Sensors and Actuators B 57 Ž1999. 268–273 www.elsevier.nlrlocatersensorb
Prussian Blue-based ‘artificial peroxidase’ as a transducer for hydrogen peroxide detection. Application to biosensors Arkady A. Karyakin ) , Elena E. Karyakina Faculty of Chemistry, M.V. LomonosoÕ Moscow State UniÕersity, 119899, Moscow, Russian Federation Accepted 17 February 1999
Abstract The present investigation of a novel electrochemical transducer is defined by the requirements of the one of the most significant fields of modern analytical biotechnology, i.e., electrochemical biosensors. Prussian Blue, which has been deposited on the surface of glassy carbon electrode under certain conditions, was found to be a selective electrocatalyst for H 2 O 2 reduction in the presence of O 2 . In its reduced form ŽPrussian White. the inorganic polycrystal is known to be partially soluble in aqueous solution. To stabilize the electrocatalyst at cathodic potentials and preventing loss from the electrode surface, an independent investigation was performed. As a result, a completely stable electrocatalyst was achieved. The kinetics of H 2 O 2 reduction onto the surface of Prussian Blue modified electrodes was investigated. Due to the high catalytic activity and selectivity which were comparative with biocatalysis, the specially deposited Prussian Blue was denoted as ‘artificial peroxidase’. The biosensors were made by enzyme immobilization on the top of the Prussian Blue modified electrodes. The electrochemical transducer and biosensors were suitable for detection of low analyte levels and were practically independent of potentially interfering reductants. q 1999 Elsevier Science S.A. All rights reserved. Keywords: Prussian Blue; Modified electrodes; Hydrogen peroxide electroreduction; Amperometric biosensors
1. Introduction Accurate, rapid, cheap and selective analysis is required nowadays for clinical and industrial laboratories. Electrochemical biosensors seem to accomplish this function. Starting from the work of Clark and Lyons w1x, amperometric biosensors have been intensively studied for three decades. Among different types of enzymes, the oxidases Žglucose, cholesterol, amino acid oxidases. found wide practical application. Operation of the biosensor requires the successive coupling of the enzyme and electrochemical reactions. The first-generation biosensors are based on direct electrochemical detection of the substrate or the product of enzyme reaction. In case of oxidases, they are oxygen and hydrogen peroxide correspondingly. Amperometric detection of these substances was usually done at platinum or platinised electrodes w1–3x. Detection of oxygen consumption at negative potentials Žy0.6 V vs. AgrAgCl. w1x was )
Corresponding author.
[email protected]
Fax:
q 7-095-939-27-42;
E-mail:
the most simple procedure. Nevertheless such biosensors were not able to detect low analyte concentrations. The reasons were: Ži. a great excess of oxygen, Žii. variation of oxygen concentrations in biological liquids and Žiii. reduction of hydrogen peroxide at similar potentials. The detection of hydrogen peroxide in the presence of oxygen was done by its oxidation at anodic potentials Ž) 0.6 V, AgrAgCl. w3x. These systems were found to be the most progressive. However all biological liquids contained a lot of reductants, for example, ascorbate, bilirubins etc. Such reductants were able to be oxidized at similar potentials and produced noise in the added to the anodic current w4x. That also reduced the sensitivity of biosensors. To provide electron transfer pathways between the enzyme active sites and the electrode one can use soluble or immobilized artificial mediators. This approach is realized in so-called second-generation biosensors w5x. The disadvantage of the oxidase-based mediator systems is the influence of reductants. Moreover, some oxidases were reported not to react with these artificial mediators. One of the advantages of the first-generation biosensors is that the enzyme can use its own ‘natural’ reagent, i.e., molecular
0925-4005r99r$ - see front matter q 1999 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 9 9 . 0 0 1 5 4 - 9
A.A. Karyakin, E.E. Karyakinar Sensors and Actuators B 57 (1999) 268–273
oxygen. An absence of artificial mediators avoids the problem of competition between different electron acceptors. Taking into account the above discussion one can conclude that for optimal amperometric oxidase-based biosensors, a selective electrocatalyst for hydrogen peroxide reduction is required. Using a peroxidase enzyme electrode Žperoxidases are responsible in nature for reduction of hydrogen peroxide. w6x, one can develop a selective method for the detection of H 2 O 2 . However, the enzymes, being biological macromolecules, obviously cannot provide long-term stability due to their inherent instability because of the denaturation process. Moreover, some commonly found reducing agents in ‘real’ samples may compete with the electrode as the source for electrons for the oxidized form of peroxidase resulting in erratic signals. An approach to low potential detection of hydrogen peroxide was made using carbon electrodes with deposited noble metals w7–9x. The highest activity in both reduction and oxidation of hydrogen peroxide was peculiar to the electrodes covered with platinum w9x. However, the best selectivity to hydrogen peroxide reduction in the presence of oxygen was shown for rhodium w7,8x, which exhibited approximately three times lower electrocatalytic activity as compared with Pt w9x. Unfortunately, in general, noble metals were not suitable for selective reduction of H 2 O 2 in the presence of O 2 . At rhodium-coated carbon fiber electrodes, the cathodic current was 10 times increased after addition of 100-fold excess of hydrogen peroxide as compared with oxygen content w7x. As a result, the corresponding biosensors did not exhibit high sensitivity. An alternative way for a low-potential and selective detection of hydrogen peroxide was recently demonstrated by our group w10x. Prussian Blue polycrystals deposited in a defined way onto the electrode surface were shown to be active and selective electrocatalysts for H 2 O 2 reduction w11x. Due to their catalytic ability, which remind biological catalysts, the specially deposited Prussian Blue was denoted as ‘artificial peroxidase’ w12x. In the present paper, we report the stabilization of Prussian Blue modified electrodes at negative potentials as well as their application for development of biosensors. Application of ‘artificial peroxidase’ enables the sensing of H 2 O 2 at around 0 V vs. SCE.
2. Experimental 2.1. Materials All inorganic salts were obtained at highest purity. Hydrogen peroxide was purchased from Aldrich ŽSteinheim, Germany. as a concentrated solution and was titrated before use. The solutions throughout this work were prepared using water from a Milli-Q system ŽMilli-
269
pore, Bedford, MA.. Absolute ethanol was prepared by distillation of sodium alcoholate and used immediately. Nafion Ž5% solution in 90% light alcohols. was obtained from Aldrich ŽGermany.. Glucose oxidase ŽE.C. 1.1.3.4. from Aspergillus niger ŽVII-S 180 IUrmg. was produced by Sigma ŽUSA.. Glutamate oxidase ŽE.C.1.4.3.11, from Streptomyces sp., 8 IU mgy1 . was given by Dr. H. Kusakabe of Yamasa Shoyu, Japan. Glucose, L-Glutamic acid Žmonosodium salt., D-glutamic acid, D,L-aspartic acid and ascorbic acid Žsodium salt. were obtained from Sigma. Stock solutions of these reagents were prepared in buffers immediately prior to use. 2.2. Instrumentation Electrochemical measurements were performed using EG and G potentiostat–galvanostat system PAR 273 ŽPrinceton, NJ.. Hydrodynamic experiments were carried out with a home-made low-noise potentiostat ŽZata ¨ Elektronik, Lund, Sweden.. Solution flow was maintained by digital Gilson peristaltic pump Žtype SVA, Buchs, Switzerland.. 2.3. Electrochemical methods A three-compartment electrochemical cell contained a platinum net auxiliary electrode and an AgrAgCl reference electrode in 1 M KCl. The cell construction allowed deaeration of the working electrode space. Glassy carbon disk electrodes Ždiameter 1.5 mm. were used as working electrodes. Prior to use, glassy carbon electrodes were mechanically polished with alumina powder ŽAl 2 O 3 , 1 m. to a mirror finish observed. The flow-through cell w13x was of the confined wall-jet type. The flow-injection and flow-through set up and experiments were done according to our previous report w11x. The flow rates used were of 0.7–0.8 mlrmin for H 2 O 2 and glucose analysis and 0.5 mlrmin for glutamate detection. The dispersion coefficient of about 1.5 was reached by injection of 50 ml of the sample. Flow-injection hydrogen peroxide and glutamate or glucose analysis was carried out in 0.05 M phosphate buffer with 0.1 M KCl at y50 mV or 0 mV ŽAgrAgClr0.1 M KCl.. 2.4. Electrodeposition of Prussian Blue Electrodeposition of Prussian Blue was done by applying a constant potential of 0.4 V within 60 s. The initial solution contained 2 mM K 3 wFeŽCN. 6 x and 2 mM FeCl 3 . The supporting electrolyte was 0.1 M KCl with various amount of HCl. After deposition the Prussian Blue films were activated in the same supporting electrolyte solution, which was used for film growth, by cycling the applied
270
A.A. Karyakin, E.E. Karyakinar Sensors and Actuators B 57 (1999) 268–273
potential in a range of y0.05 0.35 V at a sweep rate of 50 mVrs. The total amount of Prussian Blue was f 6 nmolrcm2 , if it is assumed a transfer of 4 electrons per unit cell w14x. 2.5. Stability of Prussian Blue Stability of Prussian Blue was investigated in 0.1 M KCl. The modified electrodes were cycled several times between y0.05 and 0.35 V ŽAgrAgClr1 M KCl. and then exposed to a constant potential of y0.05 V for a certain time whereafter cyclic voltammograms in the same potential region were recorded. The sum of the anodic and cathodic peak currents Žpeak-to-peak current. found from cyclic voltammograms after poising the potential at y0.05 V was related to its value before applying the potential at y0.05 V. 2.6. Enzyme containing Nafion membranes Enzyme containing Nafion membranes were prepared according to our recently reported method w15x from enzyme suspensions in 90% ethanol and 10% water. For this aim the lyophilised enzyme samples were dissolved in water to a final concentration for glutamate oxidase of 15 mgrml and glucose oxidase of 40 mgrml. Nafion stock solution Ž5%. was diluted by absolute ethanol 5 times and neutralized by alcohol alkaline. The enzyme–polyelectrolyte complex in 90% ethanol was made by further dilution of Nafion solution by absolute alcohol and subsequent mixing with the enzyme water solution. Final enzyme concentrations in the complex were 1.25 " 0.5 mgrml. Enzyme containing Nafion membranes were prepared by syringing 5 l of the enzyme–polyelectrolyte complex to the surface of the Prussian Blue modified electrode and allowing the solvent to evaporate as was described in our work w11x.
3. Results and discussion 3.1. Stability of Prussian Blue electroactiÕe layers Prussian Blue was deposited onto the electrode surface by reduction of ferrous ions in the presence of ferrocyanide at a constant potential as described in Section 2. The chosen deposition method seemed to give the most regular structure of the inorganic polycrystal w11,16x. Indeed cyclic voltammograms of Prussian Blue modified electrodes in the supporting electrolyte solution ŽFig. 1. exhibited sharp peak currents indicating a regular structure of the inorganic polycrystal and a waveform indicating the rapid electrochemistry of a surface immobilised redox active compound. This particular inorganic polycrystal was found in our previous investigation to be a selective electrocatalyst for H 2 O 2 reduction in the presence of oxygen w16x.
Fig. 1. Cyclic voltammograms of Prussian Blue modified electrodes in 0.1 M KCl, sweep rate 50 mVrs.
The Prussian Blue modified electrodes prepared as reported w11x were quite stable in neutral and acidic supporting electrolyte solutions under cyclic voltammetric conditions. No decrease in peak currents was observed while cycling from 10 to 50 times in a range of y0.05 and 0.35 V ŽAgrAgClr1 M KCl. at a sweep rate of 50 mVrs. However, at cathodic potentials corresponding to the redox state of Prussian White, the inorganic polycrystal leaked from the electrode surface. Among the several approaches to achieve stabilization of Prussian White on the electrode surface, the most successful one was to grow more regular crystals during the deposition. When the electrodes were grown from the solution contained 0.1 M HCl, the resulting polycrystal showed excellent stability at y0.05 V in 0.1 M KCl. Fig. 2 presents related peak-to-peak current values found from cyclic voltammograms after poising of the modified electrodes to negative potentials. It is seen that during first 3 h, no leaking of Prussian Blue from the electrode surface was observed. 3.2. Prussian Blue as actiÕe and selectiÕe electrocatalyst of H2 O2 reduction Hydrodynamic current–potential curves of hydrogen peroxide reduction at Prussian Blue modified electrodes are presented in Fig. 3. To find the half-wave potential Ž E1r2 . and the number of electrons transferred per catalytic cycle Ž n., the experimental data were fit to equation:
ž
i s i lim 1 q exp
½
Ž E y E1r2 . nF RT
y1
5/
where i lim is limiting current density. Taking into account the difference between Ag
