Electrochemically deposited iridium oxide reference electrode integrated with an electroenzymatic glutamate sensor on a multi-electrode arraymicroprobe

Abstract

An implantable micromachined multi-electrode array (MEA) microprobe modified for utilization as a complete electrochemical biosensor for rapid glutamate detection is described. A post-fabrication method for electrochemical deposition of an iridium oxide (IrOx) film onto a designated microelectrode enabled incorporation of an IrOx reference electrode (RE) on the microprobe. The on-probe IrOx RE provides an alternative to the commonly utilized Ag/AgCl wire RE, which has been shown to be unstable and to cause an inflammatory response in living tissue. The IrOx film electrodeposited onto a platinum site was tested as part of a complete chemical sensing system that included a platinum counter electrode and enzymatic glutamate sensing electrodes all on a single silicon-based MEA platform. The thin film IrOx was mechanically robust enough to endure conditions of repeated heating and wetting during the MEA fabrication process. The pH dependence of the IrOx open circuit potential (OCP) was measured at −77±0.4 mV/pH and remained stable over a two-week period. The on-probe IrOx RE was tested in a two- and three-electrode system with glutamate biosensors. The biosensors were shown to detect a physiologically relevant range of glutamate concentrations and to reject the interferents, dopamine and ascorbic acid. By incorporating all of the electrodes onto a single device, baseline noise was reduced by an average of ∼61% in vitro and ∼71% in vivo with reduced tissue damage, since only a single probe needed to be implanted.

Introduction

Miniaturization, which can be accomplished by employing micromachining techniques developed for the integrated circuits industry, has been the focus of recent progress on the development of neural biosensors (Varney and Aslam, 2011, Frey and Rothe, 2011, Johnson and Franklin, 2008, Kim and Hesketh, 2004, Lowinsohn and Peres, 2006, Motta and Judy, 2005, Rospert and Mokwa, 1997, Yoon and Hwang, 2000). Smaller electrodes provide improved spatial and temporal resolution as well as reduced tissue damage. The reference electrode (RE) is an important component of an electrochemical system; however, it usually is not integrated into the same micromachined probe with the biosensing electrode. Currently, most biosensors used for in vivo studies rely on a separate RE, usually a Ag/AgCl wire (Hu and Mitchell, 1994, Lee and Katarina, 2007, Thomas and Grandy, 2009, Walker and Wang, 2007, Wassum and Tolosa, 2012). Yet, there are key advantages to combining both the working and REs of an electrochemical biosensing system onto a single microprobe in a multi-electrode array (MEA) format. Such an integrated MEA biosensor for in vivo application simplifies surgery and reduces tissue damage, as only a single foreign body has to be implanted in the brain (Karp et al., 2008). Use of an on-probe RE also is expected to result in reduced noise arising from external sources (e.g., 60 Hz noise) and from within the brain, which can lead to improved detection limits (Clark et al., 2009, Peteu et al., 1996). Clearly, these are compelling reasons to combine the reference and working electrodes on a MEA microprobe for in vivo sensing applications.

Although separate wire Ag/AgCl REs are used commonly in electroanalytical applications as mentioned above, incorporation of a miniature Ag/AgCl RE onto microprobes has been achieved through techniques such as electrochemical wet processing or plasma deposition (Park and Jun, 2003, Suzuki and Hirakawa, 1998). However, these techniques are less than optimal due either to the cumbersome nature of the method and/or instability of the deposited film. AgCl film instability through delamination or dissolution of the chloride salt (Franklin and Johnson, 2005, Yang et al., 2003) makes these REs non-ideal for long-term in vivo studies, as AgCl film loss causes a drift in electrode potential resulting in inaccurate signal readings (Yang et al., 2003). In addition to the compromised signal, the dissolved film is toxic and causes significant inflammatory responses (Dymond and Kaechele, 1970, Tivol and Agnew, 1987, Yuen and Agnew, 1987). Successful implementation of an implantable RE in a MEA format requires several criteria to be met: (1)the RE fabrication method must be compatible with the MEA platform; (2)the RE must exhibit sufficient chemical and mechanical stability; (3)the RE must provide a stable reference potential over the applicable range of conditions associated with its intended use; and (4)the chosen RE should be biocompatible.

Many of the negative issues encountered with Ag/AgCl REs can be avoided by using iridium oxide (IrOx) as the reference electrode material, and in fact, IrOx has unique properties that make it attractive for in vivo neuroscience applications. Recent studies have addressed use of IrOx as a pH sensor, an electrode for electrophysiological recording and stimulation, and as a quasi-RE (Ges and Dzhura, 2008, Johnson and Franklin, 2008, Karp and Bernotski, 2008, Li and Du, 2007, Meyer et al., 2001, Meyer et al., 2001, VanHoudt and Lewandowski, 1992, Wipf and Ge, 2000, Yang and Kang, 2004). Although the IrOx potential shows strong pH dependence, the small dynamic range of normal brain pH (7.15–7.4) makes this issue unimportant in most cases (Franklin et al., 2005). More importantly, IrOx is stable over a range of potentials and in the presence of the ions and other components of brain extracellular fluid (Marzouk et al., 1998). Finally, IrOx films exhibit excellent mechanical stability and biocompatibility, both of which allow for long-term implantation with minimal damage to living tissue (Slavcheva and Vitusshinsky, 2004, Weiland and Anderson, 2000).

IrOx films can be fabricated by several different methods. Some of the most popular protocols entail electrochemical activation of either a bulk Ir metal electrode or an Ir film sputtered onto an appropriate substrate (Glab and Hulanicki, 1989, Katsube and Lauks, 1982, Kreider and Tarlov, 1995, Slavcheva and Vitusshinsky, 2004). However, incorporation of an IrOx reference site on a MEA microprobe by these methods would require major changes to our current MEA probe microfabrication process requiring costly materials and additional processing steps. A simple and cost-effective method for IrOx film deposition would be desirable for the development of a practical on-probeRE.

In this paper, we describe IrOx deposition on specified platinum (Pt) microelectrodes of an MEA by a simple one-step electrochemical method (Yamanaka, 1989) to give fully functional REs. We investigated IrOx film robustness during the microprobe manufacturing process, temporal stability in solution, reproducibility, as well as working electrode noise when an on-probe IrOx RE is used as opposed to a separate Ag/AgCl reference. Finally, in an effort to demonstrate practical utility, we conducted preliminary testing of an integrated working and RE microprobe for glutamate biosensing both in vitro and in vivo. Glutamate is the primary excitatory neurotransmitter in the brain and has been linked to many neurological disorders and diseases, including autism, schizophrenia, Huntington's and Parkinson's disease (Carlsson and Carlsson, 1990, Cha and Kosinski, 1998, Purcell and Jeon, 2001) and therefore constitutes a relevant test case for thisstudy.