Construction Of Functionalized Metal-Organic Frameworks Enabled Electrochemical Biosensor And Its Analytical Application

The level of disease marker in human body can indicate the occurrence of disease,therefore,the development of accurate and sensitive detection method for disease marker is one of the important research topics in analytical chemistry field.Electrochemical biosensor has the advantages of high sensitivity,fast response,low cost and good selectivity,which plays an important role in disease marker detection.Researchers have introduced novel functional materials and signal amplification techniques to improve the performance of the biosensors.Among them,metal-organic frameworks（MOFs）possess the characteristics of large specific surface area,high porosity,tunable structure and easy to functionalize,which hold great application potential as nanocarriers in biosensing field.However,MOFs still have some shortcomings,such as the lack of recognition ability torwards the target and the difficulty in realizing amplified signal output.In addition,the utilization approaches of the metal center of MOFs are relatively simple,which limit the further application of MOFs in electrochemical biosensing field.Therefore,in this thesis,different functional molecules were introduced into the structure of MOFs,which endow the MOFs with molecular recognition and signal output ability.Taking advantage of functionalized MOFs and efficient DNA nanotechnology,multiple signal amplified electrochemical biosensor was constructed for ultrasensitive and highly selective detection of disease marker,which provided new idea for early clinical diagnosis of diseases.The main research contents are as follows:1.Gold nanoclusters-graphene@ZIF-8 coupling hybridization chain reaction for electrochemical detection of interferon-gammaTaking advantage of the large specific surface area and abundant pores of MOFs,different nanomaterials can be introduced into the structure of MOFs to effectively improve the electrical conductivity,which are contributed to the construction of biosensing interfaces with high sensitivity.In this chapter,gold nanoclusters（Au NCs）were synthesized by utilizing the confinement effect of two-dimensional graphene@ZIF-8 composites（GR@ZIF-8）,and an electrochemical biosensor was constructed for interferon gamma（IFN-γ）detection based on Au NCs-GR@ZIF-8 and layer-branched hybrid chain reaction（LB-HCR）for signal amplification.Using Au NCs-GR@ZIF-8 as the DNA assembly platform,LB-HCR was designed on the basis of traditional HCR,which was completed by the cascaded self-assembly of four hairpins.In which,HP1 and HP2 participated in traditional HCR process to form long double-helix DNA,AD1 and AD2 assembled alternately to promote the layer-branched growth of DNA nanostructures.The presence of target IFN-γtriggered the LB-HCR process and resulted in the generation of dendritic DNA nanostructures on the electrode,which integrated with numerous in-situ formed hemin/G quadruplex DNAzyme as amplifying labels.The hemin/G quadruplex DNAzyme catalyzed the reduction of H₂O₂ with thionine acted as the electron mediator,which realized the signal output.The amplified electrochemical biosensor based on Au NCs-GR@ZIF-8,LB-HCR and hemin/G quadruplex DNAzyme achieved ultrasensitive detection of IFN-γranging from 1 f M to 50 p M with the detection limit of 0.6 f M.This strategy demonstrated an amplified sensing platform and efficient DNA assembly mode,holding potential application in disease diagnosis and monitoring.2.Responsive DNA/MOFs enabled electrochemical biosensor for cancer biomarker detectionThe abundant pores of MOFs can realize highly efficient loading of functional molecules,and DNA can be further used for MOFs modificaton,which endows MOFs with stimuli-responsive ability,achieving controllable release of functional molecules.In this chapter,a responsive electrochemical biosensor was fabricated for carcinoembryonic antigen（CEA）detection based on DNA-functionalized MOFs and target-driven cascaded signal amplification.By using MOFs（Ui O-66-NH₂）as nanocarrier for electroactive molecules（methylene blue,MB）and the programmably assembled DNA acted as the gatekeeper,the functionalized MOFs（MB@DNA/MOFs）were not only worked as three-dimensional biosensing tracks,but also acted as amplified signal labels.In the presence of target CEA,the nicking endonuclease cleavage process was triggered,leading to the generation of two strands（S1 and S2）.Both S1 and S2 acted as secondary target to participate in the strand displacement reaction on MB@DNA/MOFs,which caused the unlocking of the pore and the release of MB,resulting in the decrease of the signal.The proposed cascade-amplified biosensor presented good performance,which realized ultrasensitive CEA detection ranging from 50 fg/m L to 10 ng/m L with the detection limit of 16 fg/m L.This strategy demonstrated a label-free and responsive three-dimensional sensing platform,which provided an efficient method for the detection of cancer marker.3.DNA/MOFs degradation boosted single-component DNA assembly for electrochemical detection of enzymatic activityDNA can endow MOFs with stimuli-responsive ability,but the assembly process of DNA on the surface of MOFs is usually complicated and time-consuming,which increase the cost.The controllable release of functional molecules can be achieved by directly utilizing the MOFs with responsive properties,which is simple and highly efficient.In this chapter,by using acid-responsive MOFs as carriers of single-stranded DNA,an electrochemical biosensor based on S1/ZIF-67 was constructed for the detection of acetylcholinesterase（ACh E）activity.Target ACh E catalyzed the hydrolysis of the substrate acetylcholine to generate thiocholine and acetic acid,the presence of protonic acid induced the dissociation of S1/ZIF-67 to release S1 and large amount of Co²⁺.The released S1 cascade self-assembled to form DNA network structure,which was rapidly immobilized on the surface of polydopamine-reduced graphene oxide（PDA-r GO）by Co²⁺-mediated DNA adsorption.Using the formed DNA network structures as template,silver nanoclusters were generated by a simple and fast UV light reduction method to realize signal output.The proposed biosensor achieved sensitive detection of ACh E in the range of 0.01-100 m U/m L with the detection limit of 3.5×10^-3 m U/m L.This strategy took full advantage of the acid-responsive property,large specific surface area and abundant metal centers of MOFs,while avoiding the introduction of multiple DNA strands,which exhibited a simple and novel electrochemical sensing platform,providing new method for disease diagnosis and drug screening.4.DNA nanomachine coupling electrochemical conversion of MOFs for micro RNA detectionMOFs can be used as nanocarriers to load functional molecules,but the utilization of metal centers is still insufficient.The metal centers of MOFs can be further converted into electroactive nanomaterials,which achieve signal amplification of the sensing system.In this chapter,an electrochemical biosensor was constructed for micro RNA detection based on polydopamine nanoparticles-DNA（PDANs-DNA）nanomachine coupling electrochemical conversion of MOFs（NH₂-MIL-88（Fe））.The PDANs-DNA nanomachine was designed based on Ca²⁺-mediated DNA adsorption and target-triggered catalytic hairpin assembly（CHA）,which not only maintained the DNA immobilization simplicity but also possessed a high walking efficiency.PDANs-DNA nanomachine could walk fast on the electrode via multiple legs under exonuclease III driving,resulting in the formation of dendritic DNA nanostructures through two hairpins assembly.The MOFs probe was decorated on the dendritic DNA nanostructures to act as a porous metal precursor and converted into electroactive Prussian Blue by a simple and mild electrochemical approach,which realized the amplified signal output.Using micro RNA-21（mi RNA-21）as the model target,the proposed biosensor achieved ultrasensitive mi RNA-21 detection ranging from 10 a M to 10 p M with the detection limit of 5.8 a M.This strategy presented a highly efficient DNA nanomachine with the ingenious electrochemical conversion of MOFs,providing more options for the design of electrochemical platform and holding potential applications in disease diagnosis and clinical analysis.