Fabrication of calcium ion sensitive diamond field effect transistors (FETs) based on immobilized calmodulin

Materials Letters 64 (2010) 2321–2324

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Materials Letters j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t l e t

Fabrication of calcium ion sensitive diamond field effect transistors (FETs) based on immobilized calmodulin Jung-Hoon Yang a,c, Munenori Degawa b, Kwang-Soup Song a,b, Chunlei Wang c, Hiroshi Kawarada a,b,⁎ a b c

Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Waseda Tsurumaki-cho 513, Shinjuku-ku, Tokyo 162-0041, Japan School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan Department of Mechanical and Materials Engineering, Florida International University, Miami, FL 33174, USA

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Article history: Received 1 January 2010 Accepted 25 February 2010 Available online 3 March 2010 Keywords: Diamond FET Calmodulin Calcium ion sensor Functionalization

a b s t r a c t We have developed calcium (Ca2+) ion sensitive solution-gate field effect transistors (SGFETs) on polycrystalline diamond by using partial amination and immobilization of calmodulin (CaM), a specific protein to calcium ions. The CaM is covalently immobilized on functionalized diamond surface, and the functionalized surface was analyzed by X-ray photoelectron spectroscopy, respectively. Also, the high performance of the CaM-immobilized diamond SGFET for detecting Ca2+ were studied with respect to high selectivity and sensitivity in various concentrations and pH. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Since diagnostics by biosensors and bioelectronics has become a popular choice in clinical medicine due to their easy minutarization, fast response and better selectivity, the application of biomaterials such as DNA, enzyme, and protein for recognizing a specific target has been widely used for detecting different chemical species [1], predict disease progression [2], cancer monitoring [3], and food pathogens [4]. Calcium ion of these chemical species is one of the essential chemical for its functions such as regulation of enzyme activity, neuronal activity, muscle contraction and vesicle exocytosis [5]. Most conventional calcium detection is based on a potentiometric sensor which consists of an ion selective membrane on the electrode. Although these sensors have good sensitivity and fast response, they require additional surface modification or deposition of specific membranes on the electrode. Diamond surfaces have many attractive characteristics for bioapplication, such as wide potential window, physiochemical stability, biocompatibility, and low background signal. As a result of these advantages for biological application, several diamond-based biosensors were reported for detection of various biological phenomena such as DNA hybridization [6,7], antibody–antigen binding [8], and enzyme reaction [9]. In our previous work, we introduced the several advantages of diamond solution-gate field effect transistors (SGFETs) like the presence of conductive layer of p-type on the diamond surface

⁎ Corresponding author. School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan. Tel.: +1 305 348 1217; fax: +1 305 348 1932. E-mail address: [email protected] (H. Kawarada). 0167-577X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2010.02.057

without doping, simple functionalization, and high sensitivity without insulating layer [10]. A calmodulin (CaM), containing four binding sites of Ca2+ ion, is a small multifunctional regulatory acidic protein [11]. The main idea of this paper is to detect Ca2+ on CaM-modified diamond SGFETs after direct functionalization and subsequent immobilization on the diamond surface. The CaM was covalently immobilized by treating with glutaraldehyde (GA) on aminated diamond surface. X-ray photoelectron spectroscopy (XPS) spectra of the diamond film was compared and analyzed after each process step such as amination, GA treatment and immobilization of CaM. In addition, the electrical performance by positive charge of Ca2+ on CaMmodified diamond SGFETs with respect to sensitivity in two different pH solutions is also described and discussed.

2. Experimental details All the chemicals and solvents used in this experiment were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). The Calmodulin was purchased from Sigma Genosys Japan (Hokkaido, Japan). Polycrystalline diamond film was synthesized on a silicon substrate in 0.1% methane diluted hydrogen gas by the microwave-plasma-assisted chemical vapor deposition (MPCVD) method. After synthesizing the diamond film, surface was treated with hydrogen plasma in the MPCVD for hydrogentermination. The sheet resistance of the as prepared hydrogen-terminated (H-terminated) diamond surface was approximately 10–20 kΩ/sq and the thickness was 8 µm. The diamond solution-gate FETs (SGFETs) were then fabricated on these H-terminated diamond film. High-purity gold was evaporated through a metal mask on the surfaces to form ohmic contacts for source and drain. Ar+ ions were then implanted through a metal mask (Mo) to form an insulating region. Then the drain and source

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electrodes were passivated with epoxy resin to protect the metal regions from the electrolyte solution. The channel length and width were 500 μm and 8 mm, respectively. Finally, the gate surface on the diamond was directly exposed to the electrolyte solution. The schematic diagram of immobilization and detection procedure on diamond SGFETs is illustrated in Fig. 1. Before amination, all the Hterminated diamond SGFETs were rinsed with deionized (DI) water, and then dried under a stream of nitrogen gas (a). Direct amination on the gate surface of SGFETs was performed with ultraviolet (UV) irradiation in an ammonia gas (99.99%) environment for introducing amino group on the surface (b). At first, nitrogen gas was introduced in amination chamber before UV irradiation for 5 min to remove other activated gases. Then, the samples were irradiated with UV light in ammonia gas for 4 h. The wavelength of UV light used was about 253.7 nm. The process of amination was carried out at room temperature. For the chemical binding between the amino group and GA as linker, directly aminated gate of diamond SGFETs were treated with a solution of 5% GA in water for 1 h and washed three times with water (c). The CaM, a calcium-binding multifunctional regulatory protein, was diluted with 10 mM phosphate buffer solution (PBS) to a final concentration of 4 mg/ml. For immobilization of CaM, 30 μl of the diluted solution was treated on the GA linked gate surface and then incubated at 38 °C for 5 h (d). After immobilization, the gate surface was washed three times with PBS and then dried. Mixed buffer solution with 10 mM NaCl and 10 mM KH2PO4 was used as the supporting electrolyte for compensation of chlorine ion and ionic strength. Also, CaCl2 was used to adjust the concentration of chlorine ion. The pH of the buffer solution used was 4.8 and 8.0. Experiment of the diamond SGFETs characteristics were performed with two source measure units (Keithley Instruments Inc., source meter model 2400) under nitrogen atmosphere at room temperature (e). 3. Results and discussion To confirm the functionalization of direct amination and immobilization of CaM on diamond substrate, XPS was used at 400 W, and analyzed by AlKα line with monochrometer at a pressure of under 10− 9 Torr. Also, the binding energy scale was calibrated to 285.0 eV for the main C 1s (C–C bond) feature. As shown in Fig. 2(a), the survey scans on treated substrate primarily used to observe N 1s, O 1s and C 1s peaks according to the corresponding functionalization process; (1) partially aminated diamond surface, (2) GA linked with amino group on surface, (3) CaM immobilized with linked GA and (4) CaM-immobilized diamond surface after immersing 10 μM CaCl2 solution for 10 min. The O 1s spectrum observed a peak at 537.5 eV in this figure due to the increased hydrophilicity on the

functionalized diamond surface by UV irradiation and thus the surface easily absorbs water molecules. In addition, we can attribute the increased O 1s and N 1s peaks to the immobilized CaM. However, the Ca2p3s peak of the CaM-immobilized surface immersed in CaCl2 solution is not observed in the survey scan as the CaM has only four Ca2+ binding sites, and compared to the molecular size, the Ca2p3s peak would be very low. Also, as the O 1s peak area also includes several other oxygen molecules, such as C–O and C O bonding, and from oxygen atoms in water molecules, it is difficult to do quantitative analysis of the functionalized surface [12]. However, the adsorbed water on the diamond surface cannot be neglected on these hydrophilic surfaces such as oxygen- and amine-terminated ones. XPS spectra of N 1s, shown in Fig. 2(b) were investigated to evaluate the modified surface. A significant peak centered at 399.9 was observed in the spectrum and the corresponding coverage of nitrogen on the diamond surface was calculated by comparing the main carbon peak in the high resolution XPS scan. The nitrogen coverage after direct amination is 0.37 mono-layer (ML) with UV irradiation for 4 h, but its coverage decreased to 0.23 ML after GA treatment. It is expected that the ratio between carbon and nitrogen changed due to the chemical combined amino group with GA as linker on the diamond surface. Also, nitrogen coverage after CaM immobilization has increased two times higher than GA modified surface because of the immobilized CaM. As the CaM has numerous amino terminations, its coverage is increased on the modified surface and it is indicated that the CaM is chemically immobilized with GA on the aminated diamond surface from this result. After immersing 10 μM CaCl2 solution, nitrogen coverage is similar with CaM-immobilized surface because the CaM just reacted with Ca+ ion without any other reaction. Fig. 3 shows the response of a CaM-immobilized SGFETS on diamond in CaCl2 solution. The solution-gate field effect transistors (SGFETs) on the partial aminated diamond surface for other bioapplication such as urea and DNA sensor has been previously described [9,13]. The change in Vgs was measured at Ids =−12 μA with Vds =−0.1 V in various concentrations of CaCl2. To understand CaM activity, we have carried out the measurement in two different pH buffer solutions because isoelectric point (pI) of CaM is 3.9–4.3. Although the gate potential of the CaM-immobilized SGFETs did not change in pH 8 solution in accordance to the change in Ca2+ ion concentration, observed in the negative direction over a wide range of concentration values from 10− 6 M to 10− 9 M, as shown in Fig. 3(a). These observations indicated that the immobilized CaM is strongly combined with Ca2+ ion in pH 4.8 and its positive charge was reflected on the modified diamond SGFETs [Fig. 3(a) 1]. However, immobilized CaM shows no binding in pH 8 due to structural

Fig. 1. A schematic of the immobilization process of a calmodulin (CaM) for detection of calcium ion on the diamond surface: the CaM was immobilized with treated glutaraldehyde on the gate of diamond solution-gate field effect transistors (SGFETs).

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Fig. 3. (a) The real time detection and (b) the sensitivity of calcium ion on calmodulinimmobilized diamond SGFETs in the different pH solutions; pH 4.8 (1) and pH 8.0 (2).

Fig. 2. (a) Wide scan spectra and (b) nitrogen coverage by XPS on the diamond surface according to functionalization; partial amination (1), glutaraldehyde treatment (2), calmodulin immobilization (3), and immersing 10 μM CaCl2 solution for 10 min (4).

inactivity [Fig. 3(a)]. Thus, the CaM is one of the most extensively recognized Ca2+ binding proteins because the primary structure of CaM is highly conserved in all cell types and shares strong sequence and structure homology to troponin C, which is involved solely in the Ca2+-dependent regulation of skeletal and heart muscle contraction [14]. Because the diamond SGFET is only sensitive to halogen ions due to its characteristics of P-type surface conductive layer, the selectivity of CaM-immobilized calcium sensor also depends on immobilized calmodulin affinity with calcium ion [15]. Fig. 3(b) shows the changed gate potential, Vgs, of the CaMmodified diamond SGFETs according to the different concentrations of Ca2+. The sample volume used for the diamond SGFETs was 0.5 ml as it is important to note that the sensitivity of FET based sensor is independent of the gate area although sensitivity of amperometric sensor theoretically depends on the electrode area. The detection limits corresponds from 10− 9 M to 10− 5 M, and the sensitivity of the device is 1.9 ± 0.3 mV/pCa2+ in pH 4.8. In addition, it is observed that the shift of Vgs is saturated at more than 10− 5 M of Ca2+ since under the saturated condition of CaM, the immobilized CaM molecule is only capable of combining four Ca2+ ions. These results indicated that CaM-immobilized diamond SGFETs shows high sensitivity in low concentration of Ca2+ ions. However, the device at pH 8 cannot detect any response from Ca2+ ions because of the CaM inactivity. However, it is not easy to explain the stability of immobilized CaM in the pH range because saturation of the calcium-binding sites in CaM induced conformational change and calcium-bound Calmodulin activates

various proteins that modulate physiological activities. Consequently, the CaM was successfully immobilized on aminated diamond and the detection of Ca2+ ion on CaM-immobilized diamond SGFETs are very sensitive and stable in pH 4.8. Also, the measurements could be made in small volumes (0.5 ml) thus reducing the requirements of the sample volume. 4. Conclusions By covalent immobilization of CaM on partially aminated gate surface, we successfully fabricated Ca2+ ion sensitive diamond SGFETs in electrolyte solution. All functionalization steps were critically confirmed by XPS, and nitrogen coverage during the process was calculated for estimating the density of the functional group. It is observed that the effect of positive charge from Ca2+ ion was clearly observed in the highly sensitive diamond SGFETs in pH 4.8. However, the response in pH 8 was not detected due to the inactivity of CaM. From these results, we consider that the present system including the operational range of pH4.8 can be applied for continuous and specific ion channel in artificial cell membrane because the calcium ion is an essential cofactor in the gating of potassium channels. Acknowledgments This work was supported in part by a Grant-in-Aid for Center of Excellence (COE) Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and the Consolidated Research Institute for Advanced Science and Medical Care, Waseda University (ASMeW). Also, this work was partially supported by the National Science Foundation (Grant No. OISE 0934078).

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