| With the development of genomics and clinical medicine,the understanding of the cellular biomolecular interaction networks and the key physiological pathways of some diseases has been deepening.In the course of disease development,the content of certain biomolecules in human body fluids will undergo abnormal changes.Such biomolecules are collectively referred to as biomarkers,which can be used to indicate the progress of the disease and assess the severity of the disease.Therefore,the detection of small numbers of biomarkers,including nucleic acids,small molecules and proteins,is essential for understanding their biological and physiological functions,as well as for clinical diagnosis and treatment of diseases.Biosensor is an analysis platform that integrates the dual tasks of bio-recognition and signal processing,which has the advantages of simple operation,high sensitivity,high specificity and so on.Its application in the early diagnosis,efficacy evaluation and monitoring treatment of disease has been widely concerned and explored by researchers.Since human body fluids are extremely complex and changeable,searching for target biomarkers in the bloodstream is like fishing for a needle in a haystack in the early stage of disease development.Therefore,the analytical performance of biosensor must be continuously improved.Electrochemical biosensor is the crystallization of multidisciplinary cross-fusion,and its development involves many fields such as chemistry,biology,microelectronics,medicine,materials science,etc.Compared with other biosensors that rely on large instruments,electrochemical biosensor exhibits unique advantages.It has the advantages of high sensitivity,fast response,instrument portability and low construction cost of electrochemical analysis technology,as well as the inherent high specificity of bio-recognition,and also has the the ability of easy miniaturization and multiplexing.Therefore,it has become a valuable analysis and detection tool.With the increasing demand for on-site sensing and point-of-care testing,higher requirements are put forward for electrochemical biosensing platform in terms of sensitivity,specificity,timeliness,and biosensor preparation methods.The combination of new methods and technologies with isothermal nucleic acid amplification strategies,functional materials,and microelectrode technologies has potential to provide effective answers to the detection standards of the electrochemical biosensing platform.This thesis aims to develop new biosensors in combination with electrochemical analysis technology,focusing on improving the sensitivity of the biosensors and simplifying the preparation of the biosensors.Mainly by designing new isothermal nucleic acid amplification strategies and combining functional materials with photoelectrochemical analysis techniques to construct electrochemical biosensors with high sensitivity.Purposing nucleic acid,enzyme and aptamer as biological recognition elements and designing dual recognition binding strategies to improve the specificity of biosensors.And simplifying the preparation steps of the biosensors by designing the immobilization-free sensing mode.In this paper,several electrochemical biosensors with excellent performance have been constructed to specifically and sensitivity detect disease-related small molecules,nucleic acids and proteins.In addition,the detection principles,feasibility,analytical performance and other aspects of the proposed electrochemical biosensors have been deeply explored.The main research contents of this paper are summarized as follows:1.Study on PTB7-Th/PAni Hs-based photoelectrochemical enzyme biosensorThree-dimensional polyaniline hydrogels（PAni Hs）are used to enhance the electron transfer rate and photoelectric conversion efficiency of the donor-acceptor photoelectrochemical active material（PTB7-Th）.Based on the competitive consumption of dissolved oxygen between the xanthine oxidase–guanine catalytic reaction and oxygen–sensitive PTB7-Th/PAni Hs,a photoelectrochemical enzymatic biosensor is developed for sensitive detection of guanine.With outstanding conductivity,hierarchically porous structure and high permeability to solvent molecules,three-dimensional PAni Hs can not only be directly used as the carrier of PTB7-Th and XOD to improve the molecules-loading amounts,but also facilitate the diffusion kinetics of hydrosoluble matter and electrons,greatly enhancing the initial photocurrent signal.In the presence of guanine,xanthine oxidase catalyzes its oxidation to uric acid,and the initial photocurrent decreases as the dissolved oxygen in the electrolyte solution is consumed.The reduced photocurrent is related to the concentration of guanine,so the detection of guanine is achieved.The morphology,element types and chemical bond of PAni Hs and PTB7-Th/PAni Hs are analyzed by scanning electron microscopy,energy dispersions and X-ray photoelectron spectroscopy.The Schiff base reaction between PAni Hs and enzyme is analyzed by Fourier transform infrared spectroscopy.The light absorption characteristics of PAni Hs,PTB7-Th and PTB7-Th/PAni Hs are studied by UV-Vis diffuse reflectance spectroscopy.The bandgap of PTB7-Th and PTB7-T/PAni Hs are calculated according to the Tauc equation.The electron transfer mechanism of photoactive materials is further discussed.Then the feasibility and analytical performance of the biosensor for detecting guanine are studied,and the limit of detection is 0.02μmol/L.The PAni Hs is assembled on the surface of the glass carbon electrode in 3minutes by rapid polymerization of aniline monomer,phytic acid and polymerization initiator,which greatly simplifys the electrode preparation process.Remarkably,the proposed photoelectrochemical enzymatic biosensor can not only be extended to other aerobic enzymatic bioanalyses,but also pave a horizon for the application of environmentally friendly conductive hydrogel materials in photoelectrochemical bioanalysis.2.Study on rolling circle amplification-CRISPR/Cas12a-mediated universal immobilization-free electrochemical biosensing platformAn electrochemical biosensing platform is designed to detect disease-related nucleic acid targets and nonnucleic acid targets by designing rolling circle amplification-clustered regularly interspaced short palindromic repeats（CRISPR）/Cas12a nucleic acid amplification and combining affinity mediated immobilization-free strategy.In this strategy,modular rolling circle amplification is used to achieve the transformation and amplification of target recognition events.Through the rolling circle amplification reaction,the target recognition event is transformed into a triggered DNA strand（TS）,and then TS activates the deoxyribonuclease activity of the Cas12a enzyme to further amplify the signal.Target-activated blocker probe（BP）cleavage induces the methylene blue-tagged reporter probes（RP）can be captured by the reduced graphene oxide modified electrode,leading to obviously increased electrochemical signal.We only need to simply tune the sequence for target recognition in RCA components,and this strategy can be flexibly applied to the highly sensitive and specific detection of micro RNA-21,Parvovirus B19 DNA and adenosine 5′-triphosphate,and the limit of detection is 0.83amol/L,0.52 amol/L,0.46 pmol/L,respectively.The electrochemical biosensing platform has good selectivity and anti-interference ability,and can be used for target analysis in complex serum.Besides,four DNA logic circuits（including YES,NOT,OR,AND）of DNA inputs are constructed,and polyacrylamide gel electrophoresis is implemented to experimentally prove the modularity and programmability of the stimuli-responsive rolling circle amplification-CRISPR/Cas12a system.This electrochemical biosensor does not require complex and time-consuming surface-immobilization procedure,and exhibits a"signal-on"mode.Because the rolling circle amplification-CRISPR/Cas12a system has strong programmability and integration,the proposed electrochemical biosensing platform has excellent universality,which overcomes the limitation that the existing immobilization-free electrochemical biosensors can only be used for the detection of one kind of target and has potential to be used for detection of all kinds of disease markers in clinical diagnosis.3.Study on BIDSD-RCA-CRISPR/Cas12a-based immobilization-free electrochemical aptasensorBinding-induced DNA strand displacement strategy is used to transform thrombin-aptamer interaction into nucleic acid output,which further trigger the rolling circle amplification to regulate the deoxyribonuclease activity of Cas12a enzyme.Based on the difference of affinity between ss DNA and ds DNA of graphene,an immobilization-free electrochemical aptasensor is constructed to realize sensitive detection of thrombin.Specifically,two split aptamers simultaneously bind to different sites of thrombin to trigger the DNA strand replacement reaction.The released nucleic acid initiates rolling circle amplification reaction,generating large numbers of long DNA amplicons containing sequences of trigger DNA strand（TS）.The TS activates Cas12a enzyme to cut blocker probe（BP）,thus the methylene blue-tagged reporter probes（MRP）can stably adsorb on the reduced graphene oxide modified electrode,leading to obviously increased electrochemical signal.Combined with the rolling circle amplification-CRISPR/Cas12a biological signal amplification strategy,this electrochemical aptasensor achieves sensitive detection of thrombin with a calculated limit of detection of 12 fmol/L.Thanks to the high specificity of the aptamer for thrombin and the high affinity of the two-site recognition mode,the aptasensor exhibits good selectivity and anti-interference ability,and can be used for thrombin analysis in complex serum.This aptasensor does not require complex and time-consuming surface-immobilization procedure.This work provides a paradigm for the detection of low concentration protein biomarkers in the early diagnosis of disease.4.Study on Y-motif-mediated primer-free cyclic signal amplification-based microelectrode sensing platformDesigning the Y-motif-mediated primer-free cyclic signal amplification,mading a home-made carbon fiber microelectrode,and developing an immobilization-free electrochemical sensing platform for the detection of DNA methyltransferase activity and the screening of its inhibitors.The basic idea is to convert DNA methyltransferase cleavage event into a large amount of guanine（G）-rich DNA fragments output by Y-motif-mediated primer-free cyclic signal amplification strategy.In the presence of potassium ions,G-rich DNA fragments fold into G-quadruplex framework to capture methylene blue in the solution,which reduce the diffusion current of the methylene blue in the solution,thereby realizing sensitive detection of DNA methyltransferase activity.By coupling the unique signal-transduction approach with Y-motif-mediated primer-free cyclic signal amplification,the immobilization-free electrochemical biosensor possesses high specificity and sensitivity for DNA methyltransferase activity,and the limit of detection is 2.5×10-4U/m L.In addition,gentamicin was used as an inhibitor to evaluate the ability of this sensor in screening DNA methyltransferase inhibitors.And this sensor has been successfully applied to the detection of DNA methyltransferase in serum.Different from the previously proposed immobilization-free electrochemical biosensing strategy,the immobilization-free electrochemical biosensor does not require expensive electroactive molecular markers,which reduces the analysis cost.In addition,this home-made microelectrode has the advantages of miniaturization,rapid response,reusability,and plug and play,which holding great potential in clinical testing. |
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