Nanodiamond Macro- and Microelectrode Array BioSensor Supil Raina
W. P. Kang, J. L. Davidson
Interdisciplinary Materials Science Vanderbilt University Nashville, TN 37235 USA
Dept. of Electrical Engineering and Computer Science Vanderbilt University Nashville, TN 37235 USA
[email protected]
Abstract— A thin-film nanodiamond macro-electrode and a microelectrode array (MEA) with SiO2 film as the insulator, both on a highly doped Silicon substrate were fabricated for biosensing applications. Fe(CN)63-/4- redox couple is used for electrochemical characterization of the MEA using cyclic voltammetry, which gives a sigmoidal response consistent with hemispherical diffusion limited mass transport mechanism. Using the nanodiamond MEA, we were also able to detect different concentrations of Dopamine in Phosphate Buffered Saline (pH 7.4) without any surface functionalization. The cyclic voltammograms show a steady state response and a linear relationship between the limiting current and Dopamine concentration. In contrast, the nanodiamond macro-electrode shows a peak shaped response due to semi-infinite linear diffusion of the analytes. Overall, the nanodiamond MEA has a larger analyte flux and thereby larger current density by virtue of its small area as compared to the macro-electrode making it more sensitive for detection of dopamine and other bio-analytes.
I.
INTRODUCTION
Bio-analyte such as dopamine (DA) is an important catecholamine neurotransmitter in the mammalian central nervous system and has been linked to diseases such as schizophrenia, Parkinson’s and Alzheimer’s [1]. Electrochemical approach is one of the common techniques used to detect DA, but the interference from ascorbic acid (AA) and uric acid (UA) under physiologic conditions is a major problem for conventional electrodes. Techniques using surface modification and functionalization to improve the electrode selectivity have shown some success but with no promise for long-term and real-time detection. CVD diamond, such as boron-doped diamond and nanodiamond, has shown interesting electrochemical behavior and candidacy for use in bio-sensing due to properties such as being chemically inert, a wide working potential window, low background noise, resistance to fouling, bio-compatibility and mechanically stable [2,3].
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A microelectrode can be defined as an electrode with atleast one dimension smaller than 25µm, the critical dimension. The advantages of the small geometry include a small ohmic drop (iR), low double layer capacitance, enhanced rate of mass transport of electroactive analyte to the microelectrode surface and excellent spatial and temporal resolution [4]. This paper reports new findings for bio sensing utilizing nitrogen-incorporated nanodiamond macro-electrode and micropatterned electrode arrays. Specifically, the detection of potassium ferrocyanide and bioanalyte such as DA and selective detection DA in the interference of AA and UA are presented. II.
FABRICATION
The nitrogen incorporated nanodiamond macro-electrode was fabricated on a highly doped silicon substrate in a microwave plasma enhanced chemical vapor deposition system using a H2/CH4/N2 gas mixture. The nanodiamond MEA fabrication process steps involved thermal oxidation of a silicon substrate, sputtering of molybdenum thin film, conventional lithography to delineate the array and wet chemical etching to remove the molybdenum and SiO2 layer, leaving an array of exposed silicon elemental molds, followed by nanodiamond deposition to fill the array molds. The molybdenum layer acted as a sacrificial layer and was subsequently lifted off to expose an isolated array of nanodiamond elements separated by an insulating layer of SiO2. The MEA is comprised of an array of 20x20 (400 elements), each of radius 5µm and a pitch of 100µm. Figure 1 outlines this fabrication process.
III.
EXPERIMENTAL
Electrochemical characterization was performed using a single-chamber glass flat cell in a 3-electrode configurationAg/AgCl (3M KCl) reference electrode, Platinum counter-
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IEEE SENSORS 2009 Conference
All solutions were prepared with de-ionized water (18 MΩ). The Cyclic Voltammteric experiments were performed using a CHI 660C (CH Instruments) work station and the accompanying data acquisition software. IV.
RESULTS AND DISCUSSION
The low magnification SEM micrograph showing the microelectrode array layout can be seen in Fig. 2(a) and a high magnification image of an individual microelectrode is shown in Fig. 2(b). Electrochemical characterization was performed by first examining the Fe(CN)6-3/-4 redox couple in 0.1M KCl as the supporting electrolyte. Background scan at 100mV/s shows a wide working potential window of 3V which is sufficient to study a wide range of analytes. Cyclic voltammograms (CVs) obtained after adding potassium ferrocyanide are sigmoidal in nature, signifying steady state conditions due to hemispherical diffusion limited mass transport mechanism. Linear calibration curves were obtained for 1mM--10mM concentration range. The slope of the calibration plots gives us the sensitivity of the electrode, and in this case, the nanodiamond MEA showed 15 times higher sensitivity than the macro-electrode. (a)
(b)
Fig. 1. Schematic diagram for fabrication of a 20x20 nanodiamond microelectrode array (MEA).
electrode and the nanodiamond macro-electrode and MEA as the working electrodes. K4Fe(CN)6.3H2O and KCl were obtained from Fisher Scientific, Dopamine from Alfa Aesar, Uric Acid from Acros Organics, Ascorbic Acid from J. T. Baker and a premixed powder of PBS from EMD Chemicals.
Figure 2(a). SEM micrograph of the 20x20 array showing layout of most of the microelectrodes; (b) higher magnification image of an individual microelectrode.
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For biosensing application we first studied detection of dopamine (DA) in 0.1M Phosphate Buffered Saline (PBS) as the supporting electrolyte at physiologic pH 7.4. Different DA concentrations (200µM-1mM) also gave a sigmoidal response, at a scan rate of 100mV/s, consistent with hemispherical diffusion conditions at the electrode/solution interface, Fig. 3(a), and a linear calibration curve, Fig. 3(b). Again, the nanodiamond MEA shows more than 3 fold higher sensitivity for DA detection than the corresponding macro-electrode. Ascorbic Acid (AA) is also an electrochemically active bioanalyte which normally interferes with the detection of dopamine [5]. Using our MEA, we were able to detect DA in the presence of AA. Figure 4 shows the CVs obtained for different DA concentrations in 1mM AA, which appear as a cumulative response from both 1mM AA and DA. To obtain a calibration curve for DA, we can subtract the signal component due to AA and still get a linear relationship with DA concentration. Figure 4(b) shows 2 lines, one represents the total response for AA+ DA and the second line, passing through the origin, is representative of only DA, {(AA+DA) AA}. The macro-electrode shows distinct behavior for detection of dopamine with very well defined redox peaks, including dopamine, o-quinone, dopaminechrome, leucodopaminechrome distinctively detectable in the redox reactions (1) to (4), Fig. 5.
(a)
(b)
(a) Figure 4(a) Cyclic voltammograms at 100mV/s for different concentrations of Dopamine (100µM--1mM) and 1mM AA in 0.1M PBS, at steady state current. b) Calibration Curves for AA+DA shown in blue and that for DA (in red) obtained by subtracting AA signal from the total.
(b)
Figure 3(a) Cyclic voltammograms at 100mV/s for different concentrations of Dopamine in 0.1M PBS, showing steady state current due to hemispherical mass transport of the analyte at the electrode surface. (b) Linear calibration curve
Figure 5: CV of macro-electrode at 100mV/s for detection of 1mM DA in presence of 1mM Ascorbic Acid and 1mM Uric Acid demonstrating distinct detection of DA from the interference of AA and UA.
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REFERENCES The relationship between the peak-oxidation current for the reaction (1) and dopamine concentration was linear [2]. Moreover, the CV clearly demonstrates selective detection of 1mM DA in the presence of 1mM AA and 1mM UA, showing distinct and well resolved redox peaks of AA, DA, UA and its intermediate analyte species.
[1]
[2]
[3]
V.
CONCLUSIONS
In this work, nanodiamond macro- and micro-electrode array bio-sensor were successfully fabricated to detect DA independently, as well as in the presence of interfering agents, without any surface modification, hence allowing real-time detection with long-term stability and reliability. Also, there is an enhancement in sensitivity towards detection of potassium ferrocyanide and dopamine by using a MEA as compared to the macro-electrode, due to the high flux at the electrode surface.
[4] [5]
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S. Raina, W. P. Kang, J. L. Davidson, “Optimizing nitrogen incorporation in nanodiamond film for bio-analyte sensing,” Diamond and Related Materials, Vol. 18, pp. 718-721, 2009. S. Raina, W. P. Kang, J. L. Davidson, “Nitrogen incorporated nanodiamond film with ‘ridge’ surface morphology for detection of bio-analyte,” Diamond and Related Materials, Vol. 18, pp. 574- 577, 2009. M. Hupert, A. Muck, J. Wang, J. Stotter, Z. Cvackova, S. Haymond, Y. Show, G. M. Swain, “Conductive diamond thin-films in electrochemistry,” Diamond and Related Materials, Vol. 12, pp. 1940– 1949, 2003. J. Wang, Analytical Electrochemistry, 3rd ed., Wiley, New Jersey, 2006, pp 149-151. A. Salimi, H. MamKhezri, R. Hallaj, “Simultaneous determination of ascorbic acid, uric acid and neurotransmitters with a carbon ceramic electrode prepared by sol–gel technique,” Talanta, Vol. 70, pp. 823– 832, 2006.
