Assembly Of Nanostructures On Electrode Surface For Biosensing Application

The widely use of nanomaterials and nanotechnologies in electrochemical sensing has provided unique opportunities for biosensor design.Besides,with the fast developments of bioanalytical chemistry,bioactive molecules are introduced to the sensing surface.Biomolecule recognition and its synergistic effects at the nanometer scale have attracted much attention from many research fields.So the fabrication of desired nanostructures that enable high selectivity and sensitivity would be a hot area in bioanalytical and biomedical engineering research.In this thesis,we focus on the fabrication of new nanostructures to facilitate target recognition and signal amplification.So we have fabricated a series of nanostructures at the electrode surface.Based on the design of functional nanostructures,target recognition and signal production have been greatly improved for biomolecule detection,including small molecules,proteins,nucleic acid,cells etc.Details of the designed electrochemical biosensors are shown as follows:1.Preparation of dual-thiolated hairpin DNA nanostructure on electrode surface for ultrasensitive and highly selective detection of nucleic acidsDual-thiolated hairpin DNA immobilized on electrode surface can not only efficiently bind target and signal probe but also process impressive protein-repelling ability,which allows direct detection as few as attomolar targets(?300 copies in 100?L sample)with single-base discrimination ability.Meanwhile,the preparation procedure of functional electrode surface becomes simple(one step),fast(30 min)and homogeneous(just one probe modified surface without small molecules co-assembled).These advantages are attributed to the unique probe design,where the stem of the capture probe can act as rigid scaffold to keep it upright,and the loop of the capture probe may provide an enclosed platform for target and signal probe binding.More importantly,through tuning the distance between enzyme and the electrode surface(from 8.3 to 13.6 nm),we find that the performance of the sensor can be favorably controlled.Furthermore,taking advantage of this new binding model,different complex samples including polymerase chain reaction(PCR)product,messenger RNA,and micro RNA can be conveniently analyzed,which may hold great potential for practicle application.2.Fabrication of protein-aptamer assembled nanowire in "hand-in-hand"nanostructure for the determination of tumor-assosiated proteinsAn electrochemical assay based on the protein-aptamer assembled nanowire has been developed to detect human platelet derived growth factor BB(PDGF-BB)that is a tumor-related protein.In this work,two aptamer molecules bind two sites of one PDGF-BB molecule,undergoing conformational changes to form intact hairpin structures.The resulted "aptamer-PDGF-aptamer" units can then hybridize through the aptamer terminal strands,and finally form protein-aptamer linear nanostructure.The formed nanowire can be captured by probe DNA that is immobilized at the electrode.Large amounts of[Ru(NH3)6]3+are attracted onto the surface of electrode through the electrostatic adsorption between positively-charged electrochemical species and negatively-charged phosphate backbone,leading to signal amplification.Results have indicated high selectivity and sensitivity of this method,which can detect PDGF-BB as low as 100 fM with impressive selectivity.3.Assembly of dynamic DNA "walker" nanostructure at the electrode surface for the determination of disease-related proteinsAn electrochemical assay based on DNA walker has been developed to detect multiple disease-related proteins.Here,DNA walker that consists of target protein as body and difunctional DNA probes containing DNAzyme sequence and target recognition element can move on the surface of electrode.In the absence of target protein,DNAzyme of difunctional DNA probe cannot efficiently hybrid and cleave the short substrate DNA immobilized on the electrode due to the low melting temperature between them,resulting in electrochemical signal through the redox species labeled on the substrate DNA.Once in the presence of target protein,DNA walker assembled by one protein molecule and several difunctional DNA probes can effectively hybrid and cleave the surface-tethered substrate DNA into two short pieces with the help of proximity effect removing the redox label from the electrode surface and thus reducing electron transfer efficiency.After cleaving,the single dissociated leg will quickly explore neighbouring sites until it finds another intact substrate to reattach and cleave.This ensures the walker machine keep moving towards the substrate-contained region on the electrode surface to eliminate many redox labels,thus leading to considerable signal amplification eventually.The results demonstrate that this method is able to detect fg/mL concentrations of platelet derived growth factor BB(PDGF-BB),vascular endothelial growth factor(VEGF),prostate-specific antigen(PSA),carcino embryonic antigen(CEA),a-fetoprotein(AFP),and troponin I with impressive selectivity.Furthermore,diagnosing acute myocardial infarction(AMI)in patients can be improved with the DNA walker-based electrochemical assay compared with traditional ELISA.4.Enzyme-controlled formation of doubled-stranded DNA nanostructure for electrochemical determination of MTH1 activityIn this work,a mismatch-based("8-oxoG:A" mismatch)DNA chain elongation strategy(MB-DCE)is firstly proposed based on the control of a protein enzyme,MutT Homolog 1(MTH1).Specifically,in the absence of MTH1,T-DNA is locked in the double-stranded DNA,which cannot hybridize with the single-stranded capture DNA(C-DNA)that is immobilized on the electrode surface.Therefore,signal production is inhibited.However,in the presence of MTH1,the single-stranded T-DNA can hybridize with C-DNA on the electrode surface.Electrochemical signals can be produced.Then,further coupled with the inherent activity of MTH1 to prevent 8-oxo-dGTP misincorporation,a relationship can be established to reveal the activity of MTH1 through MB-DCE.As the method is designed directly towards the cellular function of MTH1,activity of MTH1 in different breast cancer cell lines has been detected,implying the potential application of this MTH1 assay method for biomedical research and clinical diagnostic in the future.5.Dynamic electrochemical control of cell capture-and-release based on redox-responsive host-guest interactionsBased on a redox-controlled host-gest interaction system,an electrochemical system for cell capture-and-release has been developed at the electrode surface.The host-gest interaction is highly sensitive to electrochemical stimulus.When applying a reduction voltage,the uncharged guest molecule(Fc)can bind to host molecule(?-CD)that is immobilized at the surface.Otherwise,it is disassociated from the surface as a result of electrochemical oxidation,thus releasing the captured cells.The cell capture-and-release process on this voltage-responsive surface is noninvasive with cell viability higher than 86%.Moreover,Fc molecule can act as an electrochemical probe for signal readout.The integration of this property further extends the ability of this system to cancer cell detection.By introducing branched polymer scaffold that carrying large quantities of Fc moieties,sensitive detection can be achieved.It is anticipated that such voltage-responsive surface can significantly impact biological and biomedical applications including tissue engineering,cell-based clinical diagnosis,etc.