1. New electrode materials for H 2 O 2 New electrode materials for H 2 O 2 based sensors and biosensors Francesco Ricci 1, Carla Gonçalves 2, Giuseppe Palleschi 1, Aziz Amine 3, Danila Moscone 1 1 Dipartimento di Scienze e Tecnologie Chimiche, Università “Tor Vergata”, Roma, Italia 2 Departamento de Química, Faculdade de Ciências e Technologia, Universidade de Coimbra, Coimbra, Portugal 3 Facultè de Sciences et Techniques, Universitè de Mohammadia, Mohammadia, Morocco Conclusions The novel Prussian Blue deposition on Screen Printed Electrodes and Glassy Carbon Paste Electrodes resulted in effective sensors for H 2 O 2 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. Prussian Blue Modification by chemical deposition: FeCl 3 + K 3 Fe(CN) 6 20 µl K 3 Fe(CN) 6 10 mmol l -1 + 20 µl FeCl 3 10 mmol l -1 90 min at 100 0 C 1 g. of Glassy Carbon powder in a solution of: FeCl 3 5 mmol l -1 + K 3 Fe(CN) 6 5 mmol l -1 Filtration 90 minutes at 100 0 C INTRODUCTION: Prussian Blue or Exacyanoferrate ferric has been selected as catalyst for the H 2 O 2 reduction in order to obtain a reliable, selective and interferences free tool for H 2 O 2 detection. Two electrode materials have been used: Screen Printed Electrodes (SPE’s) Glassy Carbon Paste Electrodes (GCPE’s)  Disposable  Cheap  For “in situ” use  Ag Internal pseudo-reference electrode;  Graphite working electrode;  Ag Counter-electrode; 3 mm INTRODUCTION: Prussian Blue (Ferric Hexacyanoferrate) has been selected as catalyst of the H 2 O 2 reduction to obtain a reliable, selective and interferences free device for H 2 O 2 detection. Two new electrode materials have been used: 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 Teflon Tube Pt Connector Improved stability at alkaline pH’s for PB modified electrodes E (V) vs. int. ref. i (mA) -0.3 -0.2 -0.1 0 0.30.2 0.1 0.4 -0.075 0.075 0.05 -0.025 0.025 0 -0.05 Continuous cycling in phosphate buffer 0.1 mol l -1 + KCl 0.1 mol l -1 First cycle 250 th cycle pH 9 Amperometric signal for H 2 O 2 10 µmol l -1 after 250 cycles 90% of the initial value -0.03 -0.02 -0.01 0 0.01 0.02 - 0.03 E (V) - 0.100 0 0.1000.2000.3000.4000.5000.600 i (mA) 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%) First cycle 250 th cycle pH 9 Amperometric signal for H 2 O 2 10 µmol l -1 after 250 cycles 75% of the initial value Analytical parameters of PB-SPE to H 2 O 2 Batch analysis applied potential -50 mV vs int. ref. Detection limit0.1 µmol l -1 Linearity range0.1-50 µmol l -1 RSD% (n= 7 elctrodes) 5% Response time (90%) 5 sec Sensitivity (µA mmol l -1 cm -2 ) 324 Ascorbic acid/H 2 O 2 signal ratio 0.02 0.000.010.020.030.040.050.06 0.00 0.25 0.50 0.75 1.00 1.25 1.50 i (  A) [H 2 O 2 ] (mmol l -1 ) 0.0000.0010.0020.003 0.00 0.02 0.04 0.06 PB modified Screen Printed Electrodes Prussian Blue modified electrode Prussian Blue modified electrode in H 2 O 2 (10 mM) Non modified electrode 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 H 2 O 2 an increase of the cathodic peak at potential near to 0.0 V is observed due to the reduction of H 2 O 2 CV in phosphate buffer 0.1 mol l -1 + KCl 0.1 mol l -1, pH 6 - 0.5 -0.25 0 0.25 0.5 0.75 E (V) i (A) 0 CV in phosphate buffer 0.1 mol l -1 + KCl 0.1 mol l -1, pH 6 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 1  A g 0.1  A h 5  A e 1  A f 10  A d 25  A b a c -0.1 0.2 0.6 E (V) 10  A Best paste composition: 90/10 GC/PB-GC w/w % (e)  Low  Ep value  High peak currents  High response to H 2 O 2 Applied for H 2 O 2 detection and oxidase enzyme modification 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 -0.1 0.2 10  A 0.5 CV in phosphate buffer 0.1 mol l -1 + KCl 0.1 mol l -1, pH 6 E (V) i (µA) Detection limit0.5 µmol l -1 Linearity range0.5-500 µmol l -1 Response time15 sec. Sensitivity (µA mmol l -1 cm -2 ) 99 RSD% (n=3 electrodes)6% Analytical parameters for PBGCPEs to H 2 O 2 Batch analysis applied potential 0.0 V vs Ag/AgCl Analytical parameters Choline Biosensors A novel enzyme immobilisation technique is proposed: a solution of: Glutaraldehyde + Nafion + BSA + choline ox. is placed onto the PB modified electrode Detection limit0.5 µmol l -1 Linearity range0.5-100 µmol l -1 Response time5 sec. RSD% (n=8 biosensors)6% Sensitivity (µA mmol l -1 cm -2 ) 110 Batch analysis applied potential -50 mV vs int. ref. Glucose biosensor 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 % Detection limit50 µmol l -1 Linearity range50-200 µmol l -1 Response time30 sec. RSD% (n=3 biosensors)4 % Sensitivity (µA mmol l -1 cm -2 ) 6.4 Analytical parameters Batch analysis applied potential 0.0 V vs Ag/AgCl Storage stability Operational stability Storage condition Numberof sensors Time Measurements frequency Residual activity (%) 4°C20 30 weeks Every 2 weeks 90  11 4°C49monthsEvery month 87  3 RT37daysEvery day 90  5 RT228days Every 3 days 95  10 number of measurements 0102030405060 % of initial signal 0 25 50 75 100 , , ,  = 4 different choline biosensors