Construction Of Biosensor Based On Graphene Nanomaterials

Graphene (RGO) is a single-layer carbon sheet with sp2-hybridized ofcarbon atoms arranged tightly honeycomb crystal structure. Graphene hasbeen widely applied in nanoscale electronic devices, sensors, drug carrier，super capacitor and energy storage due to its high specific surface area,outstanding thermal, electrical and mechanical properties.Objective: We developed a highly electrical conductive, goodbiocompatibile graphene nanocomposites with strong affinity for enzymes bycombining graphene materials with other nano materials, and also explored theapplication of graphene in glucose biosensor. On this basis, we proposed adirect co-electrodeposition approach to synthesis of graphene oxide–goldnanoparticles composite for efficiently fixed acetylcholinesterase (AChE) tobuild a pesticide biosensor with high sensitivity, good stability, fastelectrochemical response and good reproducibility. The purpose of theresearches is to establish sensitive and scientific methods for the rapiddetection of pesticide, and provide a theoretical basis for the construction ofother similar biosensors.Methods:Part I: Preparation, characterization of graphene and its application inglucose biosensors.Step1: Preparation of graphene oxide (GO) and reduced graphene oxide(RGO) by Hummers and offeman method with glucose as a reducing agent.Step2: Characterization of GO and RGO by X-ray diffraction method (XRD)and ultraviolet-visible spectrophotometry.Step3: On the basis of co-electrodeposition of prussian blue (PB) and chitosan(CS), we introduce a novel nano materials－RGO to fabricate the glucosebiosensor based on RGO/PB-CS nanocmposites. The electrochemicalcharacteristics of the biosensor were studied by cyclic voltammetry (CV) and current-time (i-t) curve method. We investigated the effect of PB and RGO onthe sensitivity of sensor, and discussed anti-interference and clinicalapplication of the sensor.Part II: Construction of pesticide biosensor based on prussianblue/graphene-gold nanoparticals-β-cyclodextrin (PB/RGO-AuNPs-β-CD)composite membrane.Step1: Electrochemical deposition of RGO-AuNPs-β-CD membrane on theelectrode were carried out by controlled potential method.Step2: Electrochemical deposition of PB-CS membrane on the modifiedelectrode was carried out by CV.Step3: The CS with good biological compatibility was used as a carrier for thehighly efficient immobilization of AChE.Organophosphorus pesticide and carbamate pesticides are usuallydetected based on the inhibition of the AChE by these compounds. Theinhibition of organophosphorus pesticide and carbamate pesticides on AChEresults in decrease of produced thiocholine, and the oxidation currentdecreases accordingly. The organophosphorus pesticide and carbamatepesticides can be detected by measuring the decline of the oxidation current ofthiocholine. The electrode surface was characterized by scanning electronmicroscopy (SEM) method. The current responses of the biosensor toacetylthiocholine chloride were researched by the i-t curve method. The effectof inhibition time, AChE reactivation, stability, selectivity and repeatability ofsensor were discussed, and finally the biosensor was used to detect therecovery of the real samples.Results:1Preparation, characterization of graphene and its application in glucosebiosensors1.1Graphene prepared by this experiment was stable and could be directlyused in the modification of the electrode without any treatment. As shown inXRD, the crystal structure of graphite was destroyed, while crystal integritydeclined and the flake layer became smaller during the redox process, indicating the graphene was obtained. Ultraviolet absorbance spectra showedthat the oxygen-containing functional groups on the surface of the grapheneoxide was reducded gradually and л-л conjugate system was restored, whichdemonstrated the successful preparation of graphene once again. Theelectroconductivity of different modified electrodes surface was characterizedby CV. The results show that the response interface of the electrode has a goodelectric conductivity.1.2Time-current curve method was applied to optimize the best work system.Applied potential is0.0V. The pH of buffer solution is6.5. The concentrationof glucose oxidase is10mg mL-1and the amount of RGO is5μL. Thecatalytic current of the biosensor was linear with the glucose concentrationranging from0.01to1.05mM with a correlation coefficient of0.9999. Thebiosensor showed a high sensitivity of65.3μA mM-1cm-2with a low detectionlimit of6.00μM and apparent Michaelis-Menten constant (Kappm) of1.43mM.The proposed biosensor exhibited short response time and goodanti-interferent ability. It can be used for the diabetes blood sugardetermination.2Construction of pesticide biosensor based on prussian blue/graphene-goldnanoparticals-β-cyclodextrin composite membrane2.1Optimal conditions of the RGO-AuNPs-β-CD nanocomposite assembly onthe electrode were optimized. The concentration of chloroauric acid, β-cyclodextrin and graphene oxide are0.01%,0.025%and2mg mL-1respectively. The applied potential is–1.4V, and eleltrodeposition time is720s.2.2Time-current curve method was applied to optimize the best work system.The concentration of acetylcholinesterase is0.500mg mL-1. Applied potentialis0.2V and pH is6.5.2.3The amperometric response of the biosensor to ATCl was invetigated by i-tcurve method under the optimum experimental conditions. The oxidationcurrent of the biosensor was proportional to the concentration of ATCl in tworanges, from1.50to269μM and from344to2.22×103μM, with correlation coefficients of0.9992and0.9942. The sensitivities of the biosensor were205and134μA mM-1cm-2. The results indicated that the synergy of RGO andAuNPs effectively improved the catalysis of AChE toward ATCl and enhancedthe response signal of the nanocomposite modified electrode toward thehydrolysis products of ATCl.2.4Differential pulse voltammetry method (DPV) was applied for malathionand carbaryl determination. The linear ranges of malathion and carbaryl are8.0~2.00×103pg mL-1and4.3~1.00×103pg mL-1respectively.2.5Due to the introduction of PB, the biosensor could oxidize thiocholine, theproduct from the hydrolysis of acetylthiocholine catalyzed by AChE at lowpotential, which not only improve the sensitivity of the biosensor, but alsoimprove the selectivity. The sensor storage for28days at4℃, the enzymecould maintain the92%of original activity2.6Reactivation of the enzyme was investigated with pralidoxime iodide as anantidote. The results showed that the inhibited AChE could be regeneratedmore than94.8%of its original activity after immerging in5.00mMpralidoxime iodide for15min.2.7The recoveries of malathion and carbaryl in real samples was investigatedby standard addition method. The recovery of malathion and carbarylrespectively are92.8%~106.7%and90.3%~101.5%. The results indicatedthat the biosensor exhibited a good accuracy for the organophosphorus andcarbamate pesticides determination.Conclusion: Firstly, we explore the application of graphene prepared bychemical reduction method in glucose biosensor. The results indicated that thecombination of graphene and PB-CS could effectively promote electrontransfer between the electrode surface and analyte, and improve theperformance of the sensor. The PB-CS/RGO-AuNPs-β-CD compositemembrane prepared by electrodeposition techniques was used for theconstruction of a novel acetylcholinesterase biosensor. The synergy of RGOand AuNPs effectively improved the catalysis of AChE toward ATCl, andenhanced the response signal of the nanocomposite modified electrode toward the hydrolysis products of ATCl. The introduction of PB not only improvedthe sensitivity of the biosensor, but also reduced the applied potential (0.0Vand0.2V) and improved the selectivity of the sensor. The preparation processof the biosensors is simple and low-cost, and the biosensors achievedsatisfactory results for actual samples, which has potential application value.