Construction Of Sensing Interface Based On Carbon Nanocomposites For Biomolecules Electroanalysis

As an important substance in physiological activities,reactive small biomolecule play an important role in regulating physiological and pathological processes of organisms.The traditional analysis technology have been unable to meet the needs of broad analysis targets,complex target system and more rapid and accurate analysis results.Especially for ROS/RNS,ascorbic acid（AA）,dopamine（DA）and uric acid（UA）,it has disadvantage that the signal is not easily captured and vulnerable to influence.With the development of nanomaterials,electrochemical analysis technology has developed rapidly in past decade.The multi-functional sensing interface with high sensitivity and selectivity can be constructed by nano-technology surface engineering treatment of electrode,which can realize the rapid detection and multi-component simultaneous determination.Therefore,this paper designs and synthesizes various sensing interfaces based on synergistic effects of functional nanomaterials to construct electrochemical reactive biomolecule detection platform.The sensing interfaces show good biocompatibility and specific electrocatalytic activity toward reactive biomolecule with high selectivity and sensitivity,and the electrocatalytic mechanism is further explored.The main works are summarized as follows:（1）An electrochemical biointerface based on electrodeposition of Au nanoparticles（AuNPs）on 3D graphene aerogel（3DGA）was fabricated to realize the direct electron transfer of Cytochrome c（Cyt c）for H₂O₂ detection.AuNPs was uniformly distributed on 3DGA networks and AuNPs can effectively adsorb Cyt c with good biocompatibility.The synergistic effects of these two kinds materials with good conductivity and biocompatibility can realize the direct electron transfer of Cyt c in electrochemistry.The results show that 3DGA-AuNPs/Cyt c/GCE revealed excellent catalytic activity toward H₂O₂ with high sensitivity of 351.57μA·mM^-1·cm^-2.The detection limit was～1.1μM and the detection linear range was 10-740μM.The interface design based on3DGA-AuNPs can establish an ideal contact interface for proteins to expose their electroactive centers and enhanced electron transfer between Cyt c and electrodes.（2）The redox protein modified electrochemical sensor has high sensitivity,but the protein is vulnerable to the environment result in poor long-term stability.In this study,a non-enzymatic H₂O₂ sensor was constructed by synthesis of biomimetic enzyme ultrasmall Fe₃O₄ decorated on three-dimensional graphene nanocomposites（Fe₃O₄/3DG）.Fe₃O₄ possess intrinsic peroxidase-like activity Fe₃O₄/3DG was prepared via a facile hydrothermal method with the size of 5-7 nm,in which Fe₃O₄ possess intrinsic peroxidase-like activity to efficiently catalyze the electrochemical reduction of H₂O₂.3DG networked structure with excellent conductivity,high specific surface area and fast charge mobility not only improved the sensitivity of the sensor,but also prevent Fe₃O₄ agglomeration and corrosion to increase catalyst durability.TEM showed that Fe₃O₄ was uniformly distributed on 3DGA networks.Fe₃O₄/3DG@GCE revealed excellent catalytic activity toward H₂O₂ with high sensitivity of 274.15μA·mM^-1·cm^-2.The detection limit was 0.78μM and the detection linear range was 0.8-334.4μM.The rationally designed sensor architecture of Fe₃O₄/3DG@GCE exhibited low detection limit,long-term stability and higher selectivity.This work demonstrates that this biosensor exhibits excellent ability to in situ detect H₂O₂ released from living cells by controlling cell numbers and stimulation drug dose.（3）A preparation of dopamine-derived N-Doped carbon Nanotubes/Fe₃O₄Composites（N-CNTs/Fe₃O₄）is demonstrated via hydrothermal route and calcination treatment.Dopamine can be self-polymerized on the surface of CNTs that improved the dispersion of CNTs.The unique molecular structure of PDA can promote Fe₃O₄nanocrystals deposition uniformly on the surface of CNTs.Carbonized polydopamine coatings effectively modulated the graphitic structure and the increased pyridinic N and graphitic N further improved electrochemical performance of carbon composites.XPS analysis suggested that the pyridinic N and graphitic N materials was effectively doped the graphite structure by high temperature calcination.In addition,the inhibited growth of the Fe₃O₄ crystal during calcination can be effectively avoided by soaking PDA-CNT/Fe₃O₄ Cs in phosphate solution before the treatment of calcination.Electrochemical result show that N-CNTs/Fe₃O₄ have an enhanced electrocatalytic activity toward hydrogen peroxide（H₂O₂）with high sensitivity(316.27μA·mM^-1·cm^-2)and wide linear range（0.006-2.057 mM）.The detection limit was 0.304μM.This research work has wide applicability in surface modification of materials.（4）The ideal bioelectrochemical detection platform should not only have excellent electrocatalytic performance and anti-interference performance,but also have good biocompatibility and surrounding environment to cell culture.Therefore,in this study,a cell-grown free-standing graphene paper detection platform were constructed toward H₂O₂ and NO with high-sensitivity real-time response.The reduced graphene paper（rGP）electrode was prepared by template method,and the conductive polymer polyaniline（PANI）and AuPt nanoparticles were sequential electrodeposition on the rGP to construct the sensing interface with electrocatalytic activity.The AuPt/PANI/rGP free-standing sensor has good electrocatalytic performance and high detection sensitivity for H₂O₂ and NO.The linear range of AuPt/PANI/rGP for H₂O₂ detection was 10-930μM and 1.13-5.63 mM with the detection sensitivity of 392.22μA·mM^-1·cm^-2 and 155.81μA·mM^-1·cm^-2 respectively.The detection limit was 0.675μM.The detection linear range of AuPt/PANI/rGP for NO was 0.36-437.2μM with the detection sensitivity of 112.00μA·mM^-1·cm^-2.The detection limit was 65.7 nM.Due to the good biocompatibility of rGO and AuPt,the cells were able to adhere and grow well after plasma surface treatment.This cell growth monitoring can contact living cells closely,shorten the diffusion distance between reactive biomolecule and electrode,which improve the accuracy of sensor electrodes with high sensitively.（5）The electrochemical activity biomolecules of ascorbic acid（AA）,dopamine（DA）and uric acid（UA）can be simultaneously determined by electrochemical analysis.Molybdenum disulfide nanotubes（MoS₂ NTs）were synthesized via hydrothermal route and calcination treatment.SEM and TEM indicated that the MoS₂ NTs is composed of ultrathin sheet-like MoS₂ subunits.The oxidation potentials of AA,DA and UA have a clear separation on MoS₂ NTs@GCE,which can realize simultaneous detection of AA,DA and UA.A combination of theoretical calculations and experiments investigated the electrooxidation mechanism of AA,DA and UA based on density functional theory（DFT）and computational hydrogen electrode（CHE）approach for the first time.The study of CV found that the catalytic oxidation of AA,DA and UA on MoS₂ NTs@GCE were proceed with a two electron–proton transfer process.The potential-dependence of reaction free energies in elementary steps was evaluated using the CHE approach.The order of oxidation of the three molecules on MoS₂ NTs surface from difficult to easy is UA>DA>AA.The corresponding oxidation potential from high to low is UA>DA>AA.The three molecular oxidation reaction formulas can be obtained according to the calculated path.In addition,the detection linear range of MoS₂ NTs@GCE for individual determination of AA,DA,and UA by DPV were 0.8-1200μM,0.3-70μM and 5-950μM.The limit of detection of AA,DA and UA were calculated to be 0.44μM,0.13μM and 0.83μM.the detection linear range of MoS₂ NTs@GCE for simultaneous determination of AA,DA,and UA were 1-400μM,0.5-40μM and 5-535μM.The limit of detection of AA,DA and UA were 0.39μM,0.11μM and 1.5μM.The sensor show high selectivity and stability.There was no significant difference between the electrochemical method and HPLC method in urine sample test,showing a satisfactory recovery results.