A New Biosensor for Glucose Based on Screen Printed Carbon Electrodes Modified with Tin (IV)-Oxide ()
1. Introduction
Nowadays electroanalytical methods are widely used for monitoring different important parameters in environment, food industry, medicine etc. because of their easy applicability and low cost. Development of amperometric sensors for determination of glucose in biological fluids is significant for the diagnosis and management of metabolic disease diabetes mellitus. Electrode materials used may vary a lot in their electrochemical properties and prices but most of the investigations are focused on low cost assays. Carbon paste electrode as an electrochemical sensing material has a lot of advantages including simple preparation and modification, as well as easy to handle. They also have a wide range potential application, which makes them very useful in electrochemical analysis for different analytes [1]. Screen-printing technology offers the advantages of miniaturization and mass fabrication for cost reduction [2]. Using oxidases as modifiers for electrochemical biosensors allows in many cases monitoring of the intermediate hydrogen peroxide, which is electroactive, but at relatively high potentials [3]. Due to its biological importance, hydrogen peroxide can be detected with chemically modified carbon paste electrodes in lower potential and make them useful in biological media without significant interference from sample matrix. Different modifiers were investigated on their ability to decrease overpotentials of hydrogen peroxide on carbon paste electrodes, such as iron and platinum metal or other transition metal oxides or complexes or organic compounds [4]. Metal oxides such as MnO2 [5], CuO [6], Fe2O3 [7], RhO2 [8], RuO2 [9], ReO2 [10] were found suitable for this purpose.Our research in this study is focused on the possibilities of carbon-based heterogeneous electrodes modified with tin (IV) oxide for the detection of hydrogen peroxide and its potential application as a glucose biosensor through glucose glucose oxidase/ Nafion entrapment on the electrode surface.
2. Experimental
2.1. Chemicals, Reagent and Solutions
Glucose oxidase (EC 1.1.3.4. from Aspergillus niger, 210 U/mg solid), Nafion® (5% m:m solution in lower aliphatic alcohols), tin (IV) oxide were purchased from Fluka. All chemicals used were of analytical reagent grade. Phosphate buffer solution (PBS) was prepared by mixing aqueous solutions of sodium dihydrogen phosphate (0.1 mol/L) and disodium hydrogen phosphate (0.1 mol/L) until the required pH was achieved. Hydrogen peroxide and glucose stock solution of 1.000 g∙L−1 were prepared and diluted appropriately; the glucose stock was left overnight at room temperature to allow equilibration of the anomers. Solutions of uric acid, ascorbic acid, xantine and hypoxantine, paracetamol were freshly prepared before use.
2.2. Apparatus
For cyclic voltammetry and hydrodynamic amperometry, a potentiostat EG&G Princeton Applied Research (Model 264A) with X-Y Recorder and a potentiostat Autolab PSTAT 10 with software GPES version 4.9 were used. The electrochemical cell consisted of a carbon paste electrode as the working electrode, an Ag/AgCl reference electrode (Metrohm 6.0733.100), and a platinum wire as the counter electrode. Argon or nitrogen was used for degassing the solutions. A magnetic stirrer provided convection of the solution. The flow injection system was assembled from a potentiostat (BAS 100B and the corresponding software, BAS 100W, version 2) as the detector, a high performance liquid chromatographic pump (510 Waters, Milford MA, USA in connection with a system controller, Waters 600E), a sample injection valve (5020 Rheodyne, Cotati, CA, USA), and a thin layer electrochemical detector (LC 4C, BAS, West Lafayette, Indiana, USA) with a flow through cell (spacer thickness 0.19 mm; CC-5, BAS). The working electrode was a screenprinted carbon electrode (modified with tin dioxide alone or in combination with a Nafion film containing glucose oxidase), the reference electrode an Ag/AgCl (3 M KCl) electrode, and the counter electrode was the steel back plate of the cell. All potentials mentioned in this paper are referred to the Ag/AgCl reference electrode.
2.2.1. Preparation of Working Electrodes
Unmodified carbon paste was prepared by mixing 1.000 g graphite powder and 360 μL paraffin oil (Uvasol®, 0.84 - 0.89 kg/L,) in an agate mortar by gently stirring with a pestle until uniformity and proper compactness was obtained. Modified carbon paste was prepared by mixing 0.950 g graphite powder with 0.050 g SnO2 and 360 μL paraffin oil. The carbon pastes were transferred to glass vials and allowed to stand overnight in a refrigerator.
Screen printed electrode modified with SnO2 (5% m: m), was prepared by weighing the desired mass of tin (IV) oxide into a glass vial, and the necessary mass of carbon ink (Electrodag 421 SS, Acheson). The mixture was stirred for 20 - 30 min with a glass-rod. After sonicating for about 20 min, the mixture was immediately printed onto ceramic supports (sintered aluminum oxide plates, COORS) using a semiautomatic screen printer (SP-200, MPM) and were dried at room temperature overnight.
Biosensors were prepared by coating the SnO2-modified SPE with a Nafion®/Glucose oxidase solution which was obtained by mixing 40 μL neutralized Nafion solution, 40 μL glucose oxidase solution (50 mg/mL) and 160 μL ethanol. 10 μL of the casting solution were drop-coated on the SPE surface (125 mm2). After the first layer had become dry at room temperature, a second layer was drop-coated similarly with 10 μL casting solution.
2.2.2. Procedures
Cyclic voltammograms were scanned between +1200 mV and −1200 mV with a scan rate of 20 mV/s except if stated otherwise. Hydrodynamic amperometric measurements were made at an operating potential of −200 mV if not mentioned otherwise; 250 mg/L H2O2 were added per step. Amperograms in flow injection analysis were recorded at potentials at −200 mV. The flow rate of the carrier (PBS 0.1) was 0.2 mL/min and the injection volume of the analyte was 200 µL
2.3. Analyses of Samples
Freshly sampled blood was transferred into a plastic tube containing an anticoagulant (EDTA) and centrifuged for 30 minutes at 4500 rpm. An amount of 1.00 mL of plasma was diluted with 9.00 mL PBS (0.1 M). The determination of the glucose level in the supernatant plasma was done in FIA mode using the standard addition method to exclude effects of the matrix and interferents. Reference determinations were made with a commercial glucometer Ascensia BRIO using whole blood.
3. Results and Discussion
Carbon paste electrodes and screen printed electrodes, unmodified and modified with SnO2, were studied to characterize the electrochemical behavior of the modifier and to evaluate its action on the analyte, hydrogen peroxide. The glucose biosensor was designed on the basis of a screen printed carbon electrode modified with tin dioxide in the electrode bulk and glucocose oxidase entrapped in a Nafion film on the electrode surface.
3.1. Studies with Hydrogen Peroxide
3.1.1. Cyclic Voltammetry
Figure 1 compares the cyclic voltammograms of a plain carbon paste electrode and a CPE modified with tin (IV) oxide. At positive potentials practically no difference between both electrodes can be noticed. At negative potentials the reduction of SnO2 can be noticed, probably to SnO, with a peak potential of around −0.8 V vs. Ag/AgCl. At this region also significant currents arise with the un-