Langmuir 2007, 23, 4675-4681
4675
Incorporation of Glucose Oxidase into Langmuir-Blodgett Films Based on Prussian Blue Applied to Amperometric Glucose Biosensor Hitoshi Ohnuki,*,† Takafumi Saiki,† Akira Kusakari,† Hideaki Endo,‡ Masaki Ichihara,§ and Mitsuru Izumi† Faculty of Marine Technology, Tokyo UniVersity of Marine Science and Technology, 2-1-6 Etchujima, Koto-ku, Tokyo 135-8533, Japan, Faculty of Marine Science, Tokyo UniVersity of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan, and Institute for Solid State Physics, The UniVersity of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8581, Japan ReceiVed October 30, 2006. In Final Form: January 23, 2007 Glucose oxidase (GOx) was immobilized in the organic-inorganic Langmuir-Bldogett (LB) films consisting of octadecyltrimethylammonium (ODTA) and nanosized Prussian blue (PB) clusters. The amperometric glucose biosensors based on the LB films were fabricated and tested. It was found that the sensors exhibited a clear response current under an applied voltage of 0.0 V (vs Ag/AgCl). The linearity of current density versus glucose concentration was confirmed below 15 mmol/L concentration. This is the first observation of biosensor function of the hybrid organicinorganic LB films. The successful preparation of glucose sensors operating at the very low potential indicates that the adsorbed PB clusters in the LB films act as an electrocatalyst for the electrochemical reduction of hydrogen peroxide, which is the final product of the enzymatic reaction sequence. The observed low potential applicability is estimated to inhibit the responses of interferants such as ascorbic acid, uric acid, and acetominophen. It was also found that an electrostatic interaction between positively charged ODTA+ and the adsorbed species of both GOx and PB provided a stabilized adsorption state in the LB films. Such stable immobilization contributes to the steady amperometric response current observed in the present ODTA/PB/GOx LB films.
1. Introduction Glucose oxidase (GOx) is the most widely employed enzyme as an analytical reagent with its relatively low cost and good stability, which makes the glucose/GOx system a very convenient model for method development for enzyme-based biosensors.1 For biosensor fabrication, the immobilization method of the enzyme is another important factor. In this sense, it is an attractive idea to employ Langmuir-Blodgett (LB) films for the immobilization of GOx, since the very thin nature in nanoscale may produce a highly sensitive sensor with ultrafast response time. From this viewpoint, several attempts of LB films comprising GOx have been reported, and their ability to detect glucose concentration has also been exhibited in electrochemical measurements.2-7 In spite of the applicability of LB films to glucose sensors, a complicated procedure for GOx immobilization in LB films, such as intramolecular cross-linking of GOx before spreading, and a relatively small response current as the detecting signal of the sensors are significant drawbacks for their application * To whom correspondence should be addressed. E-mail: ohnuki@ kaiyodai.ac.jp. Telephone/Fax: (81) 3 5245 7466. † Faculty of Marine Technology, Tokyo University of Marine Science and Technology. ‡ Faculty of Marine Science, Tokyo University of Marine Science and Technology. § The University of Tokyo. (1) Raba, J.; Mottola, H. A. Crit. ReV. Anal. Chem. 1995, 25, 1. (2) Tsuzuki, H.; Watanabe, T.; Okawa, Y.; Yoshida, S.; Yano, S.; Koumoto, K.; Komiya, M.; Nihei, Y. Chem. Lett. 1988, 1265. (3) Sriyudthsak, M.; Yamagishi, H.; Moriizumi, T. Thin Solid Films 1988, 160, 463. (4) Hanke, Th.; Wollenberger, U.; Ebert, B.; Scheller, F.; Zaitsev, S. Yu. Biosensors: Fundamentals, Technologies and Applications; Scheller, F. W., Schmid, R. D., Ed.; Wiley-VCH: Weinheim, 1992. (5) Sun, S.; Ho-Si, P.-U.; Harrison, D. J. Langmuir 1991, 7, 727. (6) Yasuzawa, M.; Hashimoto, M.; Fujii, S.; Kunugi, A.; Nakaya, T. Sens. Actuators, B 2000, 65, 241. (7) Singhal, R.; Takashima, W.; Kaneto, K.; Samanta, S. B.; Annapoorni, S.; Malhotra, B. D. Sens. Actuators, B 2002, 86, 42.
as sensors. Accordingly, the amount of work on GOx LB films has decreased in a past few years. Recently, in the field of enzyme-based electrochemical biosensors, Prussian blue (PB), whose composition is expressed as FeIII4[FeII(CN)6]3, has been found to work as a catalyst to reduce the operating potential drastically. PB strongly catalyzes the reduction of hydrogen peroxide, that is formed from an enzymatic reaction, in a negative operating potential range.8 As a result, the interference from reducing agents such as ascorbic acid, acetaminophen, or uric acid in clinical applications can be easily discriminated, offering the most sensitive and interferencefree detection. For example, an electrochemically deposited PB layer showed excellent catalysis for hydrogen peroxide and gave glucose sensors working at very low potential range (approximately 0.0 V vs Ag/AgCl) when GOx is immobilized on the PB film.9-11 During the same period, a significant development concerning PB has advanced the LB film research area: Organic-inorganic hybrid LB films containing PB nanoscale clusters can be obtained when positively charged molecules such as dimethyldioctadecylammonium are spread on a water solution of PB and transferred onto a substrate.12-14 The PB clusters are adsorbed beneath the positively charged Langmuir film through electrostatic interaction. The bond is strong enough to give stable electrochemical properties for the LB films.13,15 (8) Itaya, K.; Shoji, N.; Uchida, I. J. Am. Chem. Soc. 1984, 106, 3423. (9) Karyakin, A. A.; Gitelmacher, O. V.; Karyakina, E. E. Anal. Chem. 1995, 67, 2419. (10) Mattos, I. L.; Gorton, L.; Ruzgas, T. Biosens. Bioelectron. 2003, 18, 193. (11) Ferreira, M. F.; Fiorito, P. A.; Oliveira, O. N., Jr.; Cordoba de Torresi, S. I. Biosens. Bioelectron. 2004, 19, 1611. (12) Mingotaud, C.; Lafuente, C.; Amiell, J.; Delhaes, P. Langmuir 1999, 19, 289. (13) Ravaine, S.; Lafuente, C.; Mingotaud, C. Langmuir 1998, 14, 6347. (14) Romualdo-Torres, G.; Agricole, B.; Delhaes, P.: Mingotaud, C. Chem. Mater. 2002, 14, 4012. (15) Saliba, R.; Agricole, B.; Mingotaud, C.; Ravaine, S. J. Phys. Chem. B 1999, 103, 9712.
10.1021/la063175g CCC: $37.00 © 2007 American Chemical Society Published on Web 03/17/2007
4676 Langmuir, Vol. 23, No. 8, 2007
In order to build a new glucose sensor, it is an attractive idea to combine the two findings above to form a sensing part. For this purpose, we introduced GOx into the PB-based LB films and characterized the sensor properties. A remarkable feature of such a sensor based on LB films is the existence of nanoscale PB clusters, which are expected to show highly catalytic functionality with their large surface-to-volume ratios and the easy electron-transfer nature between the electrode and GOx. The important point to note is that a thick PB layer prepared by electrochemical deposition, which is commonly employed for PB-based sensors, will suppress electron transfer because of the intrinsic semiconducting nature of PB with a high resistivity of 107 ohm‚cm at a room temperature. From this point of view, nanoscale PB clusters can enhance the electron transfer between the electrode and the PB active surface due to a relatively short traveling distance for the charge carriers. A similar nanoscale approach has been reported with the so-called layer-by-layer method, where PB is protected by an organic polymer, and showed better sensor properties.16 In comparison with the layer-by-layer method, the advantage of the LB method is the use of bare PB clusters which are packed in the same layer of GOx; such features may lead to more effective charge transfer together with the high efficiency of the catalytic reaction, since the active sites of the PB surface are closely located to the GOx. In this paper, we employed octadecyltrimethylammonium (ODTA) as the positively charged molecule that can induce PB adsorption in LB films. Adsorption of PB was confirmed by both UV-vis spectroscopy and transmission electron microscopy (TEM). For GOx immobilization onto the LB films, a simple immersion method where LB films were dipped into a GOx solution was examined. A test for glucose sensor function was done in a conventional three-electrode electrochemical cell. 2. Experimental Section 2.1. Reagents and Materials. Bromide salt of ODTA was purchased from Aldrich. PB was obtained from Fluka. Glucose oxidase (E.C. 1.1.3.4), type VII, from Aspergillium niger, was purchased from Sigma. Potassium dihydrogen phoshate, potassium chloride, potassium hydroxide, and other chemicals were of analytical grade and obtained from Wako Chem (Japan). The glass slides (1 cm × 2 cm, Corning 7059), on which a Au electrode (thickness 250 nm) superposed onto Cr (thickness 50 nm) was predeposited by vacuum evaporation through a shadow mask, were used as substrates for electrochemical experiments. The sensing area of the evaporated electrode was 0.54 cm2. For spectroscopic experiments such as UV-vis absorption, infrared absorption, and X-ray diffraction, plates of calcium fluoride (1.3 cm × 38 cm × 0.05 cm, Oken, Japan) were employed as substrates. The substrates of 200 mesh copper grid covered with collodion film (NISSIN EM, Japan) were used for TEM experiments. 2.2. Preparation of LB Films. Spreading solutions of ODTA (9.2 × 10-4 mol/L) were prepared from chloroform (spectroscopic grade, Dojin, Japan) and were kept at -18 °C between experiments to avoid solvent evaporation. An appropriate amount of the ODTA solution was gently spread onto a 1 × 10-5 mol/L PB solution with a microsyringe. The spread ODTA molecules were laid on the PB solution surface for 50 min to enhance the PB adsorption, and they were compressed by a movable barrier to record the surface pressurearea isotherm or to prepare LB films. For the LB film preparation, the surface pressure was fixed at 30 mN/m. The Z-type LB films (transfer on the upstroke only) were obtained by the vertical deposition method with an approximate transfer ratio of 0.9-1.0 under a constant dipping speed of 2.3 cm/min.17 During the dipping cycles, the substrate was allowed to dry in air for 20 min. We named these LB films ODAT/PB LB films. (16) Zhao, W.; Xu, J.-J.; Shi, C.-G.; Chen, H.-Y. Langmuir 2005, 21, 9630. (17) Talham, R. R. Chem. ReV. 2004, 104, 5479.
Ohnuki et al.
Figure 1. Surface pressure-area isotherms of ODTA spread on the pure water and on the PB solution (10-5 mol/L). The GOx immobilization in the ODTA/PB LB film was performed by an immersion into a GOx water solution that contained 1 mg/mL GOx and 0.5 mmol/L KCl. It was found that immersion for 30 min was sufficient for substantial amounts of GOx to be adsorbed. After the immersion, the samples were rinsed in 0.5 mmol/L KCl solution for 30 min to confirm the immobilization of GOx in the LB films (named ODAT/PB/GOx LB films). 2.3. Film Characterization. Electrochemical measurements were performed with a ALS/CHI model 600B electrochemical analyzer. A conventional three-electrode system was used for the measurement: a Pt wire was employed as the auxiliary electrode, a Ag/AgCl electrode as the reference electrode, and ODTA/PB/GOx LB films built on the Au-patterned glass slide was used as the working electrode. The measurements were performed in a phosphate buffer solution containing 0.1 mol/L KCl and 0.05 mol/L KH2PO4 adjusted to be pH 7 by KOH. For the amperometric experiments, the potential of the LB films was held at the operating value (0.0 V vs Ag/AgCl) for 30 min before the measurements, which allows the background current to achieve a steady state. UV-vis and IR absorption spectra were recorded on a Shimadzu UV 2500 PC spectrometer and Nicolet Magna-IR 550 spectrometer, respectively. TEM was carried out with a JEOL-2010F microscope operating at 200 kV. X-ray diffraction profiles were obtained with Rigaku RAD 2A diffractometer using CuKR radiation at room temperature.
3. Results and Discussions 3.1. Monolayer Behavior of ODTA/PB. Formation of the Langmuir monolayer was observed by measuring the surface pressure as a function of the surface area. Figure 1 shows two surface pressure-area isotherms of ODTA spread on the pure water surface and ODTA spread on the PB solution. It was found that the ODTA spread on the pure water surface did not produce any surface pressure. This phenomenon is attributed to dissolution in the water even if the molecule has a long alkyl chain. On the
Incorporation of Glucose Oxidase into LB Films
Langmuir, Vol. 23, No. 8, 2007 4677
Figure 3. TEM image obtained from a ODTA/PB LB monolayer.
Figure 2. UV-vis absorption spectra of ODTA/PB LB films with a monolayer and 4, 6, 8, and 11 layers. The inset shows variation of the peak intensity observed at 700 nm.
other hand, for ODTA on the PB solution, the isotherm exhibits a stable Langmuir film formation on the water surface: The form of monolayer is maintained up to 40 mN/m without collapse; the occupied area per molecule in the solid phase (surface pressure ≈ 30 mN/m) corresponds to the lateral packing area for one alkyl chain (surface area ≈ 20 Å). The clear difference in the isotherms indicates that PB induces a strong stabilization effect on ODTA film; the behavior suggests that the PB introduction in the solution reacts with the ODTA monolayer. A similar phenomenon has been observed for related Langmuir films and demonstrated that the dissolved PB undergoes electrostatic interactions with the charged layer.12,18 Thus, for the present system, an electrostatic interaction between anionic PB clusters in the solution and the ODTA+ cation leads to a stable film formation on the water surface, which brings about hybrid organic-inorganic film formation of the ODTA/PB. Stabilization is essential for the LB film deposition, since it allows the film to be transferred by the vertical deposition method and leads to stable LB film formation on various substrates with optically defect-free surfaces. We confirmed the above PB adsorption phenomenon by UVvis absorption spectroscopy. Figure 2 shows absorption spectra of ODTA/PB LB films with a monolayer and 4, 6, 8, and 11 layers. A characteristic peak of PB that corresponds to charge transfer from Fe(II) to Fe(III) is detected at 700 nm for each sample.19 The observation of this peak indicates an occurrence of PB adsorption in the LB films. The variation of peak intensity is plotted as a function of the number of transferred layers in the inset of Figure 2. It is revealed that the peak intensity increases linearly as the number of LB layers is increased, which indicates that PB is homogeneously trapped in the LB films. A slight deviation from the linear relationship was observed for the thickest LB films of 11 layers; we estimate that the reason is the film (18) Yamamoto, T.; Umemura, Y.; Sato, O.; Einaga, Y. Chem. Mater. 2004, 16, 1195. (19) Robin, M. B.; Day, P. AdV. Inorg. Chem. Radiochem. 1967, 10, 247.
Figure 4. IR absorption spectra of 29 layers of ODTA/PB LB films (before GOx immobilization) and ODTA/PB/GOx LB films (after GOx immobilization).
transferred by the latter method would contain higher PB concentration due to its longer reaction period on the PB solution surface. For direct observation of the adsorbed PB clusters, we carried out TEM experiments on the LB films. Figure 3 shows a TEM image obtained from the ODTA/PB monolayer transferred onto a copper grid covered with a collodion film. It was observed that a lot of nanoscale PB particles whose diameters were approximately 10-20 nm were adsorbed onto the ODTA/PB LB films, and these particles coalesced to form bigger islands. An interesting feature is that there are two kinds of surface area in the LB films: one is covered with PB islands and the other is not. We estimate that approximately 30% of the surface is covered by PB islands and the other 70% is composed of the pure ODTA layer. 3.2. GOx Adsorption. The GOx immobilization was confirmed by IR absorption spectra through a comparison between the samples before and after GOx immobilization. These spectra are shown in Figure 4. For the sample of ODTA/PB LB films (before GOx immobilization), a strong absorption peak corresponding to the CN stretching mode of PB was observed at 2087 cm-1, and two absorption peaks of the CH2 stretching mode of the ODTA alkyl chains at 2850 cm-1 and 2917 cm-1 were also
4678 Langmuir, Vol. 23, No. 8, 2007
Ohnuki et al.
Figure 5. IR spectra of 11 layers of ODTA/PB/GOx LB films rinsed for 30 min and 90 min.
recorded.20 Hence, the spectrum is consistent with the film component molecules. For the sample of ODTA/PB/GOx (after GOx immobilization) LB films, new peaks originating from the GOx molecule appear in the spectrum: The broad peak observed around 3300 cm-1 is attributed to the stretching mode of NH (amide A band), the peak located at 1652 cm-1 is assigned to CdO stretching (amide I band), and the small absorption peak at 1540 cm-1 is the NH bending mode (amide II band).7,21 The appearance of these characteristic peaks shows an immobilization of GOx during the immersion process. In previous reports, it was determined that GOx has a strong tendency to adsorb onto positively charged monolayers through electrostatic interaction.3,4,11,16 Hence, for the present case, the GOx immobilization is brought about by the electrostatic interaction with ODTA+, which appears in the ODTA/PB LB films during the immersion process. We confirmed the stability of GOx adsorption by IR measurements, since GOx might be desorbed by the additional immersion necessary for the electrochemical measurement. Figure 5 shows the IR spectra of the two samples: one was rinsed for 30 min in the KCl solution, and the other was additionally rinsed for 60 min (total rinsing period was 90 min). No change of the amide I band was found between the IR spectra, which indicates that no GOx was desorbed by the additional rinse; the adsorbed GOx was immobilized tightly in the LB films. Furthermore, through IR measurements, we checked whether the GOx immobilization process was fast enough to reach a saturated state within 10 min. Figure 6 shows IR spectra of the samples immersed in the GOx solution for 10 min, 30 min, and 60 min. All the samples were rinsed for 30 min after immersion in GOx solution. As seen in the figure, there is no significant difference in the amide I bands among these spectra, which implies that almost the same amount of GOx is adsorbed in each condition. The result indicates that the GOx adsorption phenomenon reaches the saturation point within the immersion for 10 min. We found that such GOx adsorption induced a structural change in the LB films. Figure 7 shows the X-ray diffraction profiles obtained from 29 layers of the LB films before and after the GOx immobilization process. For ODTA/PB LB films, a series of diffraction peaks corresponding to a stacking periodicity of 23 Å was detected. The stacking periodicity is attributed to the Z-type stacking of ODTA in which the alkyl chains incline 20° (20) Romualdo-Torres, G.; Agricole, B.; Mingotaud, C.; Ravaine, S.; Delhaes, P. Langmuir 2003, 19, 4688. (21) Watanabe, N.; Ohnuki, H.; Saiki, T.; Endo, H.; Izumi, M.; Imakubo, T. Sens. Actuators, B 2005, 105, 404.
Figure 6. IR spectra of 11 layers of ODTA/PB/GOx LB films immersed in GOx solution for 10 min, 30 min, and 60 min.
Figure 7. X-ray diffraction profiles of 29 layers of ODTA/PB LB films (solid line) and ODTA/PB/GOx LB films (dotted line). The arrows indicate the calculated peak positions corresponding to 23 Å periodicity. The inset shows a schematic picture of ODTA/PB LB films estimated from the diffraction profile.
from the substrate normal. This observation of a pure ODTA stacking layer is consistent with the result of TEM experiments: there are two kinds of areas in the LB film, one contains PB clusters and the other does not (pure ODTA area). The former PB-containing area may not contribute to producing the X-ray diffraction peaks because of the disordering size and shape of PB clusters; on the other hand, the pure ODTA area has an ordered stacking structure and can produce the series of diffraction peaks corresponding to the stacking periodicity of 23 Å. Hence, the observed 23 Å periodicity is considered to originate from the pure ODTA area. After the GOx adsorption, the series of diffraction peaks disappeared completely. The abrupt change in diffraction profile indicates that the GOx adsorption induces structural disorder in the pure ODTA area. The disappearance of diffraction peaks also implies an occurrence of GOx intercalation in which GOx
Incorporation of Glucose Oxidase into LB Films
Langmuir, Vol. 23, No. 8, 2007 4679
Figure 8. Scheme of LB films preparation containing ODTA, PB, and GOx.
molecules interpose between the Z-type stacking layers through the electrostatic interaction with ODTA+, since such an intercalation process disturbs the periodic structure and will erase the diffraction peaks as is seen in Figure 7. It is expected that the sandwiched GOx between the layers is more mechanically stable than a GOx simply attached on the surface through an electrostatic force; this may interpret the good stability of GOx immobilization in the LB films. 3.3. Adsorption Mechanism. On the basis of the above experimental results, we estimate a series of models for both adsorption processes of PB to ODTA film on the water surface and also that of GOx to ODTA/PB LB films. As has been already mentioned, these adsorption processes utilize the attractive interaction of an electrostatic force working between the adsorbing species and the -N(CH3)+ cation of ODTA. These situations are depicted in Figure 8. First, for the floating monolayer of ODTA positively ionized on the water surface, the negatively charged PB clusters are attached beneath ODTA layer. The PB adsorption process brings about a stabilization and formation of an ODTA/ PB Langmuir film (Figure 8a). Second, a stable Z-type LB film is built by the vertical transfer method. On this stage, PB nanosized particles with 10-20 nm diameter adsorb in some parts of the LB films, while there are no PB particles in the other parts of the LB films (Figure 8b), which is consistent with a previous report on analogous LB films.14,20 Third, GOx adsorption into the LB films occurs during immersion of the film into a GOx solution (Figure 8c). The adsorption is considered a kind of intercalation reaction driven by the electrostatic interaction between GOx and ODTA+, and the established bond is strong enough to give a stable ODTA/PB/GOx LB film. 3.4. Amperometric Glucose Sensor. PB can act as an electrocatalyst for hydrogen peroxide reduction at low applied potential, which is capable of applying an amperometric glucose sensor when it coexists with GOx. It is well-known that PB on the electrode is reduced to form Prussian white (K4Fe(II)[Fe(II)(CN)6]3, abbreviated as PW) in low or negative potential range, and the PW is the key material to glucose sensing since it catalyzes the reduction of hydrogen peroxide produced by GOx. It has been noted that the analogous LB films containing PB clusters exhibited a typical cyclic voltammogram whose profile is consistent with the known redox behavior of PB films or PB particles.13,15 Hence, for the present ODTA/PB/GOx LB films, the redox cycles presented in Figure 9 are estimated to occur, which gives rise to a glucose sensor action for the LB films.22 At the first stage, hydrogen peroxide is produced by the enzymatic activity of GOx: the glucose is decomposed into gluconolactone (22) Haghighi, B.; Varma, S.; Alizadeh Sh., F. M.; Yigzaw, Y.; Gorton, L. Talanta 2004, 64, 3.
Figure 9. The redox cycles occurring at the electrode covered by ODTA/PB/GOx LB films.
and hydrogens, and then the latter reacts with O2 in the water to give hydrogen peroxide23
Glucose + O2 f Gluconolactone + H2O2
(1)
Second, the hydrogen peroxide produced oxidizes PW and transforms it into PB
K4Fe4(II)[Fe(II)(CN)6]3 + 2H2O2 f Fe4(III)[Fe(II)CN6]3 + 4OH- + 4K+ (2) Third, the PB is reduced again to PW via electron exchange on the electrode surface
Fe4(III)[Fe(II)CN6]3 + 4e- + 4K+ f K4Fe4(II)[Fe(II)(CN)6]3
(3)
As a result, when the series of reactions loops continuously, an electric current flow is induced proportionally with the glucose concentration. 3.5. Response to Glucose. Figure 10 shows the amperometric response for a glucose sensor built with ODTA/PB/GOx LB films (6 layers) operating at 0.0 V (vs Ag/AgCl) in a buffer solution of pH 7.0. For the experiments, we measured the response current during the successive injections of dense glucose solution that corresponds to an increase of 1 mmol/L concentration for each injection. In the figure, an occurrence of reduction current is clearly observed at each period of glucose injection. The phenomenon demonstrates the effective biofunctionality of GOx immobilized in the LB films and also exhibits that adsorbed PB clusters work as a catalyst causing the above reaction cycles to take place at the low potential. A typical response time required for reaching 90% steady-state response was within 20 s. This period seem to depend on the diffusion ratio of glucose. We also (23) Wilson, R.; Turner, A. P. F. Biosen. Bioelectron. 1992, 7, 165.
4680 Langmuir, Vol. 23, No. 8, 2007
Ohnuki et al.
Figure 12. Dependence of the current density at 5 mmol/L glucose concentration versus the number of ODTA/PB/GOx LB layers. Figure 10. Amperometric response obtained at 0.0 V in a buffer solution at pH 7.0 with ODTA/PB/GOx LB films (6 layers) deposited on a gold electrode. The arrows show the moment of glucose solution injection whose amount corresponds to an increase of 1 mmol/L glucose concentration.
Table 1. Performance of PB-Based Glucose Biosensors: Sensitivity and Linear Range of Glucose Detection electrode LB films (4 layers) LbL/ECD PBa Nafion-coated GOx/ECD PBb
sensitivity linear range (µA mmol L-1 cm-2) (mmol L-1) 0.12 16 0.18
e15 e6 e5
refs present work 11 9
a Layer-by-layer films adsorbed with poly(allylamine) hydrochloride layers on ITO substrate modified with electrochemically deposited PB. b Nafion-coated GOx onto the electrode modified with electrochemically deposited PB.
Figure 11. Plots of response current density versus glucose concentration for the biosensors built with a monolayer and 4, 6, and 11 layers of ODTA/PB/GOx LB films. For the response current density, the change in observed current density from that of 0 mmol/L glucose concentration is plotted in this figure.
measured amperometric response for other sensors with a monolayer, 4 layers, and 11 layers of LB films. Those LB films gave similar response properties to those of the 6 layers, except the amplitude of current density, which increases linearly with the number of layers. 3.6. Dependence of Number of Layers. Figure 11 shows plots of the response current density versus glucose concentration for the biosensors built with a monolayer, and 4, 6, and 11 layers of LB films. In the low concentration rage (below 8 mmol/L), the current density increases linearly as the glucose concentration is increased. In particular, the sensor with 4 layers exhibits a good linear relationship between the response current and glucose concentration. However, except the 4 layers, the linearity is gradually lost when the concentration is increased over 10 mmol/ L, and the current density inclines toward a constant value. Such a tendency is more obvious for thick LB films such as 11 layers. We estimate that the behavior can be explained by the limited access of molecular oxygen in the solution that is essential for the enzymatic reaction.24 The linearity of a glucose sensor is defined by efficiency of GOx turnover. If the GOx is not rapidly
oxidized, it will become saturated and the nonlinear response will appear following the Michaelis-Menten equation. On the basis of this explanation, it is possible to interpret the gradual loss of linearity for thick LB films: the enzyme close to the electrode surface is less able to access oxygen in the solution when the film thicknesses are increased. On the contrary, a thin layer can keep the linearity because of an easy access to oxygen. Similar behavior has already been observed in the comparative study between a device using a self-assembled monolayer and a device using a thick cross-linking layer.25 In order to determine the relationship between the output current at low concentration and the number of layers, the change of current density at 5 mmol/L glucose concentration versus the number of LB layers is plotted in Figure 12. The current density increases linearly with the number of deposited layers. This fact is attributed to the larger amount of GOx immobilized in thicker LB films; moreover, the linear increase of current density as a function of the number of layers indicates that a constant amount of GOx is homogeneously adsorbed in every LB layer, even when the total numbers of LB layers were different. 3.7. Sensitivity and Stability. The sensitivity, the upper limit of the linear range, and the stability of the sensor based on LB films were also studied. Table 1 shows the list of sensitivities and the linear range of glucose detection for the sensor with 4 LB layers. In order to evaluate an advantage of the LB films, the same parameters of the other two types of glucose sensors based on the electrochemically deposited PB are taken from literature.9,11 As the sensitivity is concerned, which is the parameter corresponding to the current density production by the 1 mmol/L glucose addition, the LB-based sensor did not show a very high performance value. This is why the 4 layers of LB film are so thin (approximately 10 nm thickness) that the total amount of GOx becomes small, and accordingly, the (24) Mattos, I. L.; Lukachova, L. V.; Gorton, L.; Laurell, T.; Karyakin, A. A. Talanta 2001, 54, 963. (25) Jung, S.-K.; Wilson, G. S. Anal. Chem. 1996, 68, 591.
Incorporation of Glucose Oxidase into LB Films
production of current density is limited. On the other hand, the LB films show a wide linear range of glucose detection. This is an advantage of using LB technique. As mentioned previously, a wide linear range is a typical phenomenon for a thin-film device: the phenomenon originates from the good interaction condition with oxygen and glucose that results in an enhancement of catalytic turnover for the GOx. The present LB film can immobilize both GOx and PB within a very thin layer, which is the ideal condition for enhancement of GOx turnover. We guess that the active nature of nanosized PB also contributes to the wide linear range. The nanosized PB has an active surface where electrons are transferred easily from the electrode to H2O2. This action enhances PB and PW redox cycles, which gives rise to an acceleration of the total redox cycles shown in Figure 9. As a result, the GOx turnover speed can be increased. Meanwhile, in the lower concentration range, we found that the minimum detection limit for the sensors (1-11 LB films) was approximately 0.1 mmol/L glucose concentration. A clear tendency was observed in which thinner LB films gave better S/N ratios for a low concentration range below 1.0 mmol/L. The sensor stability was evaluated through reproducibility of the plot of the response current density versus glucose concentration from 0 to 16 mmol/L. It was observed that the output current decreased by a factor of 0.7 after one series of measurements, while the linear relationship as a function of the concentration was retained. The decay mechanism is not identified up to now. Future studies including structural modulation and changes of electronic properties induced in the LB films will provide the answer to this point.
Langmuir, Vol. 23, No. 8, 2007 4681
4. Conclusion In this paper, we demonstrated GOx immobilization in ODTA/ PB LB films and observed glucose sensor action of the LB films at low applied potential range. The hybrid Langmuir film was prepared by adsorption of PB under a film of positively charged ODTA on the water surface; then, the Z-type LB films of ODTA/ PB were transferred by the vertical dipping method. GOx was immobilized in the ODTA/PB LB films by the simple immersion method in the GOx solution, and we obtained ODTA/PB/GOx LB films. Glucose sensors based on the ODTA/PB/GOx LB films showed an amperometric sensor action at the applied potential of 0.0 V (vs Ag/AgCl) with a wide range of linear relationship between the response current density and the glucose concentration. The effective immobilization of GOx and PB clusters in the LB films is accomplished, and the immobilized film can be used for glucose sensor application. Acknowledgment. We thank Dr. Christophe Mingotaud (Laboratoire des Interactions Moleculaires et Reactivites Chimique et Photochimique - CNRS, France) and Dr. Pierre Delhaes (Centre de Recherche Paul Pascal - CNRS, France) for valuable and informative discussions on the LB film preparation technique. Financial support by the Tokyo University of Marine Science and Technology and the Ministry of Education, Science and Culture of Japan (Grant-in-Aid for Scientific Research (C), 18560005, 2006) is gratefully acknowledged. LA063175G
