J. Phys. Chem. C 2007, 111, 8667-8670
8667
Quantitative Detection of Protein Using a Top-gate Carbon Nanotube Field Effect Transistor Masuhiro Abe,†,‡ Katsuyuki Murata,*,†,‡,§ Atsuhiko Kojima,§ Yasuo Ifuku,§,| Mitsuaki Shimizu,⊥ Tatsuaki Ataka,†,‡ and Kazuhiko Matsumoto‡,§,⊥,# Future Creation Laboratory, Olympus Corporation, Shinjuku Monolith, 3-1 Nishi-Shinjuku 2-chome, Shinjuku, Tokyo 163-0914, Japan, NEDO c/o Olympus Corporation, Shinjuku Monolith, 3-1 Nishi-Shinjuku 2-chome, Shinjuku, Tokyo 163-0914, Japan, CREST, Japan Science and Technology Agency c/o National Institute of AdVanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1 Umazono, Tsukuba, Ibaraki 305-8568, Japan, Mitsubishi Kagaku Iatron, 1144 Ohwadashinden, Yachiyo, Chiba 276-0046, Japan, National Institute of AdVanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1 Umazono, Tsukuba, Ibaraki 305-8568, Japan, and The Institute of Scientific and Industrial Research, Osaka UniVersity, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan ReceiVed: February 19, 2007; In Final Form: April 19, 2007
High-stability sensing of proteins of pig serum albumin (PSA) was achieved using an insulator-covered carbonnanotube field-effect transistor (CNT-FET) with a top-gate structure. The sensitivity limit of this CNT-FET PSA sensor is at least 5 nmol/L PSA solution. A layer of silicon nitride deposited on the top-gate structure provides an n-type characteristic and a highly stable CNT-FET. Since the CNT is covered with a silicon nitride layer, it is isolated from oxygen, water, and other contaminants. The equilibrium constant of the PSA/ anti-PSA reaction, which indicates the strength of the binding energy between PSA and anti-PSA fixed on the Au top-gate electrode, was calculated to be 1.2 × 107 L/mol using the Langmuir equation fitting.
1. Introduction Nanostructured carbon materials, such as carbon nanotubes (CNTs),1,2 carbon nanohorns,3,4 carbon nanowires, and graphite nanofibers, are attractive for various applications, such as field emission,5 hydrogen generation,6 hydrogen and methane storage,7-9 and capacitors.10 The CNT field-effect transistor (FET)11,12 would be one of the most practical. An important application of CNT-FETs is in the development of biosensors, which sense living biological molecules. For home medical care, compact and simple biosensors that can perform real-time measurements should be developed. Several biosensors using chromatography,13-16 chemical luminescence,17-20 and surface plasmon resonance (SPR)21,22 have been developed. These methods, however, are not suitable for home medical care: Chromatography technology cannot detect biomaterials in real time, and chemical luminescence and SPR equipment is too heavy and too large for home use. CNT-FETs have the advantage of high transconductance owing to their high carrier mobility and high carrier velocity compared with silicon metal-oxide-semiconductor FETs. This advantage means CNT-FETs have the high charge sensitivity needed for sensing living biological molecules. Moreover, a CNT-FET is compact and can be integrated into a biosensor. Various types of protein sensors using CNT-FETs have been studied, which detect proteins on the basis of the antigen/antibody reaction. However, * Corresponding author. E-mail:
[email protected]. † Olympus Corp. ‡ NEDO. § CREST-JST. | Mitsubishi Kagaku Iatron. ⊥ AIST. # Osaka University.
two major drawbacks of CNT-FET biosensors have been their instability and the lack of a quantitative study. The instability is caused by contaminants on the CNTs. In previous studies, antigens were immobilized directly on a CNT channel, and the antigens were sensed by measuring the change in electron mobility.23,24 In this type of CNT-FET biosensor, impurities such as oxygen, water, and the photoresist adsorbed on the CNTs degrade the stability of the CNT-FET. Because of this instability, a quantitative evaluation of CNT-FET biosensors has not been possible. In our previous work, we investigated and confirmed the stability of a top-gate CNT-FET with the CNT channel covered with a silicon nitride layer to avoid contamination.25,26 In the present paper, we report successful detection of the antigen/antibody reaction using this top-gate CNT-FET, which allowed us to perform the first quantitative electrical evaluation of the antigen/antibody reaction. 2. Experimental Section The starting substrate was n-type silicon with thermally grown SiO2. CNTs were synthesized by thermal chemical-vapor deposition (CVD) using cobalt as a catalyst (5 nm). The catalyst was patterned on the substrate by photolithography. The CNT was grown under a flow of mixture gases of ethanol carried by argon (500 mL/min) and hydrogen (750 mL/min) for 10 min at 900 °C and 760 Torr. Raman measurements suggested that single-wall carbon nanotubes grew between the drain and source electrodes (See Supporting Information Figure 1). After the CNT growth, gold and titanium (40- and 10-nm-thick layers) were deposited on the CNT for the source-drain electrodes, and platinum and titanium (100- and 10-nm-thick layers) for the back-gate electrode. The spacing between the source-drain electrodes was 6 µm. After the impurities adsorbed on the CNT
10.1021/jp071420e CCC: $37.00 © 2007 American Chemical Society Published on Web 05/26/2007
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Figure 1. Structure of the CNT-FET. (a) Schematic and SEM images of PSA sensor. The a-PSAs were fixed on the CNT-FET. (b) Optical micrograph. Waterproof resist was coated on the silicon nitride and on the top-gate excluding the square area where a-PSA will be immobilized.
were removed by rinsing in dimethyl acetamide, a silicon nitride layer of 100 nm was deposited all over the prepared CNT-FET by thermal CVD. Before silicon nitride deposition, adsorbed molecules, such as oxygen and water, were removed by heating under a nitrogen gas flow (4 l/min.) up to 800 °C. The thermal CVD conditions were NH4 (1 L/min), 0.3% SiH4 (Ar balance, 50 mL/min), and nitrogen (2 L/min) for 14 min at 800 °C and 760 Torr. Gold and titanium (100/10 nm) metals were deposited on the silicon nitride layer for a top-gate electrode. The width of the top-gate electrode above the CNT channel was 2 µm. When a test solution was dropped on the CNT-FET directly, water soaked into the silicon nitride, which caused leakage between the top-gate electrode and the drain-source electrodes. To protect the CNT-FET from this leakage, the silicon nitride layer was covered with a photoresist layer (S1818, Shipley Far East Ltd.) as a waterproofing layer. The entire top-gate electrode
Abe et al. except a 100 µm × 100 µm square area for antibody immobilization was also covered with waterproof resist. The waterproof resist also prevents the adsorption of chemical species, such as proteins. By this covering of the top-gate, we can designate where proteins are immobilized. The CNT-FET device prepared in the above manner is the base of the present biosensor (Figure 1). Pig serum albumin (PSA) as antigens and anti-PSA (a-PSA) as antibodies comprised the target sample for the electrical detection by the biosensor. The a-PSA was immobilized on the top gate of the CNT-FET as follows: Phosphate buffer solution (pH ) 7.4) containing a-PSA was dropped onto the top gate.The top gate was then exposed to the wet atmosphere for 30 min. Next, the solvent was evaporated for 60 min in a drybox in a nitrogen atmosphere. After the solvent had evaporated, the surface of the top gate was immersed in casein solution to prevent a-PSA diffusing around the top gate or the adsorption of chemical species on the top gate. Finally, the CNT-FET was rinsed with water, and the a-PSA was strongly immobilized on the top gate. Hereafter, the CNT-FET with immobilized a-PSA on the top-gate is referred to as the PSA sensor. For measurements with the PSA sensor, the environmental conditions are important. For precise measurements, static electricity must be removed because it causes a margin of error in the results. Temperature and humidity in the measurement field are also important. For instance, in a low-humidity environment, a test solution will easily evaporate and its concentration will change during the measurement. Temperature influences the reaction velocity and equilibrium point of chemical reactions. Therefore, it is necessary to control the temperature and humidity of the measurement field. For these purposes, a manual prober (705B-6, Micronics Japan) and a precision semiconductor parameter analyzer (4156A, HewlettPackard) were setup in an electrically sealed room, and the temperature and humidity of the room were kept at 25 °C and more than 60%, respectively. The PSA sensor was setup as follows. A silicone-rubber wall was set around the top gate. A PSA solution was poured onto the top gate. A gate voltage was applied to the top gate from a Ag/AgCl reference electrode. Drain current was measured as a function of gate voltage at several concentrations of PSA solution. The PSA/a-PSA reaction was detected as a decrease of drain current in FET characteristics from the original drain current value without the PSA/a-PSA reaction. For the precise quantization, it is necessary for a PSA sensor to define a decrease of drain current in its “equilibrium” PSA concentration. When a PSA solution is introduced and the PSA/ a-PSA reaction occurs, PSA in a bulk solution moves to a-PSA on the top gate and the bulk PSA concentration decreases after the reaction. Therefore, the introduced PSA concentration becomes different from the equilibrium PSA concentration. We addressed this problem by exchanging the test solution on the top gate several times (see Supporting Information Figure 2). 3. Results and Discussion Before performing the sensing measurements, we examined the electrical properties of the CNT-FET. The top-gate CNTFET showed an n-type property, and the top-gate electrode worked less effectively than the back-gate electrode because of the high source/drain Schottky barrier.27 To improve the topgate characteristics, the back-gate voltage of -5, 0, or +5 V was applied at the drain bias of 0.1 V. The drain current increased with increasing back-gate voltage and so did the
Protein Detection
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Figure 2. Dependence of drain current of the CNT-FET on top-gate bias. The back-gate biases are 5 V (solid curve), 0 V (broken curve), and -5 V (dotted curve). The drain voltage was set at +0.1 V. Topgate voltage was swept from -1 to +1 V.
transconductance gm, as shown in Figure 2. At top-gate voltage VTG of 0.5 V, gm increased. At the back-gate bias of -5, 0, and +5 V, gm values were 0 S, 2.2 nS, and 2.5 nS, respectively. The stability of the CNT-FET was examined by measuring the time dependence of the drain current. The fluctuation of the drain current was less than 0.3% at the applied drain bias of 0.1 V, top-gate bias of VTG ) +1 V, and back-gate voltage of VBG ) +5 V. The back-gate voltage of +5 V provides a high drain current without causing any breakdown of the CNTs. Therefore, the back-gate voltage was kept at VBG ) +5 V in all measurements hereafter. After the electrical properties of the CNT-FET had been confirmed, the PSA sensor was prepared by immobilizing a-PSA on the top-gate of the CNT-FET. Because a-PSA has a neutral electron charge, the top-gate electrode with the immobilized a-PSA did not influence the CNT-FET properties. Here, we mention the mechanism of a-PSA immobilization on the topgate electrode. Although a-PSA is immobilized by physical adsorption, it would adsorb tightly owing to the strong interaction between thiol (including proteins) and Au. The chemical luminescence measurement showed that a-PSA did not desorb from the top-gate electrode in buffer solution. A test solution was poured on the top gate of the PSA sensor, and the sensor’s electrical properties were measured. The drainsource voltage was maintained at +1 V, and the top-gate voltage was swept from -1 to +1 V. The back-gate voltage was maintained at +5 V. The broken line in Figure 3a shows the drain current ID-top-gate voltage VTG characterizations when the buffer solution without PSA was poured. The characteristics are similar to those of the CNT-FET in air. The solid line in Figure 3a shows the modulation of the drain current when the buffer solution containing 200 nmol/L PSA was poured. Since the isoelectric point of PSA is 4.8,21 PSA has negative charges in a solution with pH of 8.0, which was used in the present study. The negative charges on the top-gate electrode influenced the FET properties and decreased the drain current. When the PSA solution with the concentration of 200 nmol/L was introduced, the drain current decreased ∆ID ) 20 nA at VTG ) +1 V. The charge of PSA, Qe, causes the threshould voltage shift ∆Vth, which is linearly proportional to Qe as
Qe ) C ∆Vth ) C ∆ID/gm
(1)
where C is the gate capacitance of the CNT-FET biosensor. Therefore, the amount of the adsorbed PSA is proportional to
Figure 3. Characteristic curve of the PSA sensor measured in Tris buffer solution containing PSA. Drain voltage was set at +1 V and top-gate voltage at -1 to +1 V. (a) Drain current (∆ID) of the PSA sensor. Solid and broken curves are 0 and 200 nmol/L of PSA introduced, respectively. (b) Relationship between ∆ID and PSA equilibrium concentration.
∆Vth. In the present CNT-FET biosensor, the drain current ID is linearly proportional to the top-gate bias VTG as shown in Figure 3a. Hence, we regard gm as a constant in our measuring range according to eq 1. Therefore, Qe is linearly proportional to ∆ID. Hereafter, the properties of the PSA sensor are characterized by the drain current decrease ∆ID. For the PSA sensor, the dependence of ∆ID on the concentration of the PSA solution is shown by solid circles in Figure 3b. Figure 3b is a kind of adsorption isotherm because ∆ID is related to the amount of PSA adsorbed. This adsorption isotherm is type I in the IUPAC classification. The mechanism would be site-selective and monolayer adsorption. The ∆ID is linearly proportional to the logarithmic scale of PSA concentration below 200 nmol/L. As PSA concentration increased, the increase of ∆ID became gentle and almost saturated. The equilibrium constant Keq of the PSA/a-PSA reaction can be calculated from the Langmuir equation. The physical meaning of Keq is important because its value indicates the strength of the binding energy between PSA and a-PSA molecules. The Langmuir adsorption isotherm was applied to the experimental results in Figure 3b. It is given by
W/Wsat ) CPSA Keq/(1 + CPSA Keq)
(2)
where W is the total weight of adsorbed PSA, Wsat is the total weight of saturated adsorbed PSA, and CPSA is the PSA concentration. The amount of electric charge of the top-gate electrode is in proportion to the amount of adsorbed PSAs on
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the top gate. Equation 1 shows ∆ID is linear to the amount of electric charge of top-gate electrode. Thus, we can assume
W/Wsat ) ∆I/∆IDsat
(3)
From eqs 2 and 3, the Langmuir adsorption isotherm of the linear form is described as
CPSA/∆I ) CPSA/∆IDsat + 1/∆IDsat Keq
(4)
Using eq 4, the Langmuir adsorption isotherm well fitted the experimental results as shown in the inset of Figure 3b. From this fitting, Keq of 1.2 × 107 L/mol is obtained, indicating that the binding energy is within the range of values in previous studies of the antigen-antibody reaction. According to infrared measurements28-30 and surface plasmon resonance studies31 for the serum albumin group, such as human and bovine, the equilibrium constant ranges from 2.5 × 106 to 5.2 × 107 L/mol. Therefore, the present experimental result for Keq can be considered to be the proper value. This is the first experimental result that has succeeded in obtaining the binding energy of the PSA/a-PSA reaction by electrical measurement using a CNT-FET. The experimental ID dependence on PSA concentration was well fitted using eqs 2 and 3 and Keq and is shown by the solid line in Figure 3b. 4. Conclusion A CNT-FET biosensor with a top-gate structure was developed and has successfully detected PSA/a-PSA reaction in a solution. The CNT-FET biosensor achieved high stability and sensitivity with a sensitivity limit of 5 nmol/L. The PSA/a-PSA reaction was detected as a decrease of the drain current of the CNT-FET. The relation between the decrease of the drain current and the PSA concentration was quantitatively analyzed using the Langmuir adsorption isotherm, and the binding energy of the PSA/a-PSA reaction was obtained for the first time from the electrical measurement. These are the first experimental results that clarify the quantitative properties of a CNT-FET biosensor. These results indicate that the highly qualitative and quantitative measurement of protein detection is possible using top-gate CNT-FET biosensors. Supporting Information Available: Characterization of the CNT channel (SEM image and Raman spectrum), experimental details of the determination of equilibrium concentration, and
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