorating lipid bilayer on the electrode surface.The MTM has a cholesterol group at one vertex and overhanging blocked swimming arms at other three,ensuring its moving freely without derail from the system.The ingenious design of target circulation process converted finite number of targets to plenty secondary promoters,which triggered MTM to swim.The methylene blue tagged hairpin fueled probe provided constant power to support MTM swim freely on lipid bilayer by liberating pedals of MTM.By this way,a new motion mode,free lateral movement of DNA machine was demonstrated,improving the moving efficiency and shorting the time.In addition,the dual reporters employed in this work elevate accurate detection of target.The linear relationship between the ratio value of two reporters and target DNA concentration was observed from 50 aM to 10 pM with a detection limit of 1.2 aM.This strategy holds great potential in electrochemical detection of analytes with wide range and be a biosensing platform for early clinical diagnosis and biomedical research.5.In the previous system,we found that in order to realize the continuous movement of the walker,additional energy input is often required,which complicates the detection procedure.In addition,the rate of branch migration in the walker process has a close relationship with its walking efficiency.To solve these problems,we proposed a new hybridization chain walker(hc-walker)mechanism,which was combined with DNA grid tetrahedron to build an electrochemical aptamer sensor for the detection of infectious disease HBV.The hairpin components of hybrid chain reaction were integrated into a single DNA tetrahedron nanodevice as DNA tracks.The target induced hc-walker reaction directly,and at the same time the signal probe branches and migrates away from the sensing interface.The DNA grid tetrahedron provided rigid support for the hairpin components,and adjusted its density and orientation spatially,so that the DNA track was highly ordered and maintained an appropriate distance,improving the hybridization efficiency between DNA probes and the branching migration rate of signal probes.This strategy was enzyme-free,simple and easy to operate and avoided the addition of additional drive chains in traditional walkers.It realized the rapid detection of HBV with a linear range of 5 fM-100 pM and a detection limit of 1.27 fM.It opens up a new way for the sensitive detection of various biomarker signal amplification.