Construction Of Sensing Interface Based On 3D Graphene For Biomolecules Analysis

The accurate and reliable determination of trace biomolecules plays a critical role in elucidating various physiological and pathological processes,thereby facilitating the evolution of the worldwide healthcare system and disease management.Although numerous ultrasensitive biosensing strategies have been proposed in recent decades,it is still challenging to analyze an d detect biomolecules at extremely low levels in complex real samples,and some biomolecules are difficult to capture the signals due to its relatively active.Electrochemical sensing analysis technology is currently gaining an increasing attention for biomolecular detection due to its high sensitivity,simple operation,fast response,low cost and easy miniaturization.Previous reports have revealed that controlling the interfacial properties of the biosensor,which are affected by the nanostructure of electrodes and the coupling manner of the immobilized biomolecules.Thus,researchers are focus on the introduction of functional nanomaterials with different structure to build more sensitive and more stable sensing interface,and they are also pay attention to enhance the interface specificity,biocompatibility,and probe hybridization rate through DNA nanotechnology.Graphene is widely used in the substrate construction of electrochemical biosensor interface owing to its good electrical conductivity,high thermal stability,large surface area and excellent mechanical properties.Nevertheless,most of graphene are used in powder form(complex and time consuming),and Nafion may be required to fix the catalyst,reducing the effective area of the reactive interface and leading to a decrease in detection efficiency.Three-dimensional(3D)graphene grown in situ can overcome the shortcomings of powdered graphene,and it also has unique advantages such as rich electrolyte diffusion channels,good stability and strong repeatability.Therefore,in this thesis we designs and synthesizes various sensing interfaces based on synergistic effects of 3D graphene with functional nanomaterials,and combined with DNA nanotechnology to construct electrochemical biological molecular detection platforms with high sensitivity,high stability and good biological compatibility.The developed biosensor platforms are employed to sensitive and selective detection of some representative biomolecules(e.g.,microRNAs,CEA,EVs,glucose and H2O2),in particular,the detection limit of miR-155 is 23 zM(2.3×10-20 M),and this sensor platform also achieved the real-time detection of released H2O2 by cells.The main research works are summarized as follows:?A sensitive electrochemical miRNA analysis platform is successfully developed by integrating polypyrrole graphene/gold nanoparticles(Au/PPy-rGO),catalytic hairpin assembly(CHA),and hybrid chain reaction(HCR)multiple signal amplification strategy.First,Au/PPy-rGO was modified on two kinds of conductive substrate(glassy carbon electrode(GCE)and carbon fiber paper(CFP)),which could greatly enhance the conductivity and biocompatibility of sensing interfaces.In addition,CHA reaction achieves the target chain circulation without enzyme and HCR reaction further realizes the amplification of DNA chain,then produced dsDNA polymers for the loading of abundant MB to achieve detected electrochemical signal.MiRNA-16(T)can be detected in two sensors with detection linear ranges were 10 fM?5 nM and 1 fM?500 pM(6 orders of magnitude),two low detection limits(LOD)of 1.57 fM(GCE)and 0.36 fM(CFP)(S/N=3),respectively.The developed multiple signal amplification platform has a great potential for the applications in the field of biomedical research and clinical analysis.Compared with GCE,the CFP conductive substrate with 3D spatial structure shows better detection performance,which is more conducive to the construction of sensing interface.(2)Based on above research,we report a three-dimensional(3D)carbon nanomaze(CAM)electrode for the simultaneous detection of miR-155 and miR-21.The CFP/GWs/AuNPs(CAM)electrode consists of an interlaced CFP on which intercrossed graphene walls(GWs)were vertically tethered in situ by radio frequency plasma enhanced chemical vapor deposition(RF-PECVD),permitting local confinement of trace molecules to increase molecular hybridization efficiency.Furthermore,a self-assembled DNA tetrahedron(DNA-T)array adopts a rigid spatial conformation to guarantee the controllable arrangement of immobilized biological probes,facilitating analytical sensitivity and reproducibility.In a proof-of-concept experiment on detecting miR-155 and miR-21,a LOD of 23 zM(2.3 ×10-20M)was achieved,which was lower than most existing electrochemical detection methods.The proposed nanoelectrode demonstrated high consistency with quantitative real-time PCR(qRT-PCR)during 30 clinical sample detection.Through simple functionalization by appending various biomolecular probes of interest(such as nucleic acids,proteins,small molecules and even cells),the developed CAM platform with ultrasensitivity could be exploited as a versatile tool in environmental,chemistry,biology,and healthcare fields.(3)Given the successful anchoring of the oligonucleotide strand on the DNA-Ts,we expected to extend the flexibility of the CAM platform by immobilizing various biomolecular recognition elements(DNA-T-AptcEA and DNA-T-AptEV)corresponding to carcino-embryonic antigen(CEA)and extracellular vesicles(EVs).Under the optimal experimental parameters,the two constructed sensors detected CEA and EVs with different concentrations in a 5 mM[Fe(CN)6]3/4 solution containing 0.1 M KCl by square wave yoltammetry(SWV).The LODs are 0.46 pg/mL and 1×103/mL for CEA and EVs,respectively.Moreover,both of the two sensors obtained relatively satisfactory detection results in real samples,which verified the universality of the constructed CAM sensor platform and showed the application potential in the detection of various types of biological molecules.?Besides microRNA,CEA and EVs,we developed an enzymatic glucose sensor based on above-mentioned sensing interface(CFP/GWs),and Cu2O NPs were evenly grew on the 3D GWs layer and skeleton through the complete thermal decomposition of copper acetate(Cu(CH3COO)2)at high temperature.The CFP/GWs/Cu2O shows a large specific surface area and can expose more catalytic active sites without Nafion fixation film,thus greatly improving the electrocatalytic performance of this glucose sensor.The constructed CFP/GWs/Cu2O sensor showed excellent catalytic performance to glucose with a linear detection range of 0.5 ?M?5166?M,LOD of 0.21?M,and response time<4s.In particular,Cu2O NPs(catalyst)can be deposited on GWs precisely and quantitatively through simple concentration regulation.In addition,the real sample test results of proposed sensor were consistent with the commercial glucometer,indicating good accuracy and reliability of CFP/GWs/Cu2O sensor.? In this study,a 3D flexible sensing interface(CC/GWs/AuPt)with direct growth of living cells was constructed for the rapid,sensitive,in-situ and real-time analysis and detection of H2O2.Similarly,GWs were vertically grew on the flexible and conductive carbon cloth(CC)by RF-PECVD,and AuPt nanoparticles were electrodeposited on CC/GWs to build an ultrasensitive sensing interface with good electrocatalytic activity.The proposed CC/GWs/AuPt biosensor shows good electrocatalytic performance for H2O2,and the detection limit was as low as 0.084 ?M(84 nM)with fast response(<5 s).In addition,the cells were able to adhere and grow well on the CC/GWs/AuPt sensing electrode,which was attributed to the good biocompatibility of CC/GWs/AuPt,providing a 3D microenvironment to the adhesion/growth of cells.Under the stimulation of drugs,this CC/GWs/AuPt biosensor achieved the real-time detection of released H2O2 by cells.This cell growth monitoring sensing interface can efficiently shorten the diffusion distance between reactive biomolecule and sensing interface,and improve the sensitivity and the accuracy of biosensor,holding great promise for live-cell activity and its metabolites of continuous dynamic monitoring.