New electrode materials for H 2 O 2 based sensors and biosensors

1. Residual Storage Number of Time Measurements frequency 1 mA 25 mA 10 mA 25 mA 0.1 mA activity condition sensors (%) 30 Every 2 ± 4°C 20 90 11 weeks weeks INTRODUCTION: Prussian Blue or Exacyanoferrate ferric has been selected as catalyst for the H2O2 reduction in order to obtain a reliable, selective and interferences free tool for H2O2 detection. Two electrode materials have been used: Screen Printed Electrodes (SPE’s) Glassy Carbon Paste Electrodes (GCPE’s) INTRODUCTION: Prussian Blue (Ferric Hexacyanoferrate) has been selected as catalyst of the H2O2 reduction to obtain a reliable, selective and interferences free device for H2O2 detection. Two new electrode materials have been used: ± 4°C 4 9 months Every month 87 3 ± RT 3 7 days Every day 90 5 Every 3 ± RT 2 28 days 95 10 days - 0.5 -0.25 0 0.25 0.5 0.75 Glassy Carbon Paste: 80% of Glassy carbon spherical powder (0.4-12 µm) + 20% of Mineral Oil  Renewable surface  Easy to modify and prepare  High electrochemical activity Ag Internal pseudo-reference electrode;  Graphite working electrode;  Ag Counter-electrode;  Disposable  Cheap  For “in situ” use 3 mm 5 mA Pt Connector Teflon Tube 20 µl K3Fe(CN)6 10 mmol l-1 + 20 µl FeCl3 10 mmol l-1 90 min at 100 0C 1 g. of Glassy Carbon powder in a solution of: FeCl3 5 mmol l-1 + K3Fe(CN)6 5 mmol l-1 Filtration 90 minutes at 100 0C Prussian Blue Modification by chemical deposition: FeCl3 + K3Fe(CN)6 Prussian Blue modified Glassy Carbon Paste Electrodes PB Glassy carbon Paste Electrode (PB-GCPE) was first obtained by hand mixing PB glassy carbon and mineral oil (80/20 w/w %) N.B.  No anodic and no cathodic peaks probably due to the high amount of deposited PB (non conducting) The composition was optimised by decreasing the amount of PB-modified GC PB modified Screen Printed Electrodes When the electrode is modifed with PB the two anodic and cathodic peaks due to the oxidation and reduction of PB are present. In presence of H2O2 an increase of the cathodic peak at potential near to 0.0 V is observed due to the reduction of H2O2 0.5 Non modified electrode 0 CV in phosphate buffer 0.1 mol l-1 + KCl 0.1 mol l-1, pH 6 i (A) Prussian Blue modified electrode 10 mA Prussian Blue modified electrode in H2O2 (10 mM) -0.1 0.2 Glassy Carbon mixture Composition GC /PB-GC a) 0/100 b) 25/75 c) 50/50 d) 75/25 e) 90/10 f) 95/5 g) 98/2 h) 100/0 w/w % Glassy carbon paste: 80% glassy carbon mixture + 20% mineral oil E (V) CV in phosphate buffer 0.1 mol l-1 + KCl 0.1 mol l-1, pH 6 Analytical parameters of PB-SPE to H2O2 1.50 0.06 a d 1.25 0.04 Detection limit 0.1 µmol l-1 0.02 1.00 h e g f i (mA) 0.00 Linearity range 0.1-50 µmol l-1 0.000 0.001 0.002 0.003 0.75 Best paste composition: 90/10 GC/PB-GC w/w % (e) RSD% (n= 7 elctrodes) 5% Batch analysis applied potential -50 mV vs int. ref. 0.50 10 mA Response time (90%) 5 sec  Low DEp value  High peak currents  High response to H2O2 0.25 1 mA 0.6 -0.1 0.2 E (V) Sensitivity (µA mmol l-1 cm-2)324 0.00 0.00 0.01 0.02 0.03 0.04 0.05 0.06 [H2O2] (mmol l-1) Ascorbic acid/H2O2signal ratio0.02 c b Applied for H2O2 detection and oxidase enzyme modification CV in phosphate buffer 0.1 mol l-1 + KCl 0.1 mol l-1, pH 6 Continuous cycling in phosphate buffer 0.1 mol l-1 + KCl 0.1 mol l-1 for PB-GCPE (Glassy carbon mixture = GC/PBGC 90/10 w/w%) Improved stability at alkaline pH’s for PB modified electrodes Continuous cycling in phosphate buffer 0.1 mol l-1 + KCl 0.1 mol l-1 0.075 0.05 pH 9 pH 9 0.025 0.03 - i (mA) 0.02 0 Amperometric signal for H2O2 10 µmol l-1 after 250 cycles Amperometric signal for H2O2 10 µmol l-1 after 250 cycles 0.01 First cycle 250th cycle First cycle 250th cycle -0.025 i (mA) 0 -0.05 -0.01 -0.02 -0.075 -0.03 -0.2 -0.1 -0.3 0 0.1 0.2 0.3 0.4 0 -0.100 0.100 0.200 0.300 0.400 0.500 0.600 E (V) vs. int. ref. 90% of the initial value 75% of the initial value E (V) Analytical parameters for PBGCPEs to H2O2 Analytical parameters Batch analysis applied potential -50 mV vs int. ref. Choline Biosensors A novel enzyme immobilisation technique is proposed: a solution of: Detection limit 50 µmol l-1 Detection limit 0.5 µmol l-1 Linearity range 50-200 µmol l-1 Linearity range 0.5-100 µmol l-1 i (µA) Detection limit 0.5 µmol l-1 Batch analysis applied potential 0.0 V vs Ag/AgCl Sensitivity (µA mmol l-1 cm-2)6.4 Sensitivity (µA mmol l-1 cm-2)110 Linearity range 0.5-500 µmol l-1 Glutaraldehyde + Nafion + BSA + choline ox. is placed onto the PB modified electrode Response time 15 sec. Response time 5 sec. Response time 30 sec. Sensitivity (µA mmol l-1 cm-2)99 RSD% (n=3 biosensors) 4 % RSD% (n=8 biosensors) 6% RSD% (n=3 electrodes) 6% Storage stability Operational stability Batch analysis applied potential 0.0 V vs Ag/AgCl Analytical parameters Glucose biosensor 100 75 % of initial signal 50 Glucose biosensors were obtained by hand mixing 7.5% PB-GC + 87.5% GC + 5% glucose oxidase and then mixing with mineral oil 80/20 w/w % 25 0 0 10 20 30 40 50 60 number of measurements ,,, = 4 different choline biosensors • New electrode materials for H2O2 based sensors and biosensors • Francesco Ricci1, Carla Gonçalves2, Giuseppe Palleschi1, Aziz Amine3, Danila Moscone1 • 1Dipartimento di Scienze e Tecnologie Chimiche, Università “Tor Vergata”, Roma, Italia • 2Departamento de Química, Faculdade de Ciências e Technologia, Universidade de Coimbra, Coimbra, Portugal • 3Facultè de Sciences et Techniques, Universitè de Mohammadia, Mohammadia, Morocco E (V) Conclusions The novel Prussian Blue deposition on Screen Printed Electrodes and Glassy Carbon Paste Electrodes resulted in effective sensors for H2O2 detection, with improved stability at alkaline pH’s, high sensitivity and low electrochemical interference. These characteristics make these sensors the ideal support of enzyme immobilisation for the assembling of an “oxidase” based biosensor. Choline Biosensors realised immobilising choline oxidase onto PB-SPE showed a low detection limit and a good reproducibility. A long-term storage and operational stability, due to the proposed immobilisation procedure, has been also observed. The new GCPEs showed after PB deposition an electrochemical behaviour better than classic Carbon Paste electrodes. Glucose biosensors prepared by hand mixing glucose oxidase within the PB-Glassy Carbon Paste mixture showed a good sensitivity and promising features for further experiments.