Available online at www.sciencedirect.com ScienceDirect Procedia Engineering 168 (2016) 84 – 88 30th Eurosensors Conference, EUROSENSORS 2016 Glucose sensing by an enzyme-modified ZnO-based FET Kazuto Koikea, Yuina Moria, Shigehiko Sasaa, Yuichi Hirofujia, Mitsuaki Yanoa* a Nanomaterials Microdevices Research Center, Osaka Institute of Technology, Asahi-ku Ohmiya, Osaka 535-8585, Japan Abstract Characteristics of an enzyme-modified field-effect transistor (EnFET) made of a ZnO-based ion-sensitive FET with immobilized glucose oxidase on the gate surface are studied for the application to healthcare chips The EnFET was found to be able to detect reversibly and repeatedly the ȕ-D glucose in solution in the range of 0.2-40 mmol/L with an apparent Michaelis constant of 3.3 mmol/L, indicating the suitability for diabetic plasma glucose sensors © Published by Elsevier Ltd This © 2016 2016The TheAuthors Authors Published by Elsevier Ltd.is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference Keywords: enzyme-modified FET; ion-sensitive FET; ZnO-based FET; glucose sinsing Introduction In order to develop plasma glucose sensors, characteristics of an enzyme-modified field-effect transistor (EnFET) were studied This EnFET detects biocatalytically yielded protons by the following reaction between the ȕ-D glucose in solution and the enzyme molecules of glucose oxidase (GOD) immobilized on the gate electrode of an ion sensitive FET (ISFET) [1,2]; ȕ-D glucose + O2 ĸ GOD ĺ gluconolactone + H2O2 ĸ H2O ĺ gluconic acid + H+ [3] The ISFET used here was composed of a Ta2O5/ZnO thin film grown on a glass substrate, and had several advantages for the application to integrated healthcare chips [4,5]; 1) high conformity to electrical circuitry with high impedance to sample solution, 2) an economical and ecological material system suitable for disposable use, 3) capability of low-temperature synthesis on low cost glass and polymer substrates, 4) transparency to visible ray enabling accurate operation with low photo-induced error and combinatorial analysis with optical techniques * Corresponding author Tel.: +81-6-6954-4313; fax: +81-6-6957-2136 E-mail address: mitsuaki.yano@oit.ac.jp 1877-7058 © 2016 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference doi:10.1016/j.proeng.2016.11.153 Kazuto Koike et al / Procedia Engineering 168 (2016) 84 – 88 In this work, we report the fabrication process and characteristics of the ZnO-based EnFET, and discuss the suitability for diabetic plasma glucose sensors Experimental Procedure In Fig 1, (a) and (b) show the schematic diagram of the EnFET together with the experimental setup and the gate modification by chemical treatment, respectively As shown by Fig 1(a), the EnFET was based on a ZnO-based ISFET which was composed of an 8-nm-thick Ta2O5 insulating film as the gate insulator layer/a 30-nm-thick Indoped ZnO semiconducting film as the channel layer/a 100-nm-thick Zn0.6Mg0.4O semi-insulating film as the buffer layer The Ta2O5 film was employed to increase the stability and durability of the gate electrode in electrolyte solution [5], and the In doping was conducted to increase the carrier density in the channel layer These films were deposited on a glass substrates at 400°C for the Ta2O5 and the Zn0.7Mg0.3O films, and at 300°C for the ZnO film, by a facing-target sputtering apparatus (FTS NFTS-3S-R0) using a set of Ta metallic targets, a set of 97 mol% ZnO mixed with mol% In2O3 ceramic targets, and a set of 70 mol% ZnO mixed with 30 mol% MgO ceramic targets, respectively [5] The deposition was conducted by a reactive sputtering method in a mixture gas of Ar:O2 = 2:1 Fig (a) schematic diagram of the EnFET together with the experimental setup; (b) modification of the Ta2O5 gate surface The sputtered film was fabricated into the ISFET using a standard photolithography technique A polyimide resin (Toray PW-1900) was utilized for the photoresist material to construct the insulating structural layer to protect the electrodes from electrolyte solution As shown by Fig 1(b), the solution-gate area of the Ta2O5 with a dimension of 25-ȝm-long and 2.4-mm-wide was modified with proton receptors of amino functions by treating the surface with a silane-coupling agent of 3-aminopropyltrimetoxysilane [6] Then, the ISFET was fabricated into the EnFET by immobilizing GOD molecules on the gate surface using a cross-linking reaction of glutaraldehyde [6] It is important to note that only a small portion of the amino functions will be consumed for the scaffolds of the GOD immobilization, and the rest ones remaining on the gate electrode will detect biocatalytically yeilded protons, since the size of GOD molecules (6.0×5.2×7.7 nm3) is much larger than that of amino functions In the following study, we used several kinds of mmol/L buffered solution at 20°C as the aqueous electrolyte for pH sensing, and mmol/L phosphate-buffered solution (PBS) at 35°C and pH = 5.6 as the aqueous electrolyte for glucose sensing By applying an electrostatic potential VG to the solution-gate electrode using a commercially available Ag/AgCl reference electrode, I-V characteristics of the ISFET and EnFET were measured by using a semiconductor device analyzer (Agilent B1500A) with simultaneous monitoring of the pH in PBS by using a commercial precision pH meter with a combination of glass electrodes 85 86 Kazuto Koike et al / Procedia Engineering 168 (2016) 84 – 88 Results and Discussion At first, we checked the Ǧ characteristics of the ISFET before immobilizing GOD molecules In consistent with a standard theory for the depletion mode operation of n-type FETs, the drain current ID dependence on drain-source voltage VDS showed a clear pinch-off region with current saturation As shown in Fig 2(a), its transfer curves of IDVG indicated a parallel shift of ΔVG § 55 mV/pH on the pH in buffered solution, in good agreement with the Fig (a) pH dependent transfer curves of the ISFET; (b) ID response of the ISFET to the step-like change of pH theoretical Nernstian response of 57 mV/pH at 20°C [7] Figure 2(b) shows the reversible ID response at VDS = 2V and VG = 1V to the step-like pH changes in buffered solution The quick ID responses to the proton increase and decrease in solution were composed of exponential decay terms with the time-constants of τ1a § 2s and τ1d § 4s, respectively, and the ID at stationary-state showed a linear pH dependence of approximately –92 ȝA/pH Then, we immobilized GOD molecules on the gate surface, and measured the characteristics of the EnFET at VDS = 2V and VG = 1V Figures 3(a) and (c) show the ID response to the step-like changes of the ȕ-D glucose concentration Cglc from to 17 mmol/L and from 17 to mmol/L, respectively Fig.3 (a) and (b) ID response of the EnFET to the step-like increase of the ȕ-D glucose concentration from to 17 mmol/L, and its normalized decay of the ΔID increase on logarithmic scale, respectively; (c) and (d) ID response of the EnFET to the step-like decrease of the ȕ-D glucose concentration from 17 to mmol/L, and its normalized decay of the ΔID decrease on logarithmic scale, respectively Kazuto Koike et al / Procedia Engineering 168 (2016) 84 – 88 Fig (a) Stationary-state ΔID values of the EnFET at VG = 1V and VDS = 2V for different ȕ-D glucose concentrations Cglc in logarithmic scale; (b) Hanes-Woolf plot of the data in Fig 4(a) The increase and decrease of ID with Cglc was found to be reversible and repeatable in consistent with the expectation from the operation mechanism It is noteworthy that the observed ID change, ΔID, roughly corresponds to the pH change of 0.4 while the actual pH in the background PBS was changed scarcely, indicating that most of the protons by the ȕ-D glucose oxidation stayed at the vicinity of the gate surface In Figs 3(b) and 3(d), the decay of the ΔID increase in Fig 3(a) and that of the ΔID decrease in Fig 3(c) are shown on logarithmic scale by normalizing the data with the saturation value at 17 mmol/L, respectively It is seen that the ΔID increase consists of a single exponential decay term with a time-constant of τ2a = 3s, and the ΔID decrease consists of a major exponential decay term with a time-constant of τ2d = 6s and a tailing exponential decay term with a time-constant of τ’2d = 17s Since these time-constants were a little longer than those of the pH response in Fig 2, we postulated that the rate-determining step of the ΔID response was the biocatalytic reaction of ȕ-D glucose oxidation In order to analyze the ΔID response to the Cglc change by using the Michaelis-Menten equation [8], we summarized in Fig 4(a) the ΔID dependence at stationary-states on Cglc, and rewrote it in Fig 4(b) of the HanesWoolf plot [9] The lower detection limit was estimated as ~0.2 mmol/L from the intercept of the straight line in Fig 4(a) The upper detection limit was estimated as ~40 mmol/L from the maximum ΔID, i.e., ΔIDmax of 39 ȝA, that was derived from the slope of the straight line in Fig 4(b) This detection range covers the normal plasma glucose concentrations, from ~3.8 to ~6.5 mmol/L [10], with enough margins required for the diagnosis of diabetes mellitus The apparent Michaelis constant Kmapp of the reaction was estimated as ~3.3 mmol/L from the intercept of the straight line in Fig 4(b) This value is close to those reported by others [11], and is consistent with that derived from the time-constants in the transient response at Cglc = 17 mmol/L as Kmapp = Cglcτ2a/(τ’2d – τ2a) § 3.6 mmol/L In the estimation by the transient response, the biocatalytic reaction was assumed to follow the reversible first-order rate equation of which solution at stationary-state corresponds to the Michaelis-Menten equation We substituted the longer time-constant of τ’2d into the rate-determining dissociation process ascribing the tailing term to the hindered out-diffusion of protons by the above located GOD molecules Acknowledgements This work was supported in part by the KAKENHI Grant Numbers JP25390033, JP24360141, and JP16K04936 from JSPS References [1] P Bergveld, IEEE Trans Biomed Eng BME 17 (1970) 70–71 [2] T Matsuo, K D Wise, IEEE Trans Biomed Eng BME 21 (1974) 485–487 87 88 Kazuto Koike et al / Procedia Engineering 168 (2016) 84 – 88 [3] A B Kharitonov, M Zayats, A Lichtenstein, E Katz, I Willner, Sens Actuators B 70 (2000) pp 222–231 [4] M Yano, K Koike, K Ogata, T Nogami, S Tanabe, S Sasa, Phys Status Solidi C (2012) pp 1570–1573 [5] M Yano, K Koike, K Mukai, T Onaka, Y Hirofuji, K Ogata, S Omatu, T Maemoto, S Sasa, Phys Status Solidi A 211 (2014) pp 2098– 2104 [6] K Koike, D Takagi, M Hashimoto, T Hashimoto, T Inoue, K Ogata, S Sasa, M Inoue, M Yano, Jpn J Appl Phys 48 (2009) pp 04C081 1–04C081-4 [7] J C Chou, Y S Li, J L Chang, Sens Actuators B 71 (2000) pp 73–76 [8] L Michaelis, M L Menten, Biochem Z 49 (1913) pp 333–369 [9] J B S Haldane, Nature 179 (1957) p 832 [10] “Definition and diagnosis of diabetes mellitus and intermediate hyperglycaemia”, Report of a WHO/IDF consultation, 2006, ISBN: 978 92 159493 [11] A Wei, X W Sun, J X Wang, Y Lei, X P Cai, C M Li, Z L Dong, W Huang, Appl Phys Lett 89 (2006) 123902-1–123902-3 ... the experimental setup and the gate modification by chemical treatment, respectively As shown by Fig 1(a), the EnFET was based on a ZnO- based ISFET which was composed of an 8-nm-thick Ta2O5 insulating... process and characteristics of the ZnO- based EnFET, and discuss the suitability for diabetic plasma glucose sensors Experimental Procedure In Fig 1, (a) and (b) show the schematic diagram of the EnFET... the aqueous electrolyte for pH sensing, and mmol/L phosphate-buffered solution (PBS) at 35°C and pH = 5.6 as the aqueous electrolyte for glucose sensing By applying an electrostatic potential VG