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Dual oxygen and Ir oxide regeneration of glucose oxidase in nanostructured thin film glucose sensors
Electrochimica Acta 55 (2010) 7683–7689
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Dual oxygen and Ir oxide regeneration of glucose oxidase in nanostructured thin film glucose sensors Amit S. Jhas a , Hanna Elzanowska b,∗ , Bri Sebastian a , Viola Birss a,∗∗ a b
Department of Chemistry, University of Calgary, 2500 University Dr. N.W., Calgary, Alberta T2N 1N4, Canada Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 26 January 2010 Received in revised form 28 March 2010 Accepted 29 March 2010 Available online 3 April 2010 Keywords: Glucose Glucose oxidase Iridium oxide Nanoparticles Direct mediation dual mechanism
a b s t r a c t A nanoparticulate iridium oxide (IrOx) thin film has been developed as a redox-active matrix material for an advanced generation glucose biosensor, in which IrOx serves as the non-physiological mediator, replacing oxygen in the enzymatic re-oxidation of glucose oxidase (GOx). Ethanolic solutions of Nafion and an Ir sol were mixed with an aqueous GOx solution and then deposited on a Au support. The Ir nanoparticles were then oxidized electrochemically to IrOx and the resulting films (IrOx–GOx–Nafion) were tested for their glucose response in both oxygen- and argon-saturated solutions, with the oxygen content in both solutions monitored by a Pt electrode. The sensors that are regenerated largely by O2 are characterized by a Michaelis–Menten Km value of ∼30 mM or more and imax values of at least 20 A cm−2 . Under fully deareated conditions, the sensors lose only ∼50% of their response to glucose, clearly indicating that a dual oxygen-regeneration and IrOx mediation mechanism is operative for the biosensor under these conditions. Under optimized conditions, involving a controlled GOx:Ir ratio, only the Ir oxide sites in the film serve to mediate GOx regeneration, giving Km (10–15 mM) and imax values that are independent of the O2 content of the solution. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Progress in biosensor research over the past decades has resulted in the development of various amperometric glucose sensors, usually referred to as first-, second-, and third-generation biosensors, as described in numerous reviews [1–5]. In 1962, Clark [6] used a metallic Pt electrode covered with a thin layer of glucose oxidase (GOx) and a dialysis membrane to monitor the decrease in the O2 concentration resulting from GOx regeneration (by O2 ) after the reaction of GOx with glucose. This electrode is considered to be the first example of a first-generation amperometric glucose biosensor [2,4]. Ten years later, Guilbault and Lubrano [7] demonstrated a similar glucose sensor, but operating via the detection of hydrogen peroxide. Both oxygen consumption and hydrogen peroxide build-up are still tracked today by first-generation glucose sensors [2,4], even in implants [8], according to the following scheme: GOx + glucose → GOx–H2 + gluconicacid
(1)
GOx–H2 + O2 → GOx + H2 O2
(2)
∗ Corresponding author. Tel.: +48 22 822 0211; fax: +48 22 822 5996. ∗∗ Corresponding author. Tel.: +1 403 220 7306; fax: +1 403 289 9488. E-mail addresses: [email protected] (H. Elzanowska), [email protected] (V. Birss). 0013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2010.03.093
Many scientific works have also been devoted to developing second-generation biosensors, in which artificial mediators [2,4,9,10] are used to replace O2 , as follows: GOx + glucose → GOx–H2 + gluconicacid
(3)
GOx–H2 + M ox → GOx + M red
(4)
M red → M ox + 2e
−
(5)
where Mox and Mred are the oxidized and the reduced forms of the mediator, respectively. Second-generation biosensors have involved the use of ferrocene and its derivatives [9,10], as well as many other substances [11], added to the enzyme layer as mediators. A wide range of matrices, such as conducting polymers [12] and sol–gel (SG)-derived films [13,14], capable of immobilizing both the enzyme and the mediators, have also been tested over the years. However, problems with mediator leakage from the biosensor layer and mixed mediator/oxygen operation of the sensors [2] have prompted researchers to focus on firmly embedding mediators in order to transfer electrons efficiently enough to compete successfully with oxygen in the regeneration of the enzyme. In 1987, Heller’s group [15] described the use of relay-modified glucose oxidase in an electrode layer responding to glucose. The electrical ‘wiring’ was realized by the covalent bonding of 12 ferrocene/ferrocinium centers to each of the enzyme molecules, leading to a so-called ‘direct electrical communication’ between the
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enzyme and the underlying Au or glassy carbon (GC) electrode support. Another well known example [16] of electron-accepting and electron-transferring relays is that of a dense array of Os complexes bound covalently to a long flexible polypyridine polymer. In this case, the biosensor signal for both glucose and lactate was almost the same under oxygen and nitrogen and the Michaelis–Menten ) for GOx was ca. 5 mM, much smaller than the solution constant (Km value for GOx [17]. Electrical ‘wiring’ of the surface-bound enzyme can also be realized using non-metallic components. A recent example of enzyme wiring, proposed by Heller’s group [17], is the use of an electronconducting cross-linked polyaniline-based hydrogel, formed by cross-linking the polyaniline/poly(2-acrylamido-2-methyl-1propane sulfonic acid (PANI/PAAMPSA) with glucose-permeable poly(ethylene glycol) diglycidyl ether (PEGDGE) to ‘co-cross-link’ GOx. In this biosensor, PANI/PAAMPSA has replaced the Os2+/3+ complex, resulting in a rapidly responding and effective biosensor = 16.8 mM, i −2) ). (Km max = 200 M cm The concept of using conductive biosensor layers to immobilize enzymes has been further extended to consideration of immobilization of metallic components. Wang’s group examined glucose oxidation at GOx-containing carbon transducers metalized with Rh [19], Ir [21–23], Ru [23], and Pd [18,20,23]. The metallic component serves as the catalyst for H2 O2 detection and also improves the conductivity of the biosensor layers. In particular, the use of Ir significantly enhanced the biosensor response [23]. Similarly, the role of Au and Ag nanoparticles in the development of these biosensors has been recognized and described in a recent review [5,24,25]. Therefore, the development of a stable and rapid electrodemediated glucose sensor, which is also biocompatible, remains the goal of a significant amount of research. We have therefore devoted our efforts towards the use of Ir/Ir oxide (IrOx) electrodes for this purpose, due to their known electrocatalytic action towards H2 O2 [26,27], excellent electronic conductivity [28–32], biocompatibility [33], and rapid charge/discharge kinetics [29,32]. In our earlier work [34], we deposited GOx simultaneously with the growth of an IrOx film, formed electrochemically on a bulk Ir wire or plate substrate. However, SEM analysis showed that most of the GOx was located on the outer surface of the IrOx film, thus losing the intended stabilizing benefits of the IrOx matrix. This may explain the lack of any evidence for IrOx-mediated glucose oxidation using this electrode [34]. In the present work, we therefore mixed an aqueous solution of GOx with an ethanolic suspension of Ir nanoparticles (2–3 nm in dia) and Nafion, and then cast this mixture as a thin, homogeneous, composite film on Au substrates. The metallic Ir nanoparticles were then oxidized electrochemically to IrOx to enhance their biocompatibility and stability. The performance of this biosensor was examined in both oxygen-saturated (aerated) and Ar-saturated (deaerated) solutions to determine whether the sensor operates as a first-generation biosensor, in which oxygen is used to regenerate GOx, or as an advanced sensor, in which the IrOx matrix re-oxidizes the enzyme. We show that, depending on the relative GOx and Ir content in the film, one of these two possible mechanisms prevails.
2. Experimental 2.1. Synthesis of Ir sols Ir sol synthesis required the dissolution of 0.2 g of sodium ethoxide (NaOC2 H5 , Aldrich, 96% pure) in 6 mL of absolute ethanol (EtOH) by stirring in a 100 mL round bottom flask. IrCl3 (Aldrich, 99.99% pure) was added to the mixture and allowed to reflux under Ar at 73 ◦ C for 2 h. The solution was cooled to 22 ◦ C, stirred for 18 h
under Ar, and then the resulting mixture was sieved through fine filter paper (Qualitative, Whatman). The suspended Ir sol filtrate, containing metallic Ir nanoparticles, was collected and then stored in a sealed glass vial in the refrigerator for future use in biosensor fabrication. The black precipitate (a mixture of Ir oxide and NaCl) was filtered off and kept for further analysis. 2.2. Enzyme immobilization The EtOH-based Ir sol (typically 8 L) was mixed with 2 L of 1% Nafion® (also dissolved in EtOH) followed by the addition, by pipet, of 0.5–4 L of an aqueous glucose oxidase (GOx) solution (60 mg mL−1 ). An aliquot (typically 4 L) of this mixture was then deposited on a Au working electrode (WE) substrate and the electrode was then allowed to dry in the refrigerator for 24 h. 2.3. Electrodes, cells and equipment All electrochemical experiments were performed using either a Solartron Analytical 1480 Multistat or an EG&G PARC 173 Potentiostat. A conventional two-compartment glass cell, containing the coated Au WE and the high surface area Pt mesh counter electrode in the main compartment, connected to the reference electrode (RE) compartment through a Luggin capillary, was used. The WE substrate consisted of glass slides, sputter-coated (Denton DV502A sputtering system) first with an undercoat of Ti to a thickness of ca. 10 nm and then with Au to ca. 80–90 nm, followed by rinsing with methanol and then water. Prior to coating with the Ir sol or Ir sol/GOx mixture, the Au substrate electrodes were cleaned electrochemically by potential cycling in 0.05 M H2 SO4 between 0 and 2.0 V vs. RHE for 35 cycles and then between 0 and 1.5 V vs. RHE until the cyclic voltammetry (CV) features of the Au substrate remained constant in size. The RE was normally a saturated calomel electrode (SCE); however, all potentials in this paper are referred to the reversible hydrogen electrode (RHE). 2.4. Solutions, reagents, and general experimental conditions All solutions were prepared using analytical-grade reagents and triply distilled water. H2 O2 (30%, v/v aqueous solution), dglucose (anhydrous), and potassium dihydrogen phosphate were purchased from BDH Chemicals. Glucose oxidase (GOx, EC 1.1.3.4, Type II-S from Aspergillus Niger, 40,300 unit/g of solid) was obtained from Sigma, while Nafion® was purchased from Fisher Scientific Co. All experiments were carried out in either aerated (with oxygen) or deaerated (with argon gas for a minimum of 25 min) 0.1 M potassium phosphate buffer solution at a pH of 7. 3. Results and discussion 3.1. Electrochemical characteristics of IrOx–GOx–Nafion films in absence of glucose Fig. 1 shows a typical set of cyclic voltammograms (CVs) of a freshly deposited Ir–GOx–Nafion film in pH 7 0.1 M phosphate buffer during the early stages of the conversion (by cycling the potential between 0 and 1.25 V vs. RHE [35,36]) of the metallic Ir component to its hydrous Ir oxide (IrOx) form. The peaks at 0–0.25 V in Fig. 1 are characteristic of the hydrogen deposition/removal process (Hupd) at metallic Ir [27–32] and can be tracked to determine the total area of Ir in the film, as well as the extent of conversion of Ir to IrOx. Indeed, when the potential was repeatedly cycled to 1.25 V, it is seen in Fig. 1 that the Hupd peaks gradually decrease in size. Also, the symmetrical anodic and cathodic peaks, centered at 0.9 V (A1 /C1 ), become more well-formed. These peaks are characteristic [31,32] of the kinetically rapid Ir(III)/Ir(IV) oxide redox
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Fig. 1. Cyclic voltammograms showing the conversion of a thin film of metallic Ir nanoparticles to Ir oxide (at 0.1 V/s in a pH 7 0.1 M phosphate buffer solution). The film was formed using an ethanol Ir sol mixed with 0.04% Nafion® and GOx with 2 L deposited on a Au substrate and then dried at room temperature in air for 24 h. Inset shows linear peak current vs. sweep rate plots for Ir oxide reduction at 0.7 V and oxidation at 1.0 V.
transition within the IrOx layer. At potentials >1.25 V, another set of redox peaks is seen (not shown in Fig. 1), representative of the Ir(IV)/Ir(V,VI) oxide redox reaction. These films exhibit a linear peak current vs. sweep rate relationship up to at least 0.2 V/s, as seen in the inset of Fig. 1. These rapid redox kinetics make IrOx films highly suitable for use as a matrix material hosting a redox-active enzyme in its three-dimensional structure. Notably, the presence of GOx in the film does not influence the electrochemical conversion of Ir to IrOx under these conditions (Fig. 1). Also, it will be shown below that potential cycling of these electrodes in phosphate buffer solution during the IrOx formation process does not harm the enzyme activity. Nafion was added to the Ir sol in order to better distribute the enzyme in the oxide matrix and also to stabilize the Ir (and later IrOx) film on the Au substrate. In fact, it is shown in parallel work that the addition of Nafion to the ethanolic or the mixed aqueous/ethanolic Ir sol has a beneficial effect on the overall IrOx charge and the reversibility of the Ir redox process. This suggests that Nafion can entrap Ir nanoparticles that would not otherwise be incorporated into the film. It is also possible that Nafion improves the Ir/IrOx particle-to-particle connectivity and the Ir/IrOx–GOx contact within the film. All of these factors would serve to increase the amount of active Ir/IrOx and improve the conductivity of the IrOx–Nafion–GOx films. This is similar to the beneficial effect reported [24] when 1.4 nm Au nanocrystals, functionalized with the flavin adenine dinucleotide cofactor, were adsorbed on a dithiol-modified Au electrode, followed by reconstitution with the apo-GOx enzyme. Excellent GOx electron transfer properties were observed in these films, with the Au nanoparticles suggested to serve as an electron relay or ‘electrical nanoplug’ for the alignment of the enzyme on the conductive support and for the electrical wiring of its redox-active center. It has also been suggested previously that Nafion, when mixed with enzymes, can change the pH inside a thin film and thus, to avoid this problem, tetrabutyl ammonium salts were used to modify the Nafion acidity [38]. However, as shown in Fig. 1, the Ir(III)/Ir(IV) oxide redox peak potentials are centered at 0.8 V vs. RHE, regardless of whether or not Nafion is present in the Ir-containing film. Thus, the IrOx–GOx–Nafion-containing film studied here is sufficiently porous to allow the appropriate flux of buffer solution in and out of the film to keep the pH inside the
Fig. 2. (a) Oxidation and reduction of H2 O2 (0.9 mM) at 0.1 V/s in a pH 7 0.1 M phosphate buffer solution, all under Ar (solid line) or oxygen (dashed line), at IrOx films formed using an ethanolic Ir sol, deposited on Au and dried at room temperature for 24 h. (b) Linear plots of H2 O2 oxidation (at 1.2 V) and reduction (at 0.2 V) currents vs. H2 O2 concentration, all under Ar, at IrOx films formed using 10% H2 O/ethanol Ir sol, with (curve 1) and without (curve 2) added 0.04% Nafion® . 2 L of mixture were deposited on Au and dried at room temperature for 24 h.
film constant. This is also a good indication that glucose has good access to GOx, embedded within these layers, and that the product of the reaction (e.g., H2 O2 , in aerated conditions) and gluconic acid will also be readily transported out of these films. To demonstrate that the IrOx–GOx–Nafion films can be used for glucose sensing in aerated phosphate buffer solutions, conditions under which H2 O2 will be produced, the IrOx sol-derived electrodes were examined for their CV response to H2 O2 (Fig. 2a). The H2 O2 oxidation and reduction currents, at 1.2 and 0.2 V, respectively, are shown in Fig. 2b to be linear with H2 O2 concentration. Notably, both of these signals are higher when Nafion is present in the Ir sol, as the amount of Ir successfully transferred into the film is also higher. As shown in our previous work, the stability of IrOx is not adversely affected by the presence of H2 O2 [34]. However, because O2 reduces at these electrodes in essentially the same potential window as does H2 O2 (from 0 to 0.6–0.7 V) at IrOx, the oxidation of H2 O2 (which occurs at 1.0 V and higher) must be tracked [34], conditions under which the sensor operates as a first-generation glucose sensor. Our sensor is quite stable, especially after an initial period of time of equilibration. For example, a 20–25%, decrease in the signal is seen in the first 4–6 days, and then the response is stable for at least 35 days. This topic will be discussed in detail in out next publication.
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Fig. 3. Chronoamperometry plot (at 1.2 V) in stirred, pH 7 0.1 M phosphate buffer solution (25 mL) at room temperature with 200 L increment additions of 1 M glucose (arrows) under saturated O2 conditions for a film formed from a IrOx/GOx mixture. Inset shows the corresponding MM plot.
3.2. Response of IrOx–GOx–Nafion electrode to glucose in aerated and deaerated environments Fig. 3 shows the effect of added glucose to the current recorded at 1.2 V for an IrOx–GOx–Nafion film in a pH 7 buffer solution, saturated with oxygen. The background current is very small (dotted line in Fig. 3), consistent with the capacitive properties of IrOx films, such that the oxidation state of Ir within the oxide film rapidly equilibrates and the current quickly drops to zero at constant potential. Also, the rapid increase (within 2 s) in the chronoamperometric currents recorded after each glucose addition indicates the fast response of our sensor. The application of relatively high positive potentials (1.2 V vs. RHE) does not change the properties of the IrOx and IrOx–GOx–Nafion films. In fact, the CV response of the sensor remains essentially the same before and after potentiostatic measurements involving glucose additions. Due to thee high pseudocapacitative IrOx currents, the currents related to glucose oxidation on IrOx are relatively small in CV experiments, but under potentiostatic conditions, the oxide is fully charged and thus only the signal for the catalytic oxidation of glucose is seen. The inset to Fig. 3 shows the corresponding Michaelis–Menten plot, giving Km and imax values of 27 mM and 46 A cm−2 , respectively, in these value is very similar to the solution aerated conditions. (This Km value for GOx, which is typically around 25 mM [17].) When the experiments described above (Fig. 3) were repeated in deaerated (with Ar) solutions, an excellent response to glucose is still seen, demonstrating that GOx re-oxidation can also occur by direct electron transfer to the Ir oxide nanoparticles within the film matrix. This type of oxygen-independent response is highly desirable for glucose detection in blood, particularly if implanted biosensors are being considered, as a highly variable oxygen concentration can then be expected [2]. The Michaelis–Menten plots for glucose oxidation, in both saturated O2 and saturated Ar environments, are shown in Fig. 4 for direct comparison. In the value for this particular sensor is 21 mM, deaerated solution, the Km i.e., less than 25 mM, and imax is 13 A cm−2 , while in aerated media, and i −2 the Km max values are both larger (29 mM and 24 A cm , respectively). Although the dissolved O2 concentration should be very low in the Ar-saturated cell solution, the maximum glucose oxidation
Fig. 4. Michaelis–Menten plots of a film formed from an IrOx/GOx mixture and tested under (squares) O2 -saturated and (triangles) Ar-saturated conditions. Glucose tests carried out at 1.2 V in stirred, pH 7 0.1 M phosphate buffer solution.
current observed in Fig. 4 at saturation (∼75 mM glucose) is only ∼45% less than in air. In fact, negligible currents would have been expected in the absence of oxygen if the regeneration of GOx were occurring only by O2 (a co-substrate), as observed for other O2 dependent enzymes, e.g., laccase [39], which reduces oxygen to water (oxygen is the substrate for the enzymatic reaction). In the case of laccase [39], when the solution changes from O2 -saturated to air-saturated, a significant decrease in the biosensor signal is observed, and the signal is almost zero (90–95% loss of signal) when the solution is fully deaerated. To verify that electron transfer is indeed occurring between the reduced flavin site in GOx and the surrounding IrOx matrix under deaerated conditions, the amount of oxygen present was carefully determined. The oxygen reduction current (ORR), monitored (not shown) using a second Pt working electrode in an O2 -saturated solution, was found to be 2.1 mA cm−2 at 0.2 V, due the oxygen reduction reaction. After 40 min of deaeration with Ar, the current at 0.2 V is only 1.3 A cm−2 , i.e., the oxygen content has been lowered by at least 1000 times. This would result in a similar decrease in the observed glucose oxidation currents if the only means of GOx regeneration is through reaction with O2 , as seen for laccase [39] As the glucose oxidation currents in deaerated conditions (Fig. 4, <0.3 M O2 ) are much too high to be explained by trace O2 regeneration of GOx, these results argue strongly that the IrOx sites are indeed mediating the GOx regeneration process. This is further supported by the absence of any H2 O2 (the product of O2 regeneration of GOx) monitored electrochemically in the deaerated medium. A chronoamperometric experiment (Fig. 5) was carried out in deoxygenated conditions (oxygen concentration < 0.3 M) at 0.2 V, before and after the addition of glucose. At 0.2 V, the current will respond to the reduction of oxygen or H2 O2 , or both. Since no difference was observed in the current response at 0.2 V before and after carrying out glucose oxidation, indicating the absence of any generated H2 O2 , these results strongly support the conclusion that regeneration of the reduced GOx sites is occurring via mediation by the IrOx nanoparticles. The question could arise as to whether, in a glucose detection experiment at 1.2 V, oxygen is being evolved catalytically on the IrOx electrode, then serving to regenerate the reduced form of GOx. This supposition is refuted by the fact that the glucose signal is still very strong at 1.1 V, and a good response was still obtained even at 1.05 V (Fig. 6a). It is impossible that the insignificant amounts
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Fig. 5. Potential step experiment illustrating the (a, left scale) current at 0.2 and 1.2 V before and after the addition of glucose in stirred, pH 7 0.1 M phosphate buffer solution deaerated with Ar. (b, right scale) Shows the applied potential over the course of the same experiment.
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Fig. 7. MM plots of electrodes composed of GOx/IrOx mixture with mass ratios of 0.25 g/g (1, ), 0.50 g/g (3, ), 1.00 g/g (4, ) and 2.00 g/g (2, 䊉). Glucose testing was performed at 1.2 V in a stirred pH 7 0.1 M phosphate buffer solution under Ar-saturated conditions. Aliquots consisted of 50 L of 2 M glucose.
of O2 that would be generated at these very low potentials could be sufficient to produce the comparatively high currents (at high glucose concentrations) seen in Fig. 4. It can be concluded, therefore, that the IrOx nanoparticles are actively participating in the communication between GOx and the underlying support (Au). This can be realized through the protein part of the enzyme, allowing for electron transfer from the enzyme redox site (the flavin) to the IrOx nanoparticles (at which the Ir(IV/V/VI) redox transitions occur) surrounding the enzyme molecule. The electrons can then be transferred through the conductive IrOx layer to the support. This type of mechanism has been proposed to explain the electrical communication in a well known biosensor system in which the enzyme is ‘wired’ by ferrocene/ferrocinium centers attached covalently to the enzyme [15]. Similarly, this oxygen-independent mechanism has been seen in biosensing layers in which the electron accepting/transferring relays are composed of a dense array of Os complexes covalently bound to a long flexible polypyridine polymer [16] and also more recently in a system based totally on an organic polymer possessing PANI redox centers [17]. The oxygen-independent mechanism of electron transfer between IrOx and GOx, demonstrated by the very similar MM plots obtained under both Ar and O2 saturation (Fig. 6b), closely resembles this ‘direct electrical communication’ concept, realized through electrical ‘wiring’ of the enzyme, as proposed by Heller’s group [15–17] and discussed above. In the present work, the Ir(III)/Ir(IV)/Ir(V,VI) oxide sites serve to form the relay that accommodates the electron flow from the active site of the enzyme, through IrOx, to the underlying Au electrode. The role of IrOx in our glucose biosensor exceeds that of a typical mobile redox-active mediator and thus our sensor is more similar to biosensors that rely on ‘wiring’ the enzyme with a redox-active polymer. 3.3. Effect of IrOx–GOx–Nafion layer properties on mechanism of GOx regeneration Fig. 6. (a) Four different electrodes (made all at the same time using the GOx:Ir ratio of 22) were oxidized by cycling to 1.25 V. Each electrode was then glucose tested at its respective voltage of either 1.05, 1.10, 1.15 or 1.20 V up to 70 mM of glucose in stirred, pH 7 0.1 M phosphate buffer solution. As the voltage decreased, so did the responding current. The currents, however, for 1.05 and 1.10 V were very similar. (b) Michaelis–Menten plots of a film (GOx:Ir ratio of 22) tested under (squares) O2 -saturated and (triangles) Ar-saturated conditions at 1.2 V in stirred, pH 7 0.1 M phosphate buffer solution.
In order to determine the optimal composition of our oxygenindependent IrOx-wired glucose biosensor, the effect of the ratio of GOx to Ir in the films was examined. Fig. 7 shows the MM plots for the sensor containing variable GOx:Ir mass ratios, ranging from 0.2 to 2. As expected, imax increases with GOx loading up to an GOx:Ir ratio of 1, as more GOx is available for reaction with glucose.
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top of this film, it was found that GOx could be regenerated only by value was typically 40–60 mM [34]. The oxygen. In this case, the Km IrOx nanoparticles used in the present work can apparently interact much more effectively with the enzyme and can compete with oxygen in the regeneration of the enzyme active site, similarly to gold nanoparticles or carbon nanotubes, as discussed in recent reviews [5,25]. In fact, in optimal cases, typically with a GOx:Ir ratio of 1 g/g (Fig. 9), the glucose sensor did not respond at all to oxygen and the signal was thus completely due to IrOx regeneration of reduced GOx. 4. Conclusions
Fig. 8. Plot of imax and Km values as a function of GOx:Ir mass ratio based on data in Fig. 7a, with points calculated using Eadie–Hofstee plots.
However, a further increase in the amount of GOx relative to IrOx present results in a dramatic decrease of the biosensor performance (Fig. 8). The signal decreases significantly, from 36 to 6 A cm−2 , likely due to insufficient IrOx sites now being available to fully ‘wire’ the enzyme. In our experiments showing the response of the IrOx values are lower regenerated glucose sensor (Figs. 7 and 8), the Km than the value of ∼25 mM, reported for the native enzyme [17]. In values range from 5 to 13 mM. Similarly, the ‘wired’ Fig. 8, the Km biosensors developed by Heller’s group [15–17] were characterized by small Km values, even as low as ca. 6 mM for the Os polymer [16] and 16.8 mM when using PANI. It can be anticipated, therefore, that the right sort of ‘wiring’ within the film will have a major impact on the pathway of the reaction and can result in a change in the enzyme kinetics vs. what occurs in the natural oxygen-dependent enzyme regeneration mechanism. In the case of these IrOx–GOx-based redox-active matrices, the role of the internal structure of the film is crucial. According to our earlier work [34], when an IrOx layer was first formed electrochemically on bulk Ir, followed by the deposition of an aliquot of GOx on
A biocompatible, kinetically rapid, nanoparticulate Ir oxide (IrOx) thin film has been developed to serve as a redox-active matrix for glucose oxidase (GOx) for the chronoamperometric detection of glucose at 1.0–1.2 V vs. RHE. The IrOx-containing layer was formed by mixing an ethanolic suspension of a metallic Ir sol plus Nafion with an aqueous GOx solution, followed by casting on a Au electrode, drying, and electrochemical conversion of the Ir particles to IrOx. The addition of Nafion to the IrOx–GOx biosensor is optional. However, it has been used here as a binder material and also to prevent the oxidation of interfering substances. These biosensors are shown to operate according to a dual mechanism, including an oxygen-dependent GOx regeneration pathway, generating hydrogen peroxide, and an oxygen-free mechanism involving IrOx mediation of the enzyme re-oxidation process. Sensors that can be at least partially regenerated by oxy value of gen are characterized by an apparent Michaelis–Menten Km >30 mM, larger than the typical values reported for GOx in solution. When oxygen is fully removed from the solution with argon, the biosensor typically gives a response approximately 50% of the signal in aerated conditions. The regeneration of GOx by direct mediation via the IrOx nanoparticles has been proven by careful removal of oxygen (monitored by a Pt electrode) and by double potential step experiments. Also, the signal is still strong at potentials as low as and i 1.0 V vs. RHE, and the Km max parameters under aerated and deaerated conditions are clearly different. It has also been found that the IrOx–GOx–Nafion films can be designed to be regenerated only by IrOx mediation by controlling the mass ratio of GOx and Ir in the film. The oxygen independence of the biosensor response is vividly demonstrated by the absence of any change in the response to glucose by the addition of oxygen to the cell during a typical chronocoulometric experiment performed to obtain the kinetic parameters of the IrOx-mediated GOx values of 10–15 mM biosensor. Under these conditions, small Km or less are normally seen. At low GOx:Ir ratios, insufficient GOx is present to generate large glucose responses, while at high ratios, GOx appears to interfere with the passage of current between IrOx nanoparticles. Acknowledgements The authors gratefully acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada and the Alberta Heritage Foundation for Medical Research for scholarship support of AJ. Thanks are also extended to Preti Pratikha (University of Calgary) for technical assistance and to Dr. E. Abu Irhayem for useful discussions. References
Fig. 9. Chronoamperometry plot at 1.2 V for an electrode containing GOx:Ir mass ratio of 1 g/g. Glucose additions (50 L of 2 M glucose) were made to a stirred, pH 7, 0.1 M phosphate buffer solution (25 mL) under Ar-saturated conditions for the first 7 min. The solution was then fully aerated and no notable change in the sensor response to glucose was seen.
[1] [2] [3] [4] [5]
S.A. Jaffari, A.P.F. Turner, Physiol. Meas. 16 (1995) 1. J. Wang, Electroanalysis 13 (2001) 938. J.D. Newman, A.P.F. Turner, Biosens. Bioelectron. 20 (2005) 2435. J. Wang, Chem. Rev. 108 (2008) 814. O.A. Sadik, A.O. Aluoch, A. Zhou, Biosens. Bioelectron. 24 (2009) 2749.
A.S. Jhas et al. / Electrochimica Acta 55 (2010) 7683–7689 [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
L. Clark Jr., C. Lyons, Ann. N. Y. Acad. Sci. 102 (1962) 29. G. Guilbault, G. Lubrano, Anal. Chim. Acta 64 (1973) 439. M. Tierney, H. Kim, J. Tamada, R. Potts, Electroanalysis 13 (2000) 666. A. Cass, G. Davis, E. Francis, H.A. Hill, W. Aston, J. Higgins, E. Plotkin, L. Scott, A.P. Turner, Anal. Chem. 56 (1984) 667. J.E. Frew, H.A. Hill, Anal. Chem. 59 (1987) 933A. A.A. Karyakin, O.V. Gitelmacher, E.E. Karyakina, Anal. Chem. 67 (1995) 2419. K. Habermuller, A. Ramanavicius, V. Laurinavicius, W. Schuhmann, Electroanalysis 12 (2000) 1383. J. Wang, P.V.A. Pamidi, D.S. Park, Electroanalysis 9 (1997) 52. F. Tian, G. Zhu, Sens. Actuators B 86 (2002) 266. Y. Degani, A. Heller, J. Phys. Chem. 91 (1987) 1285. T. Ohara, R. Rajogopalan, A. Heller, Anal. Chem. 66 (1994) 2451. N. Mano, J.E. Yoo, J. Tarver, Y.-L. Loo, A. Heller, J. Am. Chem. Soc. 129 (2007) 7006. J. Wang, M. Pedrero, P.V.A. Pamidi, X. Cai, Electroanalysis 7 (1995) 1032. J. Wang, H.J. Wu, J. Electroanal. Chem. 395 (1995) 287. H. Sakslund, J. Wang, O.J. Hammerich, J. Electroanal. Chem. 402 (1996) 149. J. Wang, G. Rivas, M.J. Chicarro, Electroanal. Chem. 439 (1997) 55. M.C. Rodriguez, G.A. Rivas, Electroanalysis 11 (1999) 558. S.A. Miscoria, G.D. Barrera, G.A. Rivas, Electroanalysis 14 (2002) 981.
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[24] Y. Xiao, F. Patolsky, E. Katz, J.F. Hainfeld, I. Willner, Science 299 (2003) 1877. [25] X. Luo, A. Morrin, A.J. Killard, M.R. Smyth, Electroanalysis 18 (2006) 319. [26] A. Cox, R. Jaworski, Anal. Chem. 61 (1989) 2176. [27] H. Elzanowska, E. Abu Irhayem, B. Skrzynecka, V. Birss, Electroanalysis 16 (2004) 478. [28] P.G. Pickup, V.J. Birss, J. Electrochem. Soc. 135 (1988) 324. [29] P.G. Pickup, V.J. Birss, J. Electroanal. Chem. 220 (1987) 83. [30] P.G. Pickup, V.I. Birss, Electrochem. Soc. 135 (1988) 126. [31] H. Elzanowska, V.I. Birss, J. Appl. Electrochem. 23 (1993) 646. [32] C. Bock, V.I. Birss, Electrochim. Acta 46 (2001) 837. [33] L.S. Roblee, T.L. Rose, in: W.F. Agnew, D.B. McCreery (Eds.), Neural Prostheses, Fundamental Studies, Prentice Hall, Englewood Cliffs, NJ, 1990, p. 25. [34] E. Abu Irhayem, H. Elzanowska, B. Skrzynecka, A.S. Jhas, V.J. Birss, J. Electroanal. Chem. 538–539 (2002) 153. [35] V. Birss, H. Andreas, I. Serebrennikova, H. Elzanowska, Electrochem. Solid-State Lett. 2 (1999) 326. [36] H. Andreas, H. Elzanowska, I. Serebrennikova, V. Birss, J. Electrochem. Soc. 147 (2000) 4598. [38] N.L. Akers, C.M. Moore, S.D. Minteer, Electrochim. Acta 50 (2005) 2521. [39] J.W. Gallaway, S.A.C. Barton, J. Electroanal. Chem. 626 (2009) 149.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Dual oxygen and Ir oxide regeneration of glucose oxidase in nanostructured thin film glucose sensors Amit S. Jhas a , Hanna Elzanowska b,∗ , Bri Sebastian a , Viola Birss a,∗∗ a b
Department of Chemistry, University of Calgary, 2500 University Dr. N.W., Calgary, Alberta T2N 1N4, Canada Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 26 January 2010 Received in revised form 28 March 2010 Accepted 29 March 2010 Available online 3 April 2010 Keywords: Glucose Glucose oxidase Iridium oxide Nanoparticles Direct mediation dual mechanism
a b s t r a c t A nanoparticulate iridium oxide (IrOx) thin film has been developed as a redox-active matrix material for an advanced generation glucose biosensor, in which IrOx serves as the non-physiological mediator, replacing oxygen in the enzymatic re-oxidation of glucose oxidase (GOx). Ethanolic solutions of Nafion and an Ir sol were mixed with an aqueous GOx solution and then deposited on a Au support. The Ir nanoparticles were then oxidized electrochemically to IrOx and the resulting films (IrOx–GOx–Nafion) were tested for their glucose response in both oxygen- and argon-saturated solutions, with the oxygen content in both solutions monitored by a Pt electrode. The sensors that are regenerated largely by O2 are characterized by a Michaelis–Menten Km value of ∼30 mM or more and imax values of at least 20 A cm−2 . Under fully deareated conditions, the sensors lose only ∼50% of their response to glucose, clearly indicating that a dual oxygen-regeneration and IrOx mediation mechanism is operative for the biosensor under these conditions. Under optimized conditions, involving a controlled GOx:Ir ratio, only the Ir oxide sites in the film serve to mediate GOx regeneration, giving Km (10–15 mM) and imax values that are independent of the O2 content of the solution. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Progress in biosensor research over the past decades has resulted in the development of various amperometric glucose sensors, usually referred to as first-, second-, and third-generation biosensors, as described in numerous reviews [1–5]. In 1962, Clark [6] used a metallic Pt electrode covered with a thin layer of glucose oxidase (GOx) and a dialysis membrane to monitor the decrease in the O2 concentration resulting from GOx regeneration (by O2 ) after the reaction of GOx with glucose. This electrode is considered to be the first example of a first-generation amperometric glucose biosensor [2,4]. Ten years later, Guilbault and Lubrano [7] demonstrated a similar glucose sensor, but operating via the detection of hydrogen peroxide. Both oxygen consumption and hydrogen peroxide build-up are still tracked today by first-generation glucose sensors [2,4], even in implants [8], according to the following scheme: GOx + glucose → GOx–H2 + gluconicacid
(1)
GOx–H2 + O2 → GOx + H2 O2
(2)
∗ Corresponding author. Tel.: +48 22 822 0211; fax: +48 22 822 5996. ∗∗ Corresponding author. Tel.: +1 403 220 7306; fax: +1 403 289 9488. E-mail addresses: [email protected] (H. Elzanowska), [email protected] (V. Birss). 0013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2010.03.093
Many scientific works have also been devoted to developing second-generation biosensors, in which artificial mediators [2,4,9,10] are used to replace O2 , as follows: GOx + glucose → GOx–H2 + gluconicacid
(3)
GOx–H2 + M ox → GOx + M red
(4)
M red → M ox + 2e
−
(5)
where Mox and Mred are the oxidized and the reduced forms of the mediator, respectively. Second-generation biosensors have involved the use of ferrocene and its derivatives [9,10], as well as many other substances [11], added to the enzyme layer as mediators. A wide range of matrices, such as conducting polymers [12] and sol–gel (SG)-derived films [13,14], capable of immobilizing both the enzyme and the mediators, have also been tested over the years. However, problems with mediator leakage from the biosensor layer and mixed mediator/oxygen operation of the sensors [2] have prompted researchers to focus on firmly embedding mediators in order to transfer electrons efficiently enough to compete successfully with oxygen in the regeneration of the enzyme. In 1987, Heller’s group [15] described the use of relay-modified glucose oxidase in an electrode layer responding to glucose. The electrical ‘wiring’ was realized by the covalent bonding of 12 ferrocene/ferrocinium centers to each of the enzyme molecules, leading to a so-called ‘direct electrical communication’ between the
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enzyme and the underlying Au or glassy carbon (GC) electrode support. Another well known example [16] of electron-accepting and electron-transferring relays is that of a dense array of Os complexes bound covalently to a long flexible polypyridine polymer. In this case, the biosensor signal for both glucose and lactate was almost the same under oxygen and nitrogen and the Michaelis–Menten ) for GOx was ca. 5 mM, much smaller than the solution constant (Km value for GOx [17]. Electrical ‘wiring’ of the surface-bound enzyme can also be realized using non-metallic components. A recent example of enzyme wiring, proposed by Heller’s group [17], is the use of an electronconducting cross-linked polyaniline-based hydrogel, formed by cross-linking the polyaniline/poly(2-acrylamido-2-methyl-1propane sulfonic acid (PANI/PAAMPSA) with glucose-permeable poly(ethylene glycol) diglycidyl ether (PEGDGE) to ‘co-cross-link’ GOx. In this biosensor, PANI/PAAMPSA has replaced the Os2+/3+ complex, resulting in a rapidly responding and effective biosensor = 16.8 mM, i −2) ). (Km max = 200 M cm The concept of using conductive biosensor layers to immobilize enzymes has been further extended to consideration of immobilization of metallic components. Wang’s group examined glucose oxidation at GOx-containing carbon transducers metalized with Rh [19], Ir [21–23], Ru [23], and Pd [18,20,23]. The metallic component serves as the catalyst for H2 O2 detection and also improves the conductivity of the biosensor layers. In particular, the use of Ir significantly enhanced the biosensor response [23]. Similarly, the role of Au and Ag nanoparticles in the development of these biosensors has been recognized and described in a recent review [5,24,25]. Therefore, the development of a stable and rapid electrodemediated glucose sensor, which is also biocompatible, remains the goal of a significant amount of research. We have therefore devoted our efforts towards the use of Ir/Ir oxide (IrOx) electrodes for this purpose, due to their known electrocatalytic action towards H2 O2 [26,27], excellent electronic conductivity [28–32], biocompatibility [33], and rapid charge/discharge kinetics [29,32]. In our earlier work [34], we deposited GOx simultaneously with the growth of an IrOx film, formed electrochemically on a bulk Ir wire or plate substrate. However, SEM analysis showed that most of the GOx was located on the outer surface of the IrOx film, thus losing the intended stabilizing benefits of the IrOx matrix. This may explain the lack of any evidence for IrOx-mediated glucose oxidation using this electrode [34]. In the present work, we therefore mixed an aqueous solution of GOx with an ethanolic suspension of Ir nanoparticles (2–3 nm in dia) and Nafion, and then cast this mixture as a thin, homogeneous, composite film on Au substrates. The metallic Ir nanoparticles were then oxidized electrochemically to IrOx to enhance their biocompatibility and stability. The performance of this biosensor was examined in both oxygen-saturated (aerated) and Ar-saturated (deaerated) solutions to determine whether the sensor operates as a first-generation biosensor, in which oxygen is used to regenerate GOx, or as an advanced sensor, in which the IrOx matrix re-oxidizes the enzyme. We show that, depending on the relative GOx and Ir content in the film, one of these two possible mechanisms prevails.
2. Experimental 2.1. Synthesis of Ir sols Ir sol synthesis required the dissolution of 0.2 g of sodium ethoxide (NaOC2 H5 , Aldrich, 96% pure) in 6 mL of absolute ethanol (EtOH) by stirring in a 100 mL round bottom flask. IrCl3 (Aldrich, 99.99% pure) was added to the mixture and allowed to reflux under Ar at 73 ◦ C for 2 h. The solution was cooled to 22 ◦ C, stirred for 18 h
under Ar, and then the resulting mixture was sieved through fine filter paper (Qualitative, Whatman). The suspended Ir sol filtrate, containing metallic Ir nanoparticles, was collected and then stored in a sealed glass vial in the refrigerator for future use in biosensor fabrication. The black precipitate (a mixture of Ir oxide and NaCl) was filtered off and kept for further analysis. 2.2. Enzyme immobilization The EtOH-based Ir sol (typically 8 L) was mixed with 2 L of 1% Nafion® (also dissolved in EtOH) followed by the addition, by pipet, of 0.5–4 L of an aqueous glucose oxidase (GOx) solution (60 mg mL−1 ). An aliquot (typically 4 L) of this mixture was then deposited on a Au working electrode (WE) substrate and the electrode was then allowed to dry in the refrigerator for 24 h. 2.3. Electrodes, cells and equipment All electrochemical experiments were performed using either a Solartron Analytical 1480 Multistat or an EG&G PARC 173 Potentiostat. A conventional two-compartment glass cell, containing the coated Au WE and the high surface area Pt mesh counter electrode in the main compartment, connected to the reference electrode (RE) compartment through a Luggin capillary, was used. The WE substrate consisted of glass slides, sputter-coated (Denton DV502A sputtering system) first with an undercoat of Ti to a thickness of ca. 10 nm and then with Au to ca. 80–90 nm, followed by rinsing with methanol and then water. Prior to coating with the Ir sol or Ir sol/GOx mixture, the Au substrate electrodes were cleaned electrochemically by potential cycling in 0.05 M H2 SO4 between 0 and 2.0 V vs. RHE for 35 cycles and then between 0 and 1.5 V vs. RHE until the cyclic voltammetry (CV) features of the Au substrate remained constant in size. The RE was normally a saturated calomel electrode (SCE); however, all potentials in this paper are referred to the reversible hydrogen electrode (RHE). 2.4. Solutions, reagents, and general experimental conditions All solutions were prepared using analytical-grade reagents and triply distilled water. H2 O2 (30%, v/v aqueous solution), dglucose (anhydrous), and potassium dihydrogen phosphate were purchased from BDH Chemicals. Glucose oxidase (GOx, EC 1.1.3.4, Type II-S from Aspergillus Niger, 40,300 unit/g of solid) was obtained from Sigma, while Nafion® was purchased from Fisher Scientific Co. All experiments were carried out in either aerated (with oxygen) or deaerated (with argon gas for a minimum of 25 min) 0.1 M potassium phosphate buffer solution at a pH of 7. 3. Results and discussion 3.1. Electrochemical characteristics of IrOx–GOx–Nafion films in absence of glucose Fig. 1 shows a typical set of cyclic voltammograms (CVs) of a freshly deposited Ir–GOx–Nafion film in pH 7 0.1 M phosphate buffer during the early stages of the conversion (by cycling the potential between 0 and 1.25 V vs. RHE [35,36]) of the metallic Ir component to its hydrous Ir oxide (IrOx) form. The peaks at 0–0.25 V in Fig. 1 are characteristic of the hydrogen deposition/removal process (Hupd) at metallic Ir [27–32] and can be tracked to determine the total area of Ir in the film, as well as the extent of conversion of Ir to IrOx. Indeed, when the potential was repeatedly cycled to 1.25 V, it is seen in Fig. 1 that the Hupd peaks gradually decrease in size. Also, the symmetrical anodic and cathodic peaks, centered at 0.9 V (A1 /C1 ), become more well-formed. These peaks are characteristic [31,32] of the kinetically rapid Ir(III)/Ir(IV) oxide redox
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Fig. 1. Cyclic voltammograms showing the conversion of a thin film of metallic Ir nanoparticles to Ir oxide (at 0.1 V/s in a pH 7 0.1 M phosphate buffer solution). The film was formed using an ethanol Ir sol mixed with 0.04% Nafion® and GOx with 2 L deposited on a Au substrate and then dried at room temperature in air for 24 h. Inset shows linear peak current vs. sweep rate plots for Ir oxide reduction at 0.7 V and oxidation at 1.0 V.
transition within the IrOx layer. At potentials >1.25 V, another set of redox peaks is seen (not shown in Fig. 1), representative of the Ir(IV)/Ir(V,VI) oxide redox reaction. These films exhibit a linear peak current vs. sweep rate relationship up to at least 0.2 V/s, as seen in the inset of Fig. 1. These rapid redox kinetics make IrOx films highly suitable for use as a matrix material hosting a redox-active enzyme in its three-dimensional structure. Notably, the presence of GOx in the film does not influence the electrochemical conversion of Ir to IrOx under these conditions (Fig. 1). Also, it will be shown below that potential cycling of these electrodes in phosphate buffer solution during the IrOx formation process does not harm the enzyme activity. Nafion was added to the Ir sol in order to better distribute the enzyme in the oxide matrix and also to stabilize the Ir (and later IrOx) film on the Au substrate. In fact, it is shown in parallel work that the addition of Nafion to the ethanolic or the mixed aqueous/ethanolic Ir sol has a beneficial effect on the overall IrOx charge and the reversibility of the Ir redox process. This suggests that Nafion can entrap Ir nanoparticles that would not otherwise be incorporated into the film. It is also possible that Nafion improves the Ir/IrOx particle-to-particle connectivity and the Ir/IrOx–GOx contact within the film. All of these factors would serve to increase the amount of active Ir/IrOx and improve the conductivity of the IrOx–Nafion–GOx films. This is similar to the beneficial effect reported [24] when 1.4 nm Au nanocrystals, functionalized with the flavin adenine dinucleotide cofactor, were adsorbed on a dithiol-modified Au electrode, followed by reconstitution with the apo-GOx enzyme. Excellent GOx electron transfer properties were observed in these films, with the Au nanoparticles suggested to serve as an electron relay or ‘electrical nanoplug’ for the alignment of the enzyme on the conductive support and for the electrical wiring of its redox-active center. It has also been suggested previously that Nafion, when mixed with enzymes, can change the pH inside a thin film and thus, to avoid this problem, tetrabutyl ammonium salts were used to modify the Nafion acidity [38]. However, as shown in Fig. 1, the Ir(III)/Ir(IV) oxide redox peak potentials are centered at 0.8 V vs. RHE, regardless of whether or not Nafion is present in the Ir-containing film. Thus, the IrOx–GOx–Nafion-containing film studied here is sufficiently porous to allow the appropriate flux of buffer solution in and out of the film to keep the pH inside the
Fig. 2. (a) Oxidation and reduction of H2 O2 (0.9 mM) at 0.1 V/s in a pH 7 0.1 M phosphate buffer solution, all under Ar (solid line) or oxygen (dashed line), at IrOx films formed using an ethanolic Ir sol, deposited on Au and dried at room temperature for 24 h. (b) Linear plots of H2 O2 oxidation (at 1.2 V) and reduction (at 0.2 V) currents vs. H2 O2 concentration, all under Ar, at IrOx films formed using 10% H2 O/ethanol Ir sol, with (curve 1) and without (curve 2) added 0.04% Nafion® . 2 L of mixture were deposited on Au and dried at room temperature for 24 h.
film constant. This is also a good indication that glucose has good access to GOx, embedded within these layers, and that the product of the reaction (e.g., H2 O2 , in aerated conditions) and gluconic acid will also be readily transported out of these films. To demonstrate that the IrOx–GOx–Nafion films can be used for glucose sensing in aerated phosphate buffer solutions, conditions under which H2 O2 will be produced, the IrOx sol-derived electrodes were examined for their CV response to H2 O2 (Fig. 2a). The H2 O2 oxidation and reduction currents, at 1.2 and 0.2 V, respectively, are shown in Fig. 2b to be linear with H2 O2 concentration. Notably, both of these signals are higher when Nafion is present in the Ir sol, as the amount of Ir successfully transferred into the film is also higher. As shown in our previous work, the stability of IrOx is not adversely affected by the presence of H2 O2 [34]. However, because O2 reduces at these electrodes in essentially the same potential window as does H2 O2 (from 0 to 0.6–0.7 V) at IrOx, the oxidation of H2 O2 (which occurs at 1.0 V and higher) must be tracked [34], conditions under which the sensor operates as a first-generation glucose sensor. Our sensor is quite stable, especially after an initial period of time of equilibration. For example, a 20–25%, decrease in the signal is seen in the first 4–6 days, and then the response is stable for at least 35 days. This topic will be discussed in detail in out next publication.
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Fig. 3. Chronoamperometry plot (at 1.2 V) in stirred, pH 7 0.1 M phosphate buffer solution (25 mL) at room temperature with 200 L increment additions of 1 M glucose (arrows) under saturated O2 conditions for a film formed from a IrOx/GOx mixture. Inset shows the corresponding MM plot.
3.2. Response of IrOx–GOx–Nafion electrode to glucose in aerated and deaerated environments Fig. 3 shows the effect of added glucose to the current recorded at 1.2 V for an IrOx–GOx–Nafion film in a pH 7 buffer solution, saturated with oxygen. The background current is very small (dotted line in Fig. 3), consistent with the capacitive properties of IrOx films, such that the oxidation state of Ir within the oxide film rapidly equilibrates and the current quickly drops to zero at constant potential. Also, the rapid increase (within 2 s) in the chronoamperometric currents recorded after each glucose addition indicates the fast response of our sensor. The application of relatively high positive potentials (1.2 V vs. RHE) does not change the properties of the IrOx and IrOx–GOx–Nafion films. In fact, the CV response of the sensor remains essentially the same before and after potentiostatic measurements involving glucose additions. Due to thee high pseudocapacitative IrOx currents, the currents related to glucose oxidation on IrOx are relatively small in CV experiments, but under potentiostatic conditions, the oxide is fully charged and thus only the signal for the catalytic oxidation of glucose is seen. The inset to Fig. 3 shows the corresponding Michaelis–Menten plot, giving Km and imax values of 27 mM and 46 A cm−2 , respectively, in these value is very similar to the solution aerated conditions. (This Km value for GOx, which is typically around 25 mM [17].) When the experiments described above (Fig. 3) were repeated in deaerated (with Ar) solutions, an excellent response to glucose is still seen, demonstrating that GOx re-oxidation can also occur by direct electron transfer to the Ir oxide nanoparticles within the film matrix. This type of oxygen-independent response is highly desirable for glucose detection in blood, particularly if implanted biosensors are being considered, as a highly variable oxygen concentration can then be expected [2]. The Michaelis–Menten plots for glucose oxidation, in both saturated O2 and saturated Ar environments, are shown in Fig. 4 for direct comparison. In the value for this particular sensor is 21 mM, deaerated solution, the Km i.e., less than 25 mM, and imax is 13 A cm−2 , while in aerated media, and i −2 the Km max values are both larger (29 mM and 24 A cm , respectively). Although the dissolved O2 concentration should be very low in the Ar-saturated cell solution, the maximum glucose oxidation
Fig. 4. Michaelis–Menten plots of a film formed from an IrOx/GOx mixture and tested under (squares) O2 -saturated and (triangles) Ar-saturated conditions. Glucose tests carried out at 1.2 V in stirred, pH 7 0.1 M phosphate buffer solution.
current observed in Fig. 4 at saturation (∼75 mM glucose) is only ∼45% less than in air. In fact, negligible currents would have been expected in the absence of oxygen if the regeneration of GOx were occurring only by O2 (a co-substrate), as observed for other O2 dependent enzymes, e.g., laccase [39], which reduces oxygen to water (oxygen is the substrate for the enzymatic reaction). In the case of laccase [39], when the solution changes from O2 -saturated to air-saturated, a significant decrease in the biosensor signal is observed, and the signal is almost zero (90–95% loss of signal) when the solution is fully deaerated. To verify that electron transfer is indeed occurring between the reduced flavin site in GOx and the surrounding IrOx matrix under deaerated conditions, the amount of oxygen present was carefully determined. The oxygen reduction current (ORR), monitored (not shown) using a second Pt working electrode in an O2 -saturated solution, was found to be 2.1 mA cm−2 at 0.2 V, due the oxygen reduction reaction. After 40 min of deaeration with Ar, the current at 0.2 V is only 1.3 A cm−2 , i.e., the oxygen content has been lowered by at least 1000 times. This would result in a similar decrease in the observed glucose oxidation currents if the only means of GOx regeneration is through reaction with O2 , as seen for laccase [39] As the glucose oxidation currents in deaerated conditions (Fig. 4, <0.3 M O2 ) are much too high to be explained by trace O2 regeneration of GOx, these results argue strongly that the IrOx sites are indeed mediating the GOx regeneration process. This is further supported by the absence of any H2 O2 (the product of O2 regeneration of GOx) monitored electrochemically in the deaerated medium. A chronoamperometric experiment (Fig. 5) was carried out in deoxygenated conditions (oxygen concentration < 0.3 M) at 0.2 V, before and after the addition of glucose. At 0.2 V, the current will respond to the reduction of oxygen or H2 O2 , or both. Since no difference was observed in the current response at 0.2 V before and after carrying out glucose oxidation, indicating the absence of any generated H2 O2 , these results strongly support the conclusion that regeneration of the reduced GOx sites is occurring via mediation by the IrOx nanoparticles. The question could arise as to whether, in a glucose detection experiment at 1.2 V, oxygen is being evolved catalytically on the IrOx electrode, then serving to regenerate the reduced form of GOx. This supposition is refuted by the fact that the glucose signal is still very strong at 1.1 V, and a good response was still obtained even at 1.05 V (Fig. 6a). It is impossible that the insignificant amounts
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Fig. 5. Potential step experiment illustrating the (a, left scale) current at 0.2 and 1.2 V before and after the addition of glucose in stirred, pH 7 0.1 M phosphate buffer solution deaerated with Ar. (b, right scale) Shows the applied potential over the course of the same experiment.
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Fig. 7. MM plots of electrodes composed of GOx/IrOx mixture with mass ratios of 0.25 g/g (1, ), 0.50 g/g (3, ), 1.00 g/g (4, ) and 2.00 g/g (2, 䊉). Glucose testing was performed at 1.2 V in a stirred pH 7 0.1 M phosphate buffer solution under Ar-saturated conditions. Aliquots consisted of 50 L of 2 M glucose.
of O2 that would be generated at these very low potentials could be sufficient to produce the comparatively high currents (at high glucose concentrations) seen in Fig. 4. It can be concluded, therefore, that the IrOx nanoparticles are actively participating in the communication between GOx and the underlying support (Au). This can be realized through the protein part of the enzyme, allowing for electron transfer from the enzyme redox site (the flavin) to the IrOx nanoparticles (at which the Ir(IV/V/VI) redox transitions occur) surrounding the enzyme molecule. The electrons can then be transferred through the conductive IrOx layer to the support. This type of mechanism has been proposed to explain the electrical communication in a well known biosensor system in which the enzyme is ‘wired’ by ferrocene/ferrocinium centers attached covalently to the enzyme [15]. Similarly, this oxygen-independent mechanism has been seen in biosensing layers in which the electron accepting/transferring relays are composed of a dense array of Os complexes covalently bound to a long flexible polypyridine polymer [16] and also more recently in a system based totally on an organic polymer possessing PANI redox centers [17]. The oxygen-independent mechanism of electron transfer between IrOx and GOx, demonstrated by the very similar MM plots obtained under both Ar and O2 saturation (Fig. 6b), closely resembles this ‘direct electrical communication’ concept, realized through electrical ‘wiring’ of the enzyme, as proposed by Heller’s group [15–17] and discussed above. In the present work, the Ir(III)/Ir(IV)/Ir(V,VI) oxide sites serve to form the relay that accommodates the electron flow from the active site of the enzyme, through IrOx, to the underlying Au electrode. The role of IrOx in our glucose biosensor exceeds that of a typical mobile redox-active mediator and thus our sensor is more similar to biosensors that rely on ‘wiring’ the enzyme with a redox-active polymer. 3.3. Effect of IrOx–GOx–Nafion layer properties on mechanism of GOx regeneration Fig. 6. (a) Four different electrodes (made all at the same time using the GOx:Ir ratio of 22) were oxidized by cycling to 1.25 V. Each electrode was then glucose tested at its respective voltage of either 1.05, 1.10, 1.15 or 1.20 V up to 70 mM of glucose in stirred, pH 7 0.1 M phosphate buffer solution. As the voltage decreased, so did the responding current. The currents, however, for 1.05 and 1.10 V were very similar. (b) Michaelis–Menten plots of a film (GOx:Ir ratio of 22) tested under (squares) O2 -saturated and (triangles) Ar-saturated conditions at 1.2 V in stirred, pH 7 0.1 M phosphate buffer solution.
In order to determine the optimal composition of our oxygenindependent IrOx-wired glucose biosensor, the effect of the ratio of GOx to Ir in the films was examined. Fig. 7 shows the MM plots for the sensor containing variable GOx:Ir mass ratios, ranging from 0.2 to 2. As expected, imax increases with GOx loading up to an GOx:Ir ratio of 1, as more GOx is available for reaction with glucose.
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top of this film, it was found that GOx could be regenerated only by value was typically 40–60 mM [34]. The oxygen. In this case, the Km IrOx nanoparticles used in the present work can apparently interact much more effectively with the enzyme and can compete with oxygen in the regeneration of the enzyme active site, similarly to gold nanoparticles or carbon nanotubes, as discussed in recent reviews [5,25]. In fact, in optimal cases, typically with a GOx:Ir ratio of 1 g/g (Fig. 9), the glucose sensor did not respond at all to oxygen and the signal was thus completely due to IrOx regeneration of reduced GOx. 4. Conclusions
Fig. 8. Plot of imax and Km values as a function of GOx:Ir mass ratio based on data in Fig. 7a, with points calculated using Eadie–Hofstee plots.
However, a further increase in the amount of GOx relative to IrOx present results in a dramatic decrease of the biosensor performance (Fig. 8). The signal decreases significantly, from 36 to 6 A cm−2 , likely due to insufficient IrOx sites now being available to fully ‘wire’ the enzyme. In our experiments showing the response of the IrOx values are lower regenerated glucose sensor (Figs. 7 and 8), the Km than the value of ∼25 mM, reported for the native enzyme [17]. In values range from 5 to 13 mM. Similarly, the ‘wired’ Fig. 8, the Km biosensors developed by Heller’s group [15–17] were characterized by small Km values, even as low as ca. 6 mM for the Os polymer [16] and 16.8 mM when using PANI. It can be anticipated, therefore, that the right sort of ‘wiring’ within the film will have a major impact on the pathway of the reaction and can result in a change in the enzyme kinetics vs. what occurs in the natural oxygen-dependent enzyme regeneration mechanism. In the case of these IrOx–GOx-based redox-active matrices, the role of the internal structure of the film is crucial. According to our earlier work [34], when an IrOx layer was first formed electrochemically on bulk Ir, followed by the deposition of an aliquot of GOx on
A biocompatible, kinetically rapid, nanoparticulate Ir oxide (IrOx) thin film has been developed to serve as a redox-active matrix for glucose oxidase (GOx) for the chronoamperometric detection of glucose at 1.0–1.2 V vs. RHE. The IrOx-containing layer was formed by mixing an ethanolic suspension of a metallic Ir sol plus Nafion with an aqueous GOx solution, followed by casting on a Au electrode, drying, and electrochemical conversion of the Ir particles to IrOx. The addition of Nafion to the IrOx–GOx biosensor is optional. However, it has been used here as a binder material and also to prevent the oxidation of interfering substances. These biosensors are shown to operate according to a dual mechanism, including an oxygen-dependent GOx regeneration pathway, generating hydrogen peroxide, and an oxygen-free mechanism involving IrOx mediation of the enzyme re-oxidation process. Sensors that can be at least partially regenerated by oxy value of gen are characterized by an apparent Michaelis–Menten Km >30 mM, larger than the typical values reported for GOx in solution. When oxygen is fully removed from the solution with argon, the biosensor typically gives a response approximately 50% of the signal in aerated conditions. The regeneration of GOx by direct mediation via the IrOx nanoparticles has been proven by careful removal of oxygen (monitored by a Pt electrode) and by double potential step experiments. Also, the signal is still strong at potentials as low as and i 1.0 V vs. RHE, and the Km max parameters under aerated and deaerated conditions are clearly different. It has also been found that the IrOx–GOx–Nafion films can be designed to be regenerated only by IrOx mediation by controlling the mass ratio of GOx and Ir in the film. The oxygen independence of the biosensor response is vividly demonstrated by the absence of any change in the response to glucose by the addition of oxygen to the cell during a typical chronocoulometric experiment performed to obtain the kinetic parameters of the IrOx-mediated GOx values of 10–15 mM biosensor. Under these conditions, small Km or less are normally seen. At low GOx:Ir ratios, insufficient GOx is present to generate large glucose responses, while at high ratios, GOx appears to interfere with the passage of current between IrOx nanoparticles. Acknowledgements The authors gratefully acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada and the Alberta Heritage Foundation for Medical Research for scholarship support of AJ. Thanks are also extended to Preti Pratikha (University of Calgary) for technical assistance and to Dr. E. Abu Irhayem for useful discussions. References
Fig. 9. Chronoamperometry plot at 1.2 V for an electrode containing GOx:Ir mass ratio of 1 g/g. Glucose additions (50 L of 2 M glucose) were made to a stirred, pH 7, 0.1 M phosphate buffer solution (25 mL) under Ar-saturated conditions for the first 7 min. The solution was then fully aerated and no notable change in the sensor response to glucose was seen.
[1] [2] [3] [4] [5]
S.A. Jaffari, A.P.F. Turner, Physiol. Meas. 16 (1995) 1. J. Wang, Electroanalysis 13 (2001) 938. J.D. Newman, A.P.F. Turner, Biosens. Bioelectron. 20 (2005) 2435. J. Wang, Chem. Rev. 108 (2008) 814. O.A. Sadik, A.O. Aluoch, A. Zhou, Biosens. Bioelectron. 24 (2009) 2749.
A.S. Jhas et al. / Electrochimica Acta 55 (2010) 7683–7689 [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
L. Clark Jr., C. Lyons, Ann. N. Y. Acad. Sci. 102 (1962) 29. G. Guilbault, G. Lubrano, Anal. Chim. Acta 64 (1973) 439. M. Tierney, H. Kim, J. Tamada, R. Potts, Electroanalysis 13 (2000) 666. A. Cass, G. Davis, E. Francis, H.A. Hill, W. Aston, J. Higgins, E. Plotkin, L. Scott, A.P. Turner, Anal. Chem. 56 (1984) 667. J.E. Frew, H.A. Hill, Anal. Chem. 59 (1987) 933A. A.A. Karyakin, O.V. Gitelmacher, E.E. Karyakina, Anal. Chem. 67 (1995) 2419. K. Habermuller, A. Ramanavicius, V. Laurinavicius, W. Schuhmann, Electroanalysis 12 (2000) 1383. J. Wang, P.V.A. Pamidi, D.S. Park, Electroanalysis 9 (1997) 52. F. Tian, G. Zhu, Sens. Actuators B 86 (2002) 266. Y. Degani, A. Heller, J. Phys. Chem. 91 (1987) 1285. T. Ohara, R. Rajogopalan, A. Heller, Anal. Chem. 66 (1994) 2451. N. Mano, J.E. Yoo, J. Tarver, Y.-L. Loo, A. Heller, J. Am. Chem. Soc. 129 (2007) 7006. J. Wang, M. Pedrero, P.V.A. Pamidi, X. Cai, Electroanalysis 7 (1995) 1032. J. Wang, H.J. Wu, J. Electroanal. Chem. 395 (1995) 287. H. Sakslund, J. Wang, O.J. Hammerich, J. Electroanal. Chem. 402 (1996) 149. J. Wang, G. Rivas, M.J. Chicarro, Electroanal. Chem. 439 (1997) 55. M.C. Rodriguez, G.A. Rivas, Electroanalysis 11 (1999) 558. S.A. Miscoria, G.D. Barrera, G.A. Rivas, Electroanalysis 14 (2002) 981.
7689
[24] Y. Xiao, F. Patolsky, E. Katz, J.F. Hainfeld, I. Willner, Science 299 (2003) 1877. [25] X. Luo, A. Morrin, A.J. Killard, M.R. Smyth, Electroanalysis 18 (2006) 319. [26] A. Cox, R. Jaworski, Anal. Chem. 61 (1989) 2176. [27] H. Elzanowska, E. Abu Irhayem, B. Skrzynecka, V. Birss, Electroanalysis 16 (2004) 478. [28] P.G. Pickup, V.J. Birss, J. Electrochem. Soc. 135 (1988) 324. [29] P.G. Pickup, V.J. Birss, J. Electroanal. Chem. 220 (1987) 83. [30] P.G. Pickup, V.I. Birss, Electrochem. Soc. 135 (1988) 126. [31] H. Elzanowska, V.I. Birss, J. Appl. Electrochem. 23 (1993) 646. [32] C. Bock, V.I. Birss, Electrochim. Acta 46 (2001) 837. [33] L.S. Roblee, T.L. Rose, in: W.F. Agnew, D.B. McCreery (Eds.), Neural Prostheses, Fundamental Studies, Prentice Hall, Englewood Cliffs, NJ, 1990, p. 25. [34] E. Abu Irhayem, H. Elzanowska, B. Skrzynecka, A.S. Jhas, V.J. Birss, J. Electroanal. Chem. 538–539 (2002) 153. [35] V. Birss, H. Andreas, I. Serebrennikova, H. Elzanowska, Electrochem. Solid-State Lett. 2 (1999) 326. [36] H. Andreas, H. Elzanowska, I. Serebrennikova, V. Birss, J. Electrochem. Soc. 147 (2000) 4598. [38] N.L. Akers, C.M. Moore, S.D. Minteer, Electrochim. Acta 50 (2005) 2521. [39] J.W. Gallaway, S.A.C. Barton, J. Electroanal. Chem. 626 (2009) 149.