Study On Electrochemical Biosensor For Analysis Of MicroRNAs And Thrombin Based On Toehold-mediated DNA Strand Displacement Reactions

With the rapid development of gene diagnosis and treatment,mechanism of drug action and new drug selection,environmental monitoring and food safety assessment other research fields,it is very important to achieve the simple,rapid and highly sensitive and high specificity detection of pathogenic proteins and specific sequences of microRNA(miRNA).DNA electrochemical biosensor has the advantages of simple structure,low cost,high sensitivity and good selectivity,which has become attractive and challenging in biochemical analysis and medical field.The Toehold-mediated strand replacement reaction(TSDR)is the basis for the construction of dynamic DNA nanomachines.In this paper,three kinds of electrochemical DNA biosensors for highly sensitive detection of microRNA(miRNA)and thrombin were proposed based on TSDR-mediated target recycling strategy for signal amplification.The specific research works were depicted as follows: Part 1 DNA-fueled molecular machine enables enzyme-free target recycling amplification for electronic detection of microRNA from cancer cells with highly minimized background noiseThe variations in microRNA(miRNA)expression levels can be useful biomarkers for the diagnosis of different cancers.In this work,on the basis of a new miRNA-triggered molecular machine for enzyme-free target recycling signal amplification,the development of a simple electronic sensor for highly sensitive detection of miRNA-21 from human breast cancer cells is described.The three-stand DNA duplex probes are self-assembled on the gold electrode surface to fabricate the sensor.The miRNA-21 target binds to the terminal toehold region of the probes,displaces one of the short strands and exposes the secondary toehold region for subsequent hybridization with the methylene blue(MB)-modified DNA fuel strand,which further displaces both the miRNA-21 target and the other short strand to activate the operation of the molecular machine.As a result,the miRNA-21 target is cyclically reused and many MB-DNA fuel strands are attached to the sensor surface,leading to significantly amplified current response for sensitive detection of miRNA-21 down to 1.4 fmol/L.The developed sensor also shows high sequence discrimination capability and can be used to monitor miRNA-21 expression levels in cancer cells.Moreover,this sensor avoids the involvement of any enzymes for target recycling amplification and features with highly minimized background noise for miRNA detection,which makes this method hold great potential for convenient monitoring of different miRNA biomarkers for early diagnosis of various cancers.Part 2 Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cellsThe monitoring of microRNA(miRNA)expression levels is of great importance in cancer diagnosis.In the present work,based on two cascaded toehold-mediated strand displacement reactions(TSDRs),we have developed a label-and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells.The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface.The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences.Subsequently,the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21.The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface,which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fmol/L.The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells.Part 3 Target-triggered catalytic hairpin assembly and TdT-catalyzed DNA polymerization for amplified electronic detection of thrombin in human serumsSpecific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications.In this work,a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly(CHA)and terminal deoxynucleotidyl transferase(TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums.The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3'-OH termini on the sensor electrode.Subsequently,TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and dATP at a ratio of 6:4.These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a completely label-free fashion.The sensor shows a linear range of 0.5 pmol/L to 10.0 nmol/L and a detection limit of 0.12 pmol/L for thrombin detection.Moreover,the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.