A Comparison of Fabrication Methods for Iridium Oxide Reference Electrodes Robert K. Franklin
Segyeong Joo
Dept. of Electrical Engineering and Computer Science The University of Michigan Ann Arbor, MI, USA
[email protected]
Medical Research Center Seoul National University Seoul, Korea
Sandeep Negi, Florian Solzbacher, and Richard B. Brown Dept. of Electrical and Computer Engineering The University of Utah Salt Lake City, UT, USA Abstract—Several methods for the manufacturing of Iridium Oxide (IrOx) electrodes have been discussed in the literature. Two commonly used fabrication methods are Sputtered Iridium Oxide Films (SIROF) and Activated Iridium Oxide Films (AIROF). Most of the studies for in vivo electrodes have reported optimizations to these methods in the context of stimulation of and recording from neural tissue. In this work we characterize three fabrication methods of IrOx films for use as reference electrodes during in vivo neurochemical recordings, and we conclude that AIROF electrodes are preferable as reference electrodes due to their superior open circuit potential (OCP) stability. I.
INTRODUCTION
In order to probe the delicate chemical interactions involved in intracellular communication[1], various methods have been explored including adaptations of traditional methods such as microdialysis[2] as well as newly developed methods such as hybrid hydrogel-optical detection systems[3,4]. Neurochemical sensors based on electrochemical detection methods such as cyclic voltammetry (CV), chronoamperometry, and potentiometry have been reported in the literature [5-8]. These electrochemical sensors require at least two electrodes, a sense electrode (referred to as either a working electrode or an indicator electrode depending on the detection method used) and a reference electrode. Electrode materials for the sense electrode have been widely studied; the electrode material should be tailored to favor a specific reaction at the surface of the electrode. Reference electrodes on the other hand are selected for stability and insensitivity to changes in the surrounding media [9]. A good reference electrode is characterized by a well defined Nernst reaction at the surface of the electrode that sets the potential difference between the electrode and the surrounding media to a predictable value. The surface reaction should also be a fast
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reaction that allows current to pass through the electrode without affecting its potential. Ag/AgCl is the most popular reference electrode used in electrochemical detection. Its Nernst reaction depends on the chloride concentration in the surrounding media which in biological media is normally well controlled. As a result, many of the neurochemical sensors reported to date have used Ag/AgCl as the reference electrode. Unfortunately, Ag/AgCl, is unsuitable for chronic implantation due to its toxicity. This has led to the reference electrode being placed in a remote location to minimize its effect of the tissue of interest, or to its being replaced by a pseudo-reference electrode such as stainless steel which lacks the long-term potential stability of a real reference electrode. We previously suggested IrOx as a suitable replacement for Ag/AgCl in the brain and presented results of an initial study that showed IrOx to be suitable for this application [10]. Here, we expand upon that work by comparing multiple fabrication methods and examining the electrochemical stability of IrOx films. II.
IRIDIUM OXIDE REFERENCE ELECTRODES
IrOx is characterized by the following reaction, Ir2O3 + H2O ⇔ 2IrO2 + 2H+ + e− which indicates a strong dependence on the H+ concentration of the surrounding media. IrOx electrodes are physically robust and have high exchange currents, but this pH dependence has made them unsuitable as reference electrodes outside of buffered solutions. In biological applications, however, the pH is normally constrained to a narrow range of pH, particularly in the brain where normal pH readings are within 7.2 and 7.4[11,12]. This tightly controlled pH
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IV.
A. pH Characterization After fabrication, all of the electrodes were initially characterized for pH response in phosphate buffer by varying pH from 6 to 7.5 (Fig. 2). The AIROF films A1 and A2 were found to have a near Nernstian response of approximately 55 mV/dec and were tightly grouped with an average σ of less than 20 mV. In contrast, the SIROF film, S1, exhibited a smaller pH response of only -38 mV/dec, and exhibited much greater variability with an average σ of more than 80 mV. The S1 electrodes had an especially large variation at the low end of the pH scale (pH 6.5).
environment coupled with IrOx’s excellent biocompatibility and wide use within the neuroscience community as a recording and stimulation electrode makes IrOx an ideal reference electrode electrode for studies in the brain. Iridium oxide films may be prepared in a variety of ways. Two common methods are sputtered IrOx films (SIROF) formed by sputtering Iridium in an oxygen rich plasma and activated IrOx films (AIROF) formed by anodically growing IrOx on Ir metal [13,14]. These methods were selected due to their use on two of the most common implantable electrode platforms—Michigan style neural probes and the Utah Electrode Array. METHODOLOGY
A.
Electrode Fabrication 500 µm diameter IrOx electrodes were fabricated on silicon wafers by depositing and patterning a platinum interconnect layer. The interconnect layer was next encapsulated with a 250 nm layer of PECVD silicon nitride followed by a 100 nm layer of parylene. Contact openings were etched in the encapsulation with RIE, and a final layer of either Iridium or IrOx was deposited and patterned. The final electrodes were examined in a SEM to verify the presence of IrOx on the fabricated electrodes (See Fig. 1).
B. Soak Testing After pH testing, the electrodes were subjected to soak testing in pH 7.4 PBS for a period of nine days. At regular intervals during the test, the electrochemical impedances of the electrodes were measured at 1 kHz (Fig. 3). The open circuit potentials (OCP) of the electrodes vs. SCE in pH 7.4 PBS (Fig. 4) were also measured for each electrode. During the soak testing, the SIROF electrodes were found to have the least variance in impedance (< 0.3% change in impedance per day) while the AIROF electrodes, A1 and A2, exhibited greater rates of change, 0.8% and 1.6% per day respectively. However, the OCP of the AIROF electrodes was more stable with A2 having the lowest rate of drift (3.2 mV/day), A1 having 10.9 mV/day of drift, and S1 having a drift of 29 mV/day. C. Dopamine detection Based on the results of the soak test, a set of 1.5×103 µm2 Ir electrodes were fabricated on probes with 30 µm diameter Platinum (Pt) working electrodes, and a 1.5×103 µm2 Pt counter electrode. The Ir electrodes were activated in Na2HPO4 using the A2 protocol and then used to perform in vitro measurements of dopamine (DA). For this experiment, powdered dopamine hydrochloride was dissolved in phosphate buffered saline (PBS) solution at low pH (3.85) just before the experiment and stored in an opaque bottle to protect the DA from light induced chemical reactions. Fig. 5 shows CV scans for 10 mM DA solution in PBS versus both
Iridium Oxide formation The IrOx was prepared using two different methods: pulsed DC sputter deposition of IrOx in an oxygen plasma (S1), DC sputter deposition of Ir followed by activation in PBS (A1), and DC sputter deposition of Ir followed by activation in Na2HPO4 (A2) [11]. For A1 and A2, the electrodes were activated by applying 1 Hz Square wave between +0.9 to -0.85 V for 200 cycles. The activation was performed using a Gamry FAS2 femtostat with respect to a Saturated Calomel Electrode (SCE). After activation the electrodes were soaked in PBS for 24 hours before further characterization.
Open Circuit pH Response of IrOx electrodes
Open Circuit Potential (mV)
Figure 1. Pictures of A1 (A), A2 (B), and S1 (C) films showing surface morphology. Picture of Michigan style neural probe (D) with on-chip AIROF electrode.
III.
RESULTS
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Figure 2. pH response of AIROF and SIROF films (n=10). An ideal Nernstian response would be 59 mV/dec.
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SCE and IrOx reference electrodes. The scans are nearly identical except for a potential difference of 9 mV between two peak potentials representing the potential difference of IrOx electrode (+232 mV vs. standard hydrogen electrode (SHE)) at pH 3.85 and SCE (+241 mV vs. SHE). Fig. 5 also shows CV scans at various scan rates from 50 mV/s to 10000 mV/s. Fig. 6 shows CV scans of DA in concentrations from 10 mM down to 0 mM at pH 3.85 with a scan rate of 1000 mV/s. The second part of Fig. 6 is the calibration curve obtained by plotting the current values at 600 mV vs. IrOx (n=3). The results show a linear increase of 5.85 nA/mM in 1.6
PBS Activated Iridium Oxide
Slope = 8.3x10-3/day
1.4 R2 = 0.050 1.2 n=6 1 Normalized Impedance
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We have also successfully measured DA electrochemically using an integrated AIROF IrOx reference electrode on a microelectrode array. The IrOx electrode showed excellent characteristics as a reference electrode under the several conditions tested. Based on these findings, we recommend that AIROF IrOx electrodes be used for in vivo electrochemical detection of neurochemicals, such as serotonin, glutamate, acetylcholine, and nitric oxide.
The authors would like to thank the staff at the NSF ERC for Wireless Integrated Microsystems (WIMS) at the University of Michigan for help in fabrication of probes for this study and Dr. Matthew Johnson for assistance with activation protocols and procedures. The authors would also like to thank the staff at the University of Utah Nanofab for assistance with fabrication of sensors for this work. REFERENCES
1
[1]
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Figure 3. Normalized Impedance graphs for each of the three fabrication methods. SIROF films exhibited the least variation over time while PBS Activated films widely
PBS Activated Iridium Oxide 0.6 Slope = 10.9mV/day 0.4 R2 = 0.160 n=6 0.2 0 0
2 3 4 5 6 Base Activated Iridium Oxide 0.6 Slope = 3.2mV/day 0.4 R2 = 0.015 n=6 0.2 0 0
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3 4 5 6 Sputtered Iridium Oxide 0.6 Slope = 28.9mV/day 0.4 R2 = 0.600 n=5 0.2 0 0
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CONCLUSION
ACKNOWLEDGMENT
1.4 R2 = 0.011 1.2 n=5
Open Circuit Potential (mV)
V.
Based on the observation reported here, we conclude that although SIROF electrodes appear to be more stable in terms of electrochemical impedance, for the purposes of establishing a stable reference potential, AIROF electrodes can be considered superior due to their improved open circuit potential stability. This finding is in agreement with the more general studies reported in the literature.
Slope = 15.5x10-3/day
1.4 R2 = 0.141 1.2 n=6
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response to an increase in DA concentration.
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6
[2]
[3]
[4]
[5]
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[8]
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Figure 4. Open Circuit potential plots for the three fabrication methods. Open circuit potentials were measured using a Gamry FAS2 femtostat in pH 7.4 PBS.
[9]
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J. Changeux, “Chemical Signaling in the Brain,” Scientific American, Nov. 1993, pp 58-62. E. Pothos, P. Rada, G.P. Mark, and B.G. Hoebel, “Dopamine microdialysis in the nucleus accumbens during acute and chronic morphine, naloxone-precipitated withdrawal and clonidine treatment,” Brain Research, vol. 566, Dec. 1991, pp. 348-350. S. Tierney, B.M. Hasle Falch, D.R. Hjelme, and B.T. Stokke, “Determination of glucose levels using a functionalized hydrogeloptical fiber biosensor: Toward continuous monitoring of blood glucose in vivo,” Analytical Chemistry, vol. 81, 2009, pp. 3630-3636. J. Mitala Jr. and A. Michael, “Improving the performance of electrochemical microsensors based on enzymes entrapped in a redox hydrogel,” Analytica Chimica Acta, vol. 556, 2006, pp. 326-332. Taek Dong Chung, Hee Chan Kim, Sang Kyung Kim, and H. Lim, “A miniaturized electrochemical system with a novel polyelectrolyte reference electrode and its application to thin layer electroanalysis,” Sensors and Actuators B (Chemical), vol. 115, May. 2006, pp. 212-19. H. Yang, S.K. Kang, C.A. Choi, H. Kim, D. Shin, Y.S. Kim, and Y.T. Kim, “An iridium oxide reference electrode for use in microfabricated biosensors and biochips,” Lab on a Chip, vol. 4, 2004, pp. 42-46. M. Johnson, R. Franklin, K. Scott, R. Brown, and D. Kipke, “Neural Probes for Concurrent Detection of Neurochemical and Electrophysiological Signals in vivo,” Engineering in Medicine and Biology Society, 2005. IEEE-EMBS 2005. 27th Annual International Conference of the, 2005, pp. 7325-7328. Haesik Yang, Sun Kil Kang, Dong-Ho Shin, Hyokyum Kim, and Youn Tae Kim, “Microfabricated iridium oxide reference electrode for continuous glucose monitoring sensor,” TRANSDUCERS, Solid-State Sensors, Actuators and Microsystems, 12th International Conference on, 2003, 2003, pp. 103-106 vol.1. Robert Franklin, Steven Martin and Timothy D. Strong, Richard B. Brown, “Chemical and Biological Systems: Chemical Sensing for
Liquids,” in Comprehensive Microsystems, Y. B. Gianchandani, O. Tabata, H. Zappe (ed.) Oxford: Elsevier Ltd, 2007, vol. 2, pp. 433-462. [10] R. Franklin, M. Johnson, K. Scottt, Jun Ho Shim, Hakhyun Nam, D. Kipket, and R. Brown, “Iridium oxide reference electrodes for neurochemical sensing with MEMS microelectrode arrays,” Sensors, 2005 IEEE, 2005, p. 4 pp. [11] M. Johnson, N. Langhals, and D. Kipke, “Neural Interface Dynamics Following Insertion of Hydrous Iridium Oxide Microelectrode Arrays,” Engineering in Medicine and Biology Society, 2006. EMBS '06. 28th Annual International Conference of the IEEE, 2006, pp. 3178-3181.
[12] M.D. Johnson, O.E. Kao, and D.R. Kipke, “Spatiotemporal pH dynamics following insertion of neural microelectrode arrays,” Journal of neuroscience methods, vol. 160, 2007, pp. 276-287. [13] S. Cogan, T. Plante, and J. Ehrlich, “Sputtered iridium oxide films (SIROFs) for low-impedance neural stimulation and recording electrodes,” Engineering in Medicine and Biology Society, 2004. IEMBS '04. 26th Annual International Conference of the IEEE, 2004, pp. 4153-4156. [14] S.F. Cogan, P.R. Troyk, J. Ehrlich, C.M. Gasbarro, and T.D. Plante, “The influence of electrolyte composition on the in vitro chargeinjection limits of activated iridium oxide (AIROF) stimulation electrodes,” Journal of Neural Engineering, vol. 4, 2007, pp. 79-86.
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Figure 5. Cyclic volatmmograms (left) of 10 mM DA in PBS at pH 3.85 using IrOx and SCE reference electrodes with scan rate of 1000 mV/s.. Cyclic voltamograms (right) of 10mM DA in PBS at pH 3.85 with scan rate of: (a) 50 mV/s, (b) 100 mV/s, (c) 500 mV/s, (d) 1000 mV/s, (e) 2500 mV/s, (f) 5000 mV/s, (g) 7500 mV/s, (h) 10000 mV/s. 90 60
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Figure 6. Cyclic voltamograms (left) of DA solution in PBS at pH 3.85 with various concentrations: (a) PBS only, (b) 0.1mM, (c) 0.5mM, (d) 1 mM, (e) 5mM, (f) 10mM with scan rate of 1000 mV/s. (Right) Calibration curve for DAbased on data
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