Study On The Electrochemical Biosensor Based On DNA Walker In Detection Of Disease-related Biomarkers

Biomarker researches serve a wide range of purposes in drug development,clinical trials,and therapeutic assessment strategies.For many diseases,early diagnosis is essential.In recent years,with the development of immunology,molecular biology and genomics technology,it is very important to achieve the simple,rapid,highly sensitive and high specificity detection of disease-related biomarkers.Electrochemical biosensors have been developed by integrating electronic transduction elements with biological recognition elements.On one hand,the electronic elements provide highperformance electrochemical biosensing platforms for detecting target molecules with high sensitivity and rapid response.On the other hand,the biorecognition elements contribute to the high selectivity of the biosensors based on the specific recognition of biomolecules.These merits make the electrochemical biosensors widely used in diverse fields,such as biochemical analysis,environment monitoring,food safety control,and so on.DNA walkers are a unique class of dynamic DNA devices that move nucleic acid walkers processively along designated one-,two-,or three-dimensional tracks.As biosensors,DNA walker nanomachines have more flexible and versatile design principles compared with traditional biosensors,and of which the features of progressivity and movability enable to achieve higher sensitivity and lower limit of detection.Base on DNA walker nanomachines,the following works were completed in this thesis:1.An enzyme-free and label-free signal-on aptasensor based on DNAzyme-driven DNA walker strategy:A signal-on electrochemical aptasensor for highly sensitive detection of thrombin（TB）was constructed based on the DNAzyme-driven DNA walker strategy.A new dual functional hairpin DNA（HP）containing a substrate sequence of the Mg²⁺-dependent DNAzyme（in the loop region）and the G-quadruplex forming segment（in the stem region）was developed.The DNA walker（TBA₂-DWs）,containing a TB aptamer and an enzymatic sequence,was introduced onto gold electrode（GE）by aptamers-target specific recognition,and thus initiated the enzymatic sequences to hybridize with the substrate sequence.Then,the DNA walker could repeatedly bind and cleave HP in the assistance of Mg²⁺,unlocking many active G-quadruplex forming sequences.Finally,hemin can further bind the G-quadruplex to form G-quadruplex/hemin complexes and generate enhanced current output.The aptasensor for TB assay showed a linear detection range from 1 p M to 60 n M with a lower detection limit of 0.58 p M.And more,the proposed detection strategy was enzyme-free and label-free2.A“signal-on”electrochemical biosensor based on DNAzyme-driven bipedal DNA walkers and Td T-mediated cascade signal amplification strategy:A dual amplified signal enhancement approach based on coupling deoxyribozyme（DNAzyme）-driven bipedal DNA walkers（BDW）and terminal deoxynucleotidyl transferase（Td T）-mediated DNA elongation signal amplification has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums.In presence of thrombin,the BDW complex,which is comprised from the target thrombin and two DNAzyme-containing probes,can exhibit autonomous cleavage behavior on the surface of the substrate DNA（SD）modified electrode,and remove the cleaved DNA fragment from the electrode surface.Subsequently,the Td T can catalyze the elongation of the SD with free 3′-OH termini and formation of many G-quadruplex sequence replicates with the presence of2’-deoxyaguanosine-5’-triphosphate（d GTP）and adenosine 5’-triphosphate（d ATP）at a molar ratio of 6:4.These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a“signal-on”and completely label-free fashion.Under optimized conditions,the response peak current was linear with the concentration of thrombin in the range from0.5 p M-100 n M with detection limit of 0.31 p M.This research provides us a sustainable idea for the hyphenated multiple amplification strategies and a stable and effective method for the detection of protein biomarkers.3.A electrochemical sensing strategy for one-step determination of micro RNA based on“cleated”DNA walker:Many types of DNA walker were develpoed in resent years.Howerver,as the substrate DNA was exhausted;these applied walkers may be prone to release prematurely or to stall in a region of locally depleted substrate.To overcome this deficiency,we have modified the walker strategy to create an even simpler single-legged walker with a“cleat”to prevent its dissociating from the track.It shows that the walker amplification strategy improves the signal amplification effect,thereby achieving the purpose of simple and sensitive detection of mi RNA.Under the best experimental conditions,the response current of biosensor has a good linear relationship with the logarithm of the mi RNA-141 concentration in the range of 0.5 f M to 5 p M.4.Double walking signal amplification strategy based on three-dimensional“cleated”DNA walker for electrochemical ultra-sensitive detection of mi RNA:The three-dimensional DNA walker has many advantages in the application of electrochemical biosensing,such as high walking efficiency,strong signal amplification ability,and effective improvement of sensor sensitivity.In this work,we designed a three-dimensional DNA walker based on gold nanoparticles.The three-dimensional DNA walker is triggered by the target mi RNA and driven by DSN,which enables the target mi RNA continuously and efficiently walking and cutting H1.After that,the product can be used as a multi-leg DNA walker,which can efficiently walk and cut the substrate nucleic acid on the surface of the gold electrode under the driving force of DSN,releasing the Fc labeled on the H2.Under optimal conditions,the electrochemical signal generated by Fc decreases linearly with the logarithm of the mi RNA-141 concentration in the range of 0.1 f M to 200 f M.The reaction of mi RNA and three-dimensional DNA walker in a homogeneous solution can effectively protect RNA from degradation.In addition,the DSN added at one time drives two walk motions.