Fabrication of a Solution-gated Indium-Tin-Oxide-based One-piece Transistor Enabling Sensitive Biosensing

Summary

This protocol describes the fabrication of a one-piece indium-tin-oxide (ITO)-based ion-sensitive field-effect transistor (ISFET), which can be constructed as a solution-gated FET sensor (e.g., pH sensor) using a short and simple process (approximately half a day). This one-piece ITO-ISFET can also be applied to biosensing.

Abstract

A simple and rapid method to fabricate a solution-gated one-piece indium-tin-oxide (ITO)-based ion-sensitive field-effect transistor (ISFET) (ITO-ISFET) for biosensing is presented in this report. Although ITO is a conductive oxide, it exhibits semiconducting properties at a depletion layer thickness of approximately 20 nm or less, making it suitable as the channel of a pH-sensitive solution-gated ISFET. Specifically, by etching the middle part of a conductive ITO single thin film with an acidic solution to make it ultrathin, a one-piece ITO-ISFET with a semiconductive channel can be fabricated. The source, drain electrodes, and channel are fully integrated without any interfaces. The ITO channel surface is then soaked in sample solutions with a reference electrode, enabling it to function as a solution-gated ISFET. Thus, the one-piece ITO-ISFET can be fabricated simply and rapidly using only sputtering followed by etching through a photolithography technique, with the sample solution conveniently serving as the gate.

Introduction

A solution-gated ion-sensitive field-effect transistor (ISFET) was proposed for detecting ions in biological environments¹. In this device, an electrolyte solution induces the interfacial potential between the solution and the gate insulator instead of a metal gate in a metal-oxide-semiconductor (MOS) transistor, even though it is essential to use a reference electrode in the solution. A gate insulator is mostly composed of oxide or nitride membranes such as Ta₂O₅, Al₂O₃, SiO₂, and Si₃N₄; therefore, hydroxyl groups at the oxide or nitride surface in a solution reach the equilibrium state with hydrogen ions through protonation (−OH + H⁺−OH₂⁺) and deprotonation (−OH −O^- + H⁺) because the change in pH is detected from the change in the surface charge on the basis of the principle of the field effect^2,3 (Supplementary Figure 1). This is why the original ISFET sensor is still utilized as a pH sensor. Such ISFET sensors mostly have a silicon substrate, but various semiconducting materials have recently been applied to pH-sensitive ISFET sensors, which require the gate insulator or channel surface in contact with the electrolyte solution to be covered by functional groups such as hydroxy groups, carboxy groups, and amino groups^4,5,6,7,8,9.

Such a solution-gated ISFET, whose gate insulator or channel surface is modified with biologically or chemically active receptors, has been applied to portable biosensors, which can be built into some electronic devices^10,11,12. This biologically coupled gate (channel) FET, often called bio-FET, must realize quantitative and selective biosensing by detecting charges of biomolecules or ions included in body fluids. In addition, various low-dimensional materials have recently been utilized to enhance the sensitivity of bio-FETs, such as MoS₂, WSe₂^13,14, graphene^15,16, and Si nanowires^17,18. However, the complexity associated with the structure and fabrication processes has hindered the widespread usage of low-dimensional bio-FETs since they usually require disparate materials for the channel, source/drain electrodes, and gate-insulating film.

Our previous work revealed that a one-piece sheet of indium tin oxide (ITO) can work as a two-dimensional (2D)-like bio-FET on the basis of the fact that ITO, which is a conductive oxide, becomes semiconductive when its thickness is as small as ~20 nm^19,20,21,22. By etching the middle part of a conductive ITO single thin film with an acidic solution to make it ultrathin, which enables the fabrication of the one-piece ITO with a semiconductive channel, the source and drain electrodes and channel without any interfaces can be fully integrated^21,22. Then, the ITO channel surface is soaked in sample solutions with a reference electrode, thus functionalizing as a solution-gated ITO-ISFET. The solution-gated one-piece ITO-ISFET shows a steeper subthreshold slope (SS), which is mainly attributed to the direct contact of the ITO channel surface with a sample solution, compared to a conventional solution-gated silicon-based ISFET (Supplementary Figure 1B). This means that a relatively large electric double-layer capacitance at the ITO channel/solution interface acts as a dominant factor that determines SS^19,20,21,22. Moreover, the solution-gated one-piece ITO-ISFET shows pH responsivity in accordance with a chemical thermodynamic relationship (i.e., Nernst equation)^4,19,21. The ITO channel surface, which is in contact with an aqueous solution, conveniently has hydroxy groups that maintain an equilibrium state with hydrogen ions with positive charges, depending on pH, in the same manner as other oxide or nitride membranes^2,3. Furthermore, by functionalizing the solution-gated one-piece ITO-ISFET, it was possible to detect biomolecules such as viruses and DNA molecules^20,22.

This article describes a simple and rapid way to fabricate a solution-gated one-piece ITO-ISFET for biosensing. In particular, photolithography and etching processes for the fabrication are simply used, and then an electrolyte solution is added to the ITO channel surface with a reference electrode; that is, a sample solution serves as a solution gate electrode. Therefore, the solution-gated one-piece ITO-ISFET works as a pH sensor obtained by the fabrication process that takes only half a day.

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Fabrication of one-piece ITO-ISFET ( Figure 1A)

1. Patterning of OFPR-800
   1. Wash the glass substrate (12 mm × 24 mm) by sonication in acetone, methanol, and water for 5 min each. Then, dry the substrate with an N2 gas blower and subsequently bake it at 110 °C on a hot plate for longer than 5 min to dry the substrate completely.
   2. Spin-coat the glass substrate with an OFPR-800 layer at 500 rpm for 5 s and 3000 rpm for 30 s. Check for uniformity of coating.
   3. Prebake the substrate at 110 °C on a hot plate for 5 min. Then, cool the substrate to room temperature.
   4. Put the photomask film on the photoresist-coated side of the substrate and fix it with tape (Supplementary Figure 2A).
   5. Expose the substrate to UV (200 W) for 40 s in a photolithography machine.
      NOTE: Exposure time depends on the equipment used and is based on the required dose of photoresist.
   6. Take the substrate out of the photolithography machine and remove the photomask.
   7. Develop the photoresist in NMD-3 for 1 min and ensure that the photoresist forms a sharp pattern. Then, wash the substrate by dipping it in water.
   8. Dry the substrate by blowing N₂ gas. Then, post-bake the substrate on a hotplate at 110 °C for 5 min.
2. Deposition of ITO
   1. Fix the substrate on a substrate holder with tape and introduce it into the vacuum chamber of a sputtering machine.
   2. Pump down the sputtering chamber below 10^-3 Pa.
   3. Deposit ITO (In₂O₃ 90 wt% and SnO₂ 10 wt%) to a thickness of ~100 nm by radio frequency sputtering at 4 nm/min under Ar gas (0.2 Pa) without heating.
3. Patterning of SU-8 (Figure 1B)
   1. Lift off the photoresist by sonication in acetone, methanol, and water for 5 min each. Ensure that no small fragments of ITO remain on the substrates. If the contamination seems severe, wipe with a clean wiper and then clean the substrate by sonication again.
   2. Blow the substrate with an N₂ gas blower. Then, bake the substrate at 110 °C on a hot plate to completely dry the substrate.
   3. Spin-coat the glass substrate with an SU-8 3005 layer at 500 rpm for 5 s and 6000 rpm for 30 s. Check for uniformity of coating.
   4. Prebake the substrate at 95 °C on a hot plate for 5 min and subsequently cool the substrate to room temperature.
   5. Put the photomask film on the photoresist-coated substrate and fix it with tape (Supplementary Figure 2B).
   6. Expose the substrate to UV (200 W) for 7 s. Then, take the substrate out of the photolithography machine and remove the photomask.
   7. Postbake the substrate at 65 °C for 2 min and 95 °C for 5 min.
   8. Develop the photoresist in the SU-8 developer for 3 min under strong agitation. Then, wash the substrate in 2-propanol for 1 min. Ensure that the photoresist is fully developed.
   9. Dry the substrate by blowing N₂ gas.
4. Formation of semiconductive ITO channel by etching (Figure 1C)
   1. Prepare 0.1 M hydrochloric acid (HCl).
   2. Connect the source and drain electrodes, as shown in Figure 1B, to a semiconductor parameter analyzer.
   3. Select Classic Test tab and then select I/V-t Sampling.
   4. Set a sampling interval to 0.5 s.
   5. Apply a voltage of 1 V between the source and drain electrodes and determine the initial current (I_DS0).
   6. Place a drop of the prepared HCl solution (30 µL) onto the exposed ITO channel area. Ensure that the drop fully covers the channel area.
   7. Monitor the change in conductivity by monitoring the current between the source and drain electrodes. Continue the etching until the current decreases to 10% of initial I_DS0.
   8. Immediately rinse the ITO channel with deionized water to stop the etching as soon as the etching current ratio reaches 10% of initial I_DS0 to control the ITO channel thickness (~10-20 nm). This is regarded as a one-piece transistor with no interfaces among the source, channel, and drain electrodes.
   9. Gently blow N₂ gas to dry the one-piece transistor using an N₂ gas blower.
   10. Store the devices in a vacuum desiccator until measurement.

2. Electrical measurements of one-piece ITO-ISFET

1. Set up of the electrical measurement system
   1. Connect the source and drain electrodes of the one-piece transistor to the semiconductor parameter analyzer via a test fixture.
   2. Place a silicone O-ring around the ITO channel surface as a well into which a sample is placed.
   3. Add 30 µL of phosphate buffer (PB, pH 7.41) or other standard pH buffer solutions (pHs 4.01, 6.86, 7.41, and 9.18) carefully into the silicone O-ring to ensure direct contact between the solution and the ITO channel surface. This solution serves as the gate electrolyte.
   4. Insert an Ag/AgCl reference electrode in a saturated KCl solution, which is connected to the gate electrolyte by a salt bridge.
   5. Connect the Ag/AgCl reference electrode to the semiconductor parameter analyzer, which works as the gate electrode.
   6. Turn on the B1500A and start the EasyEXPART software, then open Workspace.
2. I_DS-V_GS transfer characteristics
   1. Select Classic Test tab and then select I/V Sweep.
   2. Connect the source electrode to the ground.
   3. Set the parameter to apply a constant drain voltage (V_DS) of 1 V.
      NOTE: The potential of each electrode should be -1 V - 1 V to avoid electrolysis of water.
   4. Set the sweep range of gate voltages (V_GS) to -0.8 V - +0.8 V and the sweep rate to approximately 50 mV/s.
      NOTE: The sweep rate can be controlled by setting the "number of steps" to 141 and the "delay" to 10 ms, and the "A/D converter" to HR ADC in the PLC mode (Factor: 2).
   5. Set the number of iterations to 10 cycles, and use the data obtained from the last cycle for analysis. Simultaneously, the leak current between the source and gate electrodes (I_GS) is obtained.
   6. Start measurement.
      NOTE: All measurement data is automatically saved in the "Results" tab.
3. I_DS-V_DS transfer characteristics
   1. Select CMOS category in the Application Test tab, then select Id-Vd mode
   2. Set V_GS to change sequentially from 0 V to 0.8 V at 0.1 V intervals.
   3. Set V_DS for each V_GS to sweep from 0 V to 1 V at 0.01 V intervals.
   4. Start measurement.
4. Follow-up steps
   1. Remove the O-ring and rinse the channel surface with deionized water. Then, dry the one-piece ITO device by blowing N₂ gas.
   2. Store the device in a vacuum desiccator.

Results

Figure 2A shows the change in current (I_DS0) measured during etching. I_DS0 remained almost constant around 800 s after the start of etching. With further etching, I_DS0 began to decrease rapidly. This indicates that the thickness of the ITO film decreased, which was approaching the thickness of the depletion layer in the ITO film¹⁹; thereby, electrons as carriers began to be affected in the channel by the surf...

Discussion

According to the above protocol, the one-piece ITO-ISFET can be fabricated as a solution-gated FET sensor (e.g., pH sensor) through a short and simple process (approximately half a day). ITO exhibits semiconducting properties at a depletion layer thickness of approximately 20 nm or less, making it suitable as the channel of a pH-sensitive solution-gated ISFET.

By etching the middle part of a conductive ITO single thin film with an acidic solution to make it ultrathin, a one-piece ITO-ISFET wit...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1M HCl	FUJIFILM Wako Pure Chemicals Corp.	083-01095
2-propanol	FUJIFILM Wako Pure Chemicals Corp.	166-04831
Acetone	FUJIFILM Wako Pure Chemicals Corp.	019-00353
Ag wire	The Nilaco Corp.	AG-401385
Agar	FUJIFILM Wako Pure Chemicals Corp.	010-15815
Buffer Solution Standard (Phosphate pH Standard Equimolal Solution) pH6.86 (25 degrees C)	FUJIFILM Wako Pure Chemicals Corp.	025-03195
Buffer Solution Standard (Phthalate pH Standard Solution) pH4.01 (25 degrees C)	FUJIFILM Wako Pure Chemicals Corp.	028-03185
Buffer Solution Standard (Tetraborate pH Standard Solution) pH9.18 (25 degrees C)	FUJIFILM Wako Pure Chemicals Corp.	028-03205
Carbon tape	Nisshin-EM	7321
Electrochemical Analyzer	BAS Instrument	618 E
Film mask	UnnoGiken Co., Ltd.		custom-made
Hot plate	As One Corp.	ND-1
Methanol	FUJIFILM Wako Pure Chemicals Corp.	137-01823
Micro Cover Glass	Matsunami Glass Ind., Ltd	12×24 No.5	for glass substrate
Micropipette	Eppendorf SE	3123000055
NMD-3	Tokyo Ohka Kogyo Co., Ltd.	NMD-3
OFPR-800	Tokyo Ohka Kogyo Co., Ltd.	OFPR-800
Phosphate pH standard solution	FUJIFILM Wako Pure Chemicals Corp.	166-17445
Photolithography Machine	Neutronix Quintel	PhL Q-2001CT
Pottasium Chrolide	FUJIFILM Wako Pure Chemicals Corp.	163-03545
Semiconductor Device Parameter Analyzer	Keysight	B1500A	softwere verion: rev 5 and 6
Semiconductor Device Parameter Analyzer Source measurement units	Keysight	B1517A	for source, drain and gate
Semiconductor Device Parameter Analyzer Test Fixture	Keysight	B1500A Opt A5F
Siliconce O-ring	MasuokaTokyo Co., Ltd.	P-5
Spin coater	MIKASA Co., Ltd	MS-A100
Sputtering machine	ULVAC Inc.		custom-made
SU-8 3005	Nippon Kayaku Co., Ltd.	SU8-3005
SU-8 developer	Nippon Kayaku Co., Ltd.	SU-8 Developer
Ultrasonic bath	SND Co., Ltd.	US-102
White-light Interferometer	KEYENCE Corp.	VK-X3000