Extended Summary 本文は pp.317-320

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Design and Fabrication of Biosensor Device by Use of Receptor Proteins

Yoshihiko Kuwana Member (National Institute of Agrobiological Sciences, kuwana@affrc.go.jp)

Katsura Kojima Non-member (National Institute of Agrobiological Sciences, kojikei@affrc.go.jp)

Yasushi Tamada Non-member (National Institute of Agrobiological Sciences, ytamada@affrc.go.jp)

Keywords : Biosensor, Receptor proteins, Lipid bilayer membrane, Insulated-gate field-effect transistor, Silk moth

Sensory organs of insects have high sensitivity and selectivity. For example, a male silk moth can detect even a single molecule of a sex pheromone from a female moth(1). This very high sensitivity is attributed to receptor proteins present in their antennae. Thus far, a biosensor that uses receptors, i.e., a receptor-based biosensor, has not been realized, because of the receptor’s unmanageability. Since receptor proteins have a very high potential as a sensing device, a receptor-based biosensor will have a wide range of applications in a variety of fields such as protein science, drug screening, environmental studies, and agriculture. In this study, we considered pheromone receptors and their signal transduction system present in antennae of male silk moths as the model of our receptor-based biosensor.

Here, we report the development of a prototype of a receptor-based biosensor using which the sensory functions of an insect can be imitated. We propose the design of a biosensor that can be constructed by combining receptor proteins with a semiconductor device such as a field-effect transistor (FET), as shown in Figure 1. When a stimulus molecule attaches to the binding site of a receptor protein (1 in Figure 1), signal transduction reactions occur on the membrane; these reactions affect the membrane potential (2–4 in Figure 1). The change in membrane potential can be recorded using an FET (5 in Figure 1).

We also present the basic characteristics of the proposed device. This device was designed to record the changes in the membrane potential and to measure the channel activities of α-hemolysin (α-HL), which acts as a model of insect receptors.

At first, we developed a lipid bilayer membrane that can immobilize receptor proteins. The bilayer membrane was formed on a polystyrene microchamber by lipid painting. In order to confirm the presence of the bilayer, optical microscopy, membrane capacitance measurement, and ion channel current recording were carried out. Experimental results showed the existence of a lipid bilayer membrane on our biosensor system.

We have designed and fabricated an insulated-gate field-effect transistor (IGFET) to record potentials of the lipid bilayer membrane. By using this bilayer-IGFET device, we could measure changes in the bilayer membrane potential. We stimulated the bilayer membrane using a pulse current and found that the resolution of our bilayer-IGFET system was of the order of several picoamperes, which implies that our system can detect the activities of several ion channels simultaneously.

Furthermore, we introduced α-HL, which acts as a model of insect receptors. Due to the introduction of α-HL (10 µl of 0.5 mg/ml in buffer), the system could detect the membrane potential change caused by the increase of the channel current as shown in Figure 2. The gray lines in this figure show original signals and

the thick black lines show signals after the reduction of high frequency noise. This current increase is due to the increase in the channel current, which is caused by the formation of α-HL heptamers. This indicates that the developed device could be used for the biosensor using receptor proteins.

In the future, we intend to improve the sensitivity of the device in order to detect smaller potential changes by modifying the design of the system. In addition, we intend to measure the response of the insect receptors using actual insect receptor proteins. Furthermore, we intend to use recombinant insect receptors to increase the channel activities.

Fig. 1. Proposed receptor-based biosensor

Fig. 2. IGFET output after the introduction of α-HL

© 2009 The Institute of Electrical Engineers of Japan. 317

Design and Fabrication of Biosensor Device by Use of Receptor Proteins

Yoshihiko Kuwana＊ Member

Katsura Kojima＊ Non-member

Yasushi Tamada＊ Non-member

Abstract We have been studying a new type of biosensor that uses and mimics sensory functions of insects. The biosensor can be characterized in combination with immobilized signal transduction biomolecules, i.e., receptor proteins, and a semiconductor device as a transducer. We have developed a lipid bilayer membrane for receptor immobilization and combined it with an insulated-gate field-effect transistor (IGFET). By using this bilayer-IGFET device, we have measured changes in the bilayer membrane potential after introducing α-hemolysin, which acts as a model of receptors. This indicates that the developed device could be used for the biosensor using receptor proteins.

Keywords : Biosensor, Receptor proteins, Lipid bilayer membrane, Insulated-gate field-effect transistor, Silk moth

1. Introduction

Sensory organs of insects have high sensitivity and selectivity. For example, a male silk moth can detect even a single molecule of a sex pheromone from a female moth(1). This very high sensitivity is attributed to receptor proteins present in their antennae. Thus far, a biosensor that uses receptors, i.e., a receptor-based biosensor, has not been realized, because of the receptor’s unmanageability. Since receptor proteins have a very high potential as a sensing device, a receptor-based biosensor will have a wide range of applications in a variety of fields such as protein science, drug screening, environmental studies, and agriculture. In this study, we considered pheromone receptors and their signal transduction system present in antennae of male silk moths as the model of our receptor-based biosensor.

Here, we report the development of a prototype of a receptor-based biosensor using which the sensory functions of an insect can be imitated. We propose the design of a biosensor that can be constructed by combining receptor proteins with a

semiconductor device such as a field-effect transistor (FET), as shown in Fig.1. When a stimulus molecule attaches to the binding site of a receptor protein (1 in Fig.1), signal transduction reactions occur on the membrane; these reactions affect the membrane potential (2–4 in Fig.1). The change in membrane potential can be recorded using an FET (5 in Fig.1). The designed biosensor immobilizes signal transduction proteins existing in the insect sensory system rather than immobilizing a living nerve cell(2) or insect sensory organs such as a beetle antenna(3); this immobilization is carried out on the FET.

We also present the basic characteristics of the proposed device. This device was designed to record the changes in the membrane potential and to measure the channel activities of α-hemolysin (α-HL), which acts as a model of insect receptors.

2. Materials and Methods

2.1 Lipid Bilayer Membrane Several techniques such as painting, vesicle fusion, and the Langmuir-Blodgett (LB) film technique can be employed for developing a lipid bilayer membrane.

In the painting technique, several materials such as Teflon thin films with micropores(4) are used to support the lipid membrane. Glass capillaries and polymer films have been used for constructing horizontal lipid membranes(5). Further, the formation of pores with diameters of 5–200 µm, micromachined by deep reactive ion etching (DRIE)(6), and anisotropically etched apertures has been reported(7)(8).

In the vesicle fusion method, a glass substrate is usually employed to support lipid membranes(9)~(11). In the past, patterning of the lipid bilayer has been facilitated by photolithography or microcontact printing(12)~(14).

The LB film technique can also be used to form a bilayer membrane. A drawback of this technique is that it requires special and expensive apparatuses.

In this study, we employed the painting technique because it is the simplest technique for fabricating bilayers. Polystyrene microchamber plates prepared by injection moulding, each with a 200-µm-diameter aperture, were kindly donated by Starlite Co., Ltd., as shown in Fig.2 (left). * National Institute of Agrobiological Sciences

1-2, Ohwashi, Tsukuba, Ibaraki 305-8634

Paper

Fig. 1. Proposed receptor-based biosensor

318 IEEJ Trans. SM, Vol.129, No.10, 2009

The lipid, diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Inc.), was dissolved in n-decane at 20 mg/ml. A lipid bilayer was easily obtained by immersing the microchamber in a buffer solution of 10 mM Tris HCl + 100 mM NaCl (pH 7.5) and applying the lipid to the aperture. A lipid bilayer membrane surrounded by an annulus was formed at the aperture, as shown in Fig.2 (right).

2.2 Field-effect Transistor In order to detect changes in the membrane potential, we used an insulated-gate FET (IGFET) as a signal transducer. Further, we used a silicon dioxide layer and a silicon nitride layer as gate insulators.

We fabricated transistors with different gate sizes ranging from 20 × 20 µm2 to 20 × 3200 µm2. An example of an IGFET design is shown in Fig.3. We prepared p-type silicon wafers with 3–8 Ω·cm resistivity, 380 µm thickness, and a <100> crystal face. First, a thick silicon dioxide layer, having a thickness of approximately 0.3–0.4 µm, was thermally grown for surface insulation. Source and drain areas were etched by hydrofluoric (HF) acid, and then, phosphorus was thermally diffused at 1100 °C for 30 min. The sheet resistance of the diffusion area was approximately 13 Ω/sq. The field oxide layer of the gate area was stripped by HF etching. A gate oxide layer (silicon dioxide) having a thickness of approximately 25 nm was thermally grown at 1000 °C. Subsequently, silicon nitride was sputtered to a thickness of approximately 50 nm. Contact holes were etched prior to patterning of aluminum wires. The surface of the silicon wafer was covered with polyimide (CRC8300, Sumitomo Bakelite Co.) for electrical insulation and protection from chemical and physical damage. Finally, the wafer was cut into small chips, and each chip was attached to an integrated circuit (IC) package or circuit board.

I–V characteristics were measured using a data acquisition

board (NI-PCI-6229, National Instruments Co.) and LabVIEW (National Instruments Co.).

2.3 Microfluidic Device A microfluidic device with polydimethylsiloxane (PDMS) was developed for buffer perfusion, as shown in Fig.4. The buffer solution was easily perfused without breaking the bilayer. These molds were formed using a 3D plotter (Modela MDX-15, Roland DG Co.).

The IGFET was set at the bottom of the lower channel. Each layer was adhered by using PDMS. Silver wires were inserted into the groove (shown in Fig.4), and then, the groove was filled with PDMS for electrical insulation. Prior to the experiments, the silver wires were chloridized by electroplating. Chloridization was carried out by considering the inserted silver wires as positive electrodes in the 0.4 M NaCl solution contained in the upper and lower channels. Current was passed through the solution at a rate of 1 mA/cm2. These Ag/AgCl wires were used in the upper and lower channels as reference and recording electrodes, respectively.

2.4 Measurement Setup The experimental setup shown in Fig.5 was used to measure the membrane potentials using the IGFET. A lipid bilayer was formed just above the gate of the IGFET, and the bilayer membrane potentials were altered by stimulating the membrane with a stimulus buffer such as salt solution. The change in the source–drain current (Id) was dependent on the change in membrane potential. This current change was converted to voltage change by an I–V converter. The output voltage (Vout) was proportional to the drain current:

VdsRfIdVout +×= ................................................... (1)

where Id, Vout, Vds, and Rf are shown in Fig.5. Further, a commercial patch-clamp amplifier (CEZ-2400, Nihon

Kohden Co.) was used to measure the transmembrane current and the membrane potential.

Fig. 4. Design of our fluidic device

Fig. 3. Design of IGFET

Fig. 2. (Left) Microchamber plate and (right) lipid bilayer membrane formed at the microaperture

Fig. 5. Measurement setup

Design and Fabrication of Receptor-Based Biosensor

電学論 E，129 巻 10 号，2009 年 319

3. Results and Discussion

3.1 Lipid Bilayer Membrane Formation In order to confirm the membrane formation, we measured the membrane capacitance, which was found to be approximately 0.3 µF/cm2. This value was in good agreement with the theoretical value of the capacitance of the lipid bilayer (0.38 µF/cm2)(15). Furthermore, 3 µl of α-HL, whose concentration was 0.5 mg/ml in buffer, was introduced into the membrane, and the channel current was recorded by the patch-clamp amplifier, as shown in Fig.6. α-HL is one of the membrane proteins, and its heptamer forms a nano pore on the bilayer. α-HL heptamers were formed corresponding to the stepwise increase in the channel current. These results indicate that the lipid bilayer is present in our system.

3.2 IGFET Characteristics We measured the I–V characteristics of our IGFET. The Id–Vds curve of a 400-µm-gate-width IGFET used in subsequent experiments is shown in Fig.7. This curve shows that our IGFETs are capable of measuring the membrane potentials. In this experiment, gate voltage was applied via a buffer by using an Ag/AgCl electrode. The 400-µm-gate-width IGFETs had high stability and were therefore employed in our study.

3.3 Measurement of Lipid Bilayer Membrane Potentials In order to verify the capability of our bilayer-IGFET system

for measuring membrane potential changes, bilayer membrane potentials were changed by maintaining different ion strengths on both sides of the membrane. We could record changes in membrane potentials (shown in Fig.8) by adding salt solution to the upper channel. Points on the graph are obtained by taking the average of ten samples; the error bars show the standard

deviations. This result shows that the output of our bilayer-IGFET system is directly proportional to the salt concentration. The actual membrane potential change, which was recorded by the commercial patch-clamp amplifier, was approximately 180 mV when the salt concentration of the upper channel was 1 M. It is known that the action potential of neurons ranges from several tens of millivolts to 100 mV(1). The results obtained from Fig.8 show that our IGFET device can measure the change in membrane potentials of the order of several tens of millivolts and can be used as a biosensor.

3.4 Pulse Current Stimulation We stimulated the bilayer membrane using a pulse current. The relationship between the amplitude of stimulating current and the output of the bilayer-IGFET system is shown in Fig.9. Each point represents the average of ten samples. Error bars are omitted because errors were negligible. As observed in Fig.9, the output of our IGFET system is proportional to the amplitude of the pulse current and the value of Rf. When Rf was 20 kΩ (shown in red in Fig.9), we could record the stimulation using a pulse current of several picoamperes. Since the single channel current is only of the order of picoamperes, our system can detect channel activities even if several channels are open simultaneously.

3.5 Incorporation of α-Hemolysin into Bilayer Membrane In order to verify the capability of our bilayer-IGFET system,

α-HL was introduced into the bilayer membrane; this was considered to be a model of receptor proteins. By maintaining different ion strengths on both sides of a bilayer, i.e., 1 M NaCl in the upper channel and 100 mM NaCl in the lower channel, the membrane potential was generated. In the IGFET circuit, Vds and Rf were set to 1.0 V and 20 kΩ, respectively. Several minutes after

Fig. 8. Recorded changes in membrane potential

Fig. 7. Id-Vds characteristics of IGFET

Fig. 9. Pulse current stimulation

Fig. 6. α-Hemolysin channel current

320 IEEJ Trans. SM, Vol.129, No.10, 2009

the introduction of α-HL (10 µl of 0.5 mg/ml in buffer), an increase in the IGFET output was observed, as shown in Fig.10. The gray lines in this figure show original signals and the thick black lines show signals after the reduction of high frequency noise. This current increase is due to the increase in the channel current, which is caused by the formation of α-HL heptamers.

From Figs. 6 and 9, we expected that the IGFET output would vary by several tens of millivolts during this model experiment. However, the observed change was only several millivolts. A possible reason for this could be that the amount of α-HL introduced in the bilayer in this experiment was very small. Since we couldn’t control the amount of introduced α-HL, we need more sentive experimental setups to measure smaller signals.

4. Conclusion

We formed a lipid bilayer membrane on a polystyrene microchamber by applying a lipid. In order to confirm the presence of the bilayer, we carried out optical microscopy, measured membrane capacitance, and recorded α-HL channel current. Experimental results showed the presence of a lipid bilayer membrane on our biosensor system. We designed and fabricated an IGFET to record potentials of the lipid bilayer membrane. The fabricated bilayer-IGFET device could successfully record the bilayer membrane potentials. We stimulated the bilayer membrane using a pulse current and found that the resolution of our bilayer-IGFET system was of the order of several picoamperes, which implies that our system can detect the activities of several ion channels simultaneously. Furthermore, we introduced α-HL, which acts as a model of insect receptors. Due to the introduction of α-HL, the system could detect the membrane potential change caused by the channel current. These results indicate that the developed device could be used for the biosensor using receptor proteins.

In the future, we intend to improve the sensitivity of the device in order to detect smaller potential changes by modifying the design of the system. In addition, we intend to measure the response of the insect receptors using actual insect receptor proteins. Furthermore, we intend to use recombinant insect receptors to increase the channel activities.

Acknowlegdement This research was supported in part by the Ministry of

Agriculture, Forestry and Fisheries of Japan and by a Grant-in-Aid for Scientific Research (B: 18380046).

(Manuscript received Aug. 18, 2008, revised Jan. 30, 2009)

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Yoshihiko Kuwana (Member) was born in Shizuoka, Japan, on March 9, 1967. He received a Ph. D degree in information engineering from The University of Tokyo in 1996, and is presently a chief researcher at The National Institute of Agrobiological Sciences, Japan. He has worked in interdisciplinary fields between engineering and biology; robotics, biosensors, microelectromechanical systems, and molecular

biology. Member of the Robotics Society of Japan, The Japan Society of Mechanical Engineers, and The Biophysical Society of Japan.

Katsura Kojima (Non-member) was born in Nagano, Japan, on June 14, 1972. He received a Ph. D degree in agriculture from Hokkaido University in 2002, and is presently a chief researcher at The National Institute of Agrobiological Sciences, Japan. He has worked in the field of molecular biology. Member of The Molecular Biology Society of Japan and The Japanese Society of Sericultural Science.

Yasushi Tamada (Non-member) was born in Gifu, Japan, on April 22, 1957. He received a Ph. D degree in engineering from Kyoto University in 1989, and is presently the head of the Silk-Materials Research Unit at The National Institute of Agrobiological Sciences, Japan. He has worked in the fields of polymer and biomaterial sciences. Member of The Society of Polymer Science, Japan and The American Chemical Society.

Fig. 10. IGFET output after the introduction of α-HL