the electrodes and potentiostat. Once optimized, EIS based biosensors may be able to provide rapid, cheap, dependable and sensitive detection of many ...
Carbon Nanotube Array Immunosensor Development Adam Bange, H. Brian Halsall, William R. Heineman Department of Chemistry, University of Cincinnati
YeoHeung Yun, Mark J. Schulz Department of Mechanical Engineering University of Cincinnati
Vesselin Shanov, Department of Chemical and Materials Engineering, University of Cincinnati Abstract—This paper describes the development of a label-free immunosensor using carbon nanotube array electrodes. Highly aligned multi-walled carbon nanotubes were grown by chemical vapor deposition using a metallic catalyst, Fe/Al2O3/SiO2, on Si wafers. Harvested towers were cast in epoxy and polished on both ends; one end being for electrical connection and the other being the electrode array surface. The nanotubes were functionalized electrochemically to form carboxyl groups and then coupled to antibodies. EIS was used to directly monitor the antibody-antigen binding. Keywords- nanotube; impedance; immunosensor
I.
INTRODUCTION
Typical immunoassays are multi-step procedures that involve a capture step, multiple rinses, adding a label for detection and/or amplification, and finally detection.[1] These methods can be very sensitive, and are perfectly suitable for laboratory analysis, but are limited for some applications that require continuous monitoring, rapid response, and portability. Electrochemical impedance spectroscopy (EIS), coupled with the selectivity and sensitivity of biological recognition molecules such as antibodies promises to be an effective labelfree sensing technique for biomolecules. [2-5] EIS has advantages over many other label-free sensing techniques in that it can be used for continuous monitoring, it does not destroy the sample, and the only hardware needed consists of the electrodes and potentiostat. Once optimized, EIS based biosensors may be able to provide rapid, cheap, dependable and sensitive detection of many biological molecules in sample volumes from the bulk scale of lakes and rivers to submicroliter clinical samples. The physical properties of carbon nanotubes make them an excellent electode material for an EIS biosensor. [6-8] The aligned multi-walled nanotubes used in this study are highly conductive, and when functionalized the carbon surface can be attached directly to antibodies or other sensing biomolecules via covalent chemical bonds. Also, the remarkable mechanical strength of the nanotubes will enable future biosensors to be needle-thin, and the ability to control the geometry of the nanotubes at the molecular level becomes a powerful tool for creating nanoscale structure that optimizes the electrode’s analytical performance. A schematic of the sensor can be seen in figure 1.
Figure 1. Schematic of the nanotube immunosensor (not to scale)
The surface of the electrode can be seen as a forest of exposed conducting nanotubes with antibodies on the ends, immersed in a block of insulating material. The design goal was to increase the reduction in current observed per molecule bound to the sensor by limiting the electrode surface to an array of small points that can be easily obstructed by target molecules. II.
EXPERIMENTAL
A. Synthesis of nanotubes P-type 4 in. diameter Si wafers with a typical resistivity of 1-20 Ohm-cm were used. An E-beam evaporator was used to deposit an Al thin film with 10 nm thickness. Then the Al is oxidized to form a layer of Al2O3. Finally, catalytic iron films of controlled thickness between 1 and 2 nm were deposited on Si, SiO2, and Al2O3 surfaces. The evaporation was done at approximately 5 X 10-7 torr and the system was equipped with a film thickness monitor. The final substrate was cut to 5 mm x 5 mm. The nanotubes were synthesized by thermal CVD in a horizontal 2 in tube furnace, (EasyTubeTM ET1000, FirstNano), which consists of 4 mass flow controllers and a vapor delivery system. Argon was used to carry the water vapor to the reaction chamber, and to purge the reactor for 20 minutes while the CVD furnace was heated to 750°C. The gas flow was then switched to ethylene, water, and hydrogen for specific lengths of time based on process parameters. During this time the hydrocarbon precursor, ethylene, reacts with the catalyst and deposits CNT on the substrate. After the nanotube array was synthesized it was cooled to ambient temperature, completing the last process step. During cooling, ethylene, water, and hydrogen flow were stopped and the system was purged with argon to prevent back flow of air from the exhaust line. The synthesized CNT arrays were characterized by environmental scanning electron microscopy (ESEM).
5 mg sulfo-N-hydroxysuccinimide (sNHS) to stabilize the acylisourea intermediate. The activation was quenched in a 1% ethanolamine solution to stop further carbodiimide reactivity. The electrode was then incubated in an antibody solution containing 20µl of donkey anti-mouse IgG in 1 mL PBS, pH 7.0 for four hours. The resulting product of antibody covalently attached to the carbon nanotubes was rinsed with deionized water and stored at 4º C and pH 7 until used for testing. D.
Electrochemical analysis The electrochemical properties of the biosensor were evaluated by CV with a Bioanalytical Systems (BAS), Epsilon system with a C3 cell stand with a Faraday cage. A platinum wire and Ag/AgCl reference electrode were used as the auxiliary and reference electrodes, respectively. K3Fe(CN)6 (Fisher, 99%), and KNO3 (Fisher, 99%) were all prepared fresh daily. The EIS was done with three-electrode cells, with the nanotube electrode as the working electrode, an Ag/AgCl reference electrode, and a platinum wire as the counter electrode. The antigen was mouse IgG. EIS measurements were taken with a Gamry Potentiostat (Model: PCI4/750) coupled with the EIS (Gamry, EIS300) software. All testing was done at 0 V DC and 0.1 Hz to 300 KHz, and the sinusoidal potential magnitude was ±20 mV in the redox probe 5 mM K4[Fe(CN)6], K3[Fe(CN)6] with PBS (pH 7.0).
III.
Figure 2. Steps of biosensor fabrication
B. Biosensor Fabrication The aligned nanotubes were peeled off the silicon substrate and cast in epoxy using Resin 862 and the EPICURE curing agent W. The epoxy insulates the nanotubes thermally and electrically, and provides mechanical support. The bottom side of the array was then polished to expose the aligned nanotubes for connection. Conductive epoxy was used to attach copper wire to the bottom of the array, and insulating epoxy was used to seal the connection. The top side of the array was then polished to expose the nanotubes. A summary of this procedure can be seen in figure 2. C.
Immobilization of Antibody Antibodies were attached covalently to the nanotubes by first oxidizing the electrode surface to present carboxyl surface groups. The nanotubes were opened using H2SO4/70% HNO3, and HCl, followed by electrochemical treatment at 1.5 V (versus Ag/AgCl) in 1.0 M NaOH for 30 seconds. The carboxyl groups were activated with a 20 minute incubation in a 500 µL solution containing 10 mg of 1-ethyl-3(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) and
RESULTS/DISCUSSION
A. Nanotube Synthesis Figures 3(a)-(c) show the ESEM results for the waterassisted nanotube synthesis with fixed growth conditions of: 200 SCCM of H2 flow, 200 SCCM of C2H4 flow, and at 750° C with 3 hours growth time. Fe/Al2O3/SiO2/Si (5 mm × 5 mm) was cut from one wafer and Fe was patterned into 100 µm × 100 µm squares with 100 µm space in between. With the increase of growth time, the length of CNT arrays keeps increasing for up to 3 hours. This CNT array has high density which is suitable for further sensor development. Adhesion to the substrate is weak and the CNT array is easily peeled off. Figure 3(c) is the ESEM image of aligned multi-wall carbon nanotube patterned arrays showing the effect of alignment with high density. Figure 3(d) shows the final fabricated device. The nanotube average diameter was 20 nanometers, with an aspect ratio of 200,000:1. The resistivity of the nanotube tower was measured by casting a nanotube tower with epoxy and polishing both ends of nano-composite, and then connecting a copper wire to the electrode using conductive epoxy. The volume resistivity is approximately 0.11 ohm ⋅ cm.
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Figure 4. Cyclic voltammetry of 6 mM K3Fe(CN)6 in 1.0 M KNO3 with nanotube tower electrode with 100 mV/s scan rates: after (a) functionalized nanotube array, (b) immobilization of antibody and (c) antigen binding
Figure 3. ESEM images of (a) aligned multi-wall carbon nanotube patterned arrays on Si substrate, (b) side view of nanotube array, (c) high resolution view of nanotube array, and (d) final nanotube tower-epoxy electrode. Growth conditions: 200 SCCM of H2 flow, 200 SCCM of C2H4 flow, 100 SCCM Bubbler flow, 750º C growth temperature.
B. Electrochemical Analysis The results of a scan rate study using the K3Fe(CN)6 redox probe are shown in figure 4. The measured currents were low, but clearly high enough to indicate that a large number of conducting nanotubes were exposed. Immobilizing the antibody on the nanotube surface caused loss of current due to the diffusional barrier of a large insulating molecule being attached to the electrode surface. Exposure to the target antigen resulted in a further reduction in current. The Nyquist plot of EIS results in figure 5 show that the sensor response depends on analyte concentration. The presence of the target antigen (mouse IgG) bound to the immobilized antibody progressively increased the electron transfer resistance, as indicated by the radius of the curve. In figure 6, the response of the sensor was monitored as a function of time. The electron transfer resistance was shown to increase as more antigen became bound to the electrode, until a saturation point was reached.
Figure 5. Electrochemical impedance spectra of immunosensor response with the addition of different concentrations of antigen (inner to outer curve); (a) without antigen, with (b) 500 ng/ml, (c) 1µg/ml, (d) 5 µg/ml, (e) 10 µg/ml, and (f) 100 µg/ml of antigen.
This study has shown the feasibility of an EIS-based biosensor using carbon nanotubes as an electrode material. Further experiments will evaluate non-specific binding and its effect on sensor performance, different sizes, geometries, and fabrication techniques, alternative molecular recognition strategies, and further investigation of the EIS technique. Figure 6. Electrochemical impedance spectra for nanotube immunosensor with (a) donkey anti-mouse IgG immobilization, and mouse IgG binding after the incubation for half (b), one (c), two (d) and four (e) hours incubation.(inner to outer curve) EIS is done at DC potentials 0 V with frequencies between 0.1Hz and 300KHz. Simusoidal potential magnitude is ±20 mV in 5 mM K4[Fe(CN)6], K3[Fe(CN)6] with PBS (pH 7.0).
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