Poster presented at the 4th International Conference on Bio-Sensing Technology, 10-13 May 2015, Lisbon

Almeida, M. G.

Fortunato, E.

Martins, R.

Santos, L.

Silveira, C.

Todorovic, S.

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This work is funded by FEDER funds throughthe COMPETE 2020 Programme and NationalFunds throught FCT-MEC under the projectEXCL/CTM-NAN/0201/2012.WO3 Nanoparticles Probes for Direct Electron Transfer of ProteinsL. Santos1,*, C. Silveira2,3, R. Martins1, S. Todorovic3, M. G. Almeida2,4, E. Fortunato1,*1CENIMAT/I3N, Dep. de Ciência dos Materiais, FCT-UNL and CEMOP-UNINOVA, 2829-516 Caparica, Portugal 2REQUIMTE, Departamento de Química, CQFB, FCT-UNL, 2829-516 Caparica, Portugal 3ITQB, Universidade Nova de Lisboa (UNL), Av. da República, 2780-157 Oeiras, Portugal 4Instituto Superior de Ciências da Saúde Egas Moniz, Monte da Caparica, 2829-511 Caparica, Portugal *corresponding authors: ls.santos@campus.fct.unl.pt, emf@fct.unl.ptIntroductionNitrites are constantly monitored for clinical diagnosis in physiological fluids and for environmental toxicity control in soil, water and air.Electrochemical enzyme based biosensors are of particular interest due to their:- operational simplicity, - low cost fabrication,- portability,- real-time monitoring ability. The ideal approach (3rd generation biosensors) allows direct electron transfer (DET) between the oxidoreductase and the electrode, thereby:- increasing selectivity,- simplifying the manufacturing process,- requiring fewer reagents.Propose of this work: Study of the electron transfer ability of WO3nanoprobes for the development of electrochemical nitrite biosensors and future implementation in a portable and low-cost sensing platform.-0.2 0.0 0.2 0.4 0.6-150-100-50050100150W1/ITO-0.2 0.0 0.2 0.4 0.6ITO-150-100-50050100150Current /µAPotential /V vs. Ag/AgCl5 10 15-150-100-50050100150I /µAscan rate1/2 /(mV/s)1/2Current /µAPotential /V vs. Ag/AgCl5 10 15-150-100-50050100150I /µAscan rate1/2 /(mV/s)1/2Ag/AgClWorking electrodePotentiostatPt wireMagnetic stirrerArgon valve-0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1-15-10-5.00.0Current /µAPotential /V vs Ag/AgCl0 0.2 0.4 0.6 0.802468101214ΔI /µA[NO2-] /mMΔI0.8 mM [NO2-]0 mM [NO2-]W3/ITO W3/ITOExperimental section1. Hydrothermal synthesis of WO3 nanoparticlesNO2-NH4+NO2-NH4+NO2-NH4+WO3 nanoparticleNa2WO4.2H2O + NaCl[HCl] = 0.3, 1.5, 2.7 M2 h, 180 CCentrifugation, drying3. SEM and TEM characterization0.39 nm0.31 nm0.62 nm0.39 nm100 nm 5 nmW1W2W3[HCl] = 2.7 M[HCl] = 1.5 M[HCl] = 0.3 MMonoclinic WO3OrthorhombicWO3·0.33H2OMonoclinic WO3+OrthorhombicWO3·0.33H2O2. XRD and Raman characterization*10 20 30 40 50 60 70 802 (degrees)W2(002)(002)W1Intensity (a.u.)W3m-WO3* *ortho-WO3·0.33H2O(220)*300 400 500 600 700 800 900 1000806805809328327327                         627934953676713700W1W2W3Raman shift (cm-1)Intensity (a.u.) Nernst equation: Ip = 2.69 × 105 n3/2 A C D1/2 1/24. Electrochemical characterizationFerrocyanide: Fe(CN)64-/ Fe(CN)63--0.2 0.0 0.2 0.4 0.6-150-100-50050100150W1/ITO-0.2 0.0 0.2 0.4 0.6ITO-150-100-50050100150Current /µAPotential /V vs. Ag/AgCl5 10 15-150-100-50050100150I /µAscan rate1/2 /(mV/s)1/2Current /µAPotential /V vs. Ag/AgCl5 10 15-150-100-50050100150I /µAscan rate1/2 /(mV/s)1/2Fe4-NNNNNNFe4-NNNNNNCytochrome c (cyt c)5. Proof-of-concept: Nitrite biosensorConclusions• The interfacial electron transfer properties of the three WO3nanostructures were compared.• The electroactive areas of the WO3/ITO electrodes were calculated using ferrocyanide electron transfer probe and showed a significant increase due to the surface area of the nanostructured films.• The higher conductivity of the WO3·0.33H2O nanostructures contributed for faster and reversible redox reactions with cyt c.• ccNiR modified electrodes showed good electroactivity towards the reduction of nitrite. The lowest response was attained with the W2 electrodes as a result of the high capacitive current and impedance of this electrode.• ccNiR/WO3/ITO is a promising alternative for ccNiR based electrochemical biosensor as the sensitivities are similar to those obtained for single-walled carbon nanotubes.• WO3/ITO electrodes represent novel, biocompatible and efficient platforms for the development of protein electron transfer reactions.0.0 0.2 0.4-8.0-6.0-4.0-2.00.02.04.06.08.0W1/ITO0 10 20 30-2.0-1.00.01.02.03.0I (µA)0.1 0.3 0.5-8.0-6.0-4.0-2.00.02.04.06.08.00.0 0.2 0.40.1 0.3 0.5-8.0-6.0-4.0-2.00.02.04.06.08.00.0 0.2 0.4Potential (V vs. Ag/AgCl)0.1 0.3 0.50 2010 30-2.0-1.00.01.02.03.0I (µA)W2/ITO W3/ITO0.0 0.1 0.2 0.3 0.4 0.5ITO-8.0-6.0-4.0-2.00.02.04.06.08.0Current (µA)0 10 20 30-2.0-1.00.01.02.03.0I (µA)Potential (V vs. Ag/AgCl)Potential (V vs. Ag/AgCl)Potential (V vs. Ag/AgCl)Current (µA)Current (µA)Current (µA)0 20-2.0-1.00.01.02.03.0I (µA)scan rate1/2 /(mV/s)1/210 30scan rate1/2 /(mV/s)1/2scan rate1/2 /(mV/s)1/2scan rate1/2 /(mV/s)1/2• Drop casted immobilized enzyme (ccNiR)• Sensitivity between 1421 and 2143 mA/M cm2Composite transducere-e-e- e-e-Glass substratePortable and low-cost sensorKochi methodk0 = 2.18 (nFD0/RT)0.5 exp(-2nFEp/RT)k0 = 6.19  10-4 cm/s k0 = 1.34  10-3 cm/sk0 = 2.62  10-3 cm/s k0 = 1.49  10-3 cm/sMichaelis-Menten equationIcat = ImaxC / (C + KM)

WO3 nanoparticles probes for direct electron transfer of proteins

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WO3 nanoparticles probes for direct electron transfer of proteins

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