sensors

Article Design, Fabrication, Simulation and Characterization of a Novel Dual-Sided Microelectrode Array for Deep Recording and Stimulation

Zongya Zhao 1,2, Ruxue Gong 1,2, Hongen Huang 1,2 and Jue Wang 1,2,* 1 The Key Laboratory of Biomedical Information Engineering of Ministry of Education, Institute of Biomedical Engineering, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China; [email protected] (Z.Z.); [email protected] (R.G.); [email protected] (H.H.) 2 National Engineering Research Center of Health Care and Medical Devices, Xi’an Jiaotong University Branch, Xi’an 710049, China * Correspondence: [email protected]; Tel.: +86-29-8266-3497

Academic Editors: Zhiping Wang and Charles Chun Yang Received: 6 April 2016; Accepted: 20 May 2016; Published: 15 June 2016 Abstract: In this paper, a novel dual-sided microelectrode array is specially designed and fabricated for a rat Parkinson’s disease (PD) model to study the mechanisms of (DBS). The fabricated microelectrode array can stimulate the subthalamic nucleus and simultaneously record electrophysiological information from multiple nuclei of the basal ganglia system. The fabricated microelectrode array has a long shaft of 9 mm and each planar surface is equipped with three stimulating sites (diameter of 100 µm), seven electrophysiological recording sites (diameter of 20 µm) and four sites with diameter of 50 µm used for neurotransmitter measurements in future work. The performances of the fabricated microelectrode array were characterized by scanning microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry. In addition, the stimulating effects of the fabricated microelectrode were evaluated by finite element modeling (FEM). Preliminary animal experiments demonstrated that the designed microelectrode arrays can record spontaneous discharge from the striatum, the subthalamic nucleus and the globus pallidus interna. The designed and fabricated microelectrode arrays provide a powerful research tool for studying the mechanisms of DBS in rat PD models.

Keywords: Parkinson’s disease; mechanisms of deep brain stimulation; dual-sided microelectrode array

1. Introduction Parkinson’s disease (PD) is a degenerative disease with cardinal motor symptoms that correlate with the loss of dopaminergic in the substantia nigra of the brain. PD is characterized by akinesia, bradykinesia, rigidity, resting tremors, and other motor and postural impairments [1,2]. Although deep brain stimulation (DBS) of the subthalamic nucleus (STN) has proved to be an effective symptomatic treatment in advanced PD for well selected patients [3–8], its mechanisms of action are still not well understood, which will limit further clinical application of DBS. Currently, investigations of the mechanisms underlying DBS have attracted great interest in the scientific community. On the one hand, although numerous experimental studies have analyzed the effect of DBS at the single cell level using in vivo microelectrode recording in recent years [9–15], the tools used by these researchers were based on wire electrodes. On the other hand, recent studies have shown that the mechanisms underlying DBS correlate with the regulation of basal ganglia system (BGS) which consists of four interconnected nuclei: the subthalamic nucleus (STN), globus pallidus (futher divided in pars interna, GPi, and pars externa, GPe), substantia nigra (futher divided in pars

Sensors 2016, 16, 880; doi:10.3390/s16060880 www.mdpi.com/journal/sensors Sensors 2016, 16, 880 2 of 16 compacta, SNc, and pars reticulata, SNr) and striatum [16–19]. Therefore, the mechanisms underlying DBS could be better understood from the perspective of functional network interactions among different nuclei of BGS [20], which need to simultaneously obtain electrophysiological information from multiple nuclei of BGS during electrical stimulation of STN. Although wire electrodes could be used to record neuronal discharge signals from multiple nuclei, for example, Shi et al. implanted eight stainless teflon-insulated microwires into the striatum, globus pallidus and SNr to record their neuronal discharge signals simultaneously [21]. However, in this case, multiple wire electrodes need to be implanted into different brain nuclei, and in addition, another type of wire electrode should be implanted into STN for electrical stimulation, which will greatly increase the complexity and difficulty of surgical procedures. Moreover, the limitations of wire electrodes, such as difficult implantation, low spatial resolution, low reproducibility and manual construction are also well known [22,23]. Therefore, in order to better study the mechanisms of DBS, a multifunctional microelectrode which can stimulate STN and obtain electrophysiological information from multiple nuclei of BGS is urgently needed. The rapid development of micro-electromechanical systems (MEMS) technology in the biomedical field provides a feasible solution for the above-mentioned problems. With MEMS technology, the precise definition of electrode size and shape can be realized, and multiple recording/stimulation sites can be fabricated on a single probe shank [24–26]. Since the pioneering work of Wise et al. [27], a growing number of -based neural probes have been developed, with a variety of electrode geometries and methods for fabrication and assembly, including the well-known Michigan probes [28,29] and Utah electrodes [30–32]. With the development of neuroscience, especially brain science, novel microelectrode arrays are urgently needed by neuroscientists to further understand the working principles of the nervous system. On the one hand, the performances of MEMS microelectrode arrays have been improved in many aspects [33], e.g., three-dimensional arrays [34], dual-sided microelectrode arrays [35,36], microelectrode arrays with high-density stimulation/recording sites [37], integrated electronics or microfluidic channels [38–43], silicon probes for optogenetics or infrared stimulation probes [44,45]. On the other hand, according to different experimental requirements, some specially designed microelectrode arrays have been developed. For example, Xu et al. [46] designed and fabricated a multi-shank electrode which can reach multiple regions of the simultaneously according to the special neuro-anatomical structure of the hippocampus. Márton et al. [47] designed and characterized a microelectrode array system specifically for obtaining in vivo extracellular recordings from three deep-brain areas of freely moving rats simultaneously. Furthermore, rats have become a common animal model for studying the mechanisms of DBS due to their low cost and being easy to model [20,48]. However, to the best of our knowledge, there is no report about a specially designed dual-sided microelectrode array which is used for studying the mechanisms of DBS in a rat PD model, can stimulate STN and record electrophysiological information of the striatum, GPi and STN of BGS. In this paper, according to the size of a rat brain and relative positional relationship of the STN, GPi and striatum of BGS, a novel dual-sided microelectrode array was designed and fabricated. Firstly, the fabricated microelectrode array was specially designed for studying the mechanisms of DBS in a rat PD model, which could stimulate STN and record discharge signals from the striatum, GPi and STN of BGS. Secondly, conventional single-side arrays may shield spike activity of some neurons facing the other side of the array, so a dual-side electrode array was developed by placing separately stimulating/recording sites on each planar surface of the microelectrode, which increased the density of stimulating/recording sites in the region of interest by twofold. Moreover, due to the characteristics of high frequency and high stimulating intensity of DBS, the diameter of stimulating sites (100 µm) is much larger than that of recording sites (20 µm), which would greatly enhance the charge injection capability of stimulating sites. Finally, the fabricated microelectrode arrays were characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and other electrochemical methods. In addition, the stimulating effects of the fabricated microelectrode were evaluated by finite element modeling (FEM), and preliminary animal experiments showed that Sensors 2016, 16, 880 3 of 16

Sensors(EIS) 2016and, 16other, 880 electrochemical methods. In addition, the stimulating effects of the fabricated3 of 16 microelectrode were evaluated by finite element modeling (FEM), and preliminary animal experiments showed that the designed microelectrode arrays could simultaneously record the designed microelectrode arrays could simultaneously record spontaneous discharge signals of spontaneous discharge signals of multiple nuclei of BGS. multiple nuclei of BGS.

2.2. Materials Materials and and Methods Methods

2.1.2.1. MicroelectrodeMicroelectrode ArraysArrays DesignDesign andand FabricationFabrication TheThe microelectrodemicroelectrode arraysarrays werewere designeddesigned accordingaccording toto thethe sizesize ofof ratrat brainbrain andand relativerelative positionalpositional relationshiprelationship ofof thethe STN,STN, GPiGPi andand striatumstriatum ofof BGSBGS (Figure(Figure1 ),1), and and our our purpose purpose was was to to simultaneouslysimultaneously recordrecord electrophysiologicalelectrophysiological information from the striatum, GPi and STN of BGS. FigureFigure2 2shows shows the the detailed detailed design design parameters. parameters. The The designed designed microelectrode microelectrode array array with with dimensions dimensions of 9of mm 9 mmˆ 400 × µ400m ˆμm200 ×µ 200m (l μˆmw (lˆ ×t) employedw × t) employed 14 round-shaped 14 round-shaped microelectrode gold microelectrode sites distributed sites ondistributed each side. on These each sites side. could These be divided sites could into threebe di types:vided three into stimulatingthree types: sites three (diameter stimulating of 100 µsitesm), seven(diameter electrophysiological of 100 μm), seven recording electrophysiological sites (diameter recording of 20 µm) sites and (diameter four sites of with 20 μ diameterm) and four of 50 sitesµm usedwith fordiameter other experimental of 50 μm used purposes for other in theexperimental future. For purposes example, in these the 50future.µm sites For couldexample, be modified these 50 withμm sites the -exchangecould be modified resin Nafionwith the for ion-exchange detection of resin neurotransmitter Nafion for detection dopamine of [neurotransmitter49], or modified withdopamine a protein [49], matrix or modified layer containing with aL -glutamateprotein matrix oxidase layer as glutamate containing sensors L-glutamate [50]. At theoxidase front ofas theglutamate designed sensors microelectrode, [50]. At the three front stimulating of the designed sites microelectrode, and three recording three sites stimulating were mutually sites and spaced three withrecording equal sites center-to-center were mutually distance spaced of with 250 µequalm. The center-to-center distance between distance the tipof 250 and μ them. centerThe distance of the firstbetween recording the tip site and was the 300 centerµm, definedof the first by therecording fabrication site was process. 300 μ Thesem, defined four sites by withthe fabrication diameter ofprocess. 50 µm These and four four recording sites with sitesdiameter were of also 50 μ mutuallym and four spaced recording with equalsites were center-to-center also mutually distance spaced ofwith 250 equalµm. Eachcenter-to-center recording site distance was electrically of 250 μm. connected Each recording to a bonding site was pad electrically (400 µm ˆconnected400 µm) to via a 5bondingµm-wide pad gold (400 conductive μm × 400 pathsμm) via with 5 μ 5m-wideµm-wide gold gaps conductive between paths them, with and each5 μm-wide stimulating gaps between site was electricallythem, and connectedeach stimulating to a bonding site was pad electrically via 10 µm-wide connected gold to conductive a bonding paths pad withvia 10 5 µμm-widem-wide gapsgold betweenconductive them. paths with 5 μm-wide gaps between them.

FigureFigure 1.1. Schematic diagram of median sagittal plane ofof Sprague-DawleySprague-Dawley ratrat brainsbrains showingshowing thethe relativerelative positionalpositional relationshiprelationship ofof thethe STN,STN, GPiGPi and and striatum striatum (coordinate (coordinate unit: unit: mm). mm).

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Figure 2. Schematic diagram of the designed microelectrode array. Figure 2. Schematic diagram of the designed microelectrode array. Figure 2. Schematic diagram of the designed microelectrode array.

Figure 3. Schematic diagram of the process flow: (a) thermal oxidation; (b) spinning photoresist and patterning by lithography; (c) deposition of adhesion layer Cr; (d) deposition of Au layer; (e) lifting off the unpatterned metal and photoresist; (f) deposition of dielectric PECVD SiO2 layers; (g) Figure 3. Schematic diagram of the process flow: (a) thermal oxidation; (b) spinning photoresist and Figurespinning 3. Schematic photoresist diagram for patterning of the process passivation flow: layer; (a) thermal (h) exposing oxidation; electrode (b )sites spinning and bonding photoresist pads; and patterning by lithography; (c) deposition of adhesion layer Cr; (d) deposition of Au layer; (e) lifting patterning(i) spinning by lithography; photoresist for (c) DRIE; deposition (j) DRIE of adhesionand device layer release. Cr; (d) deposition of Au layer; (e) lifting off off the unpatterned metal and photoresist; (f) deposition of dielectric PECVD SiO2 layers; (g) the unpatterned metal and photoresist; (f) deposition of dielectric PECVD SiO2 layers; (g) spinning spinning photoresist for patterning passivation layer; (h) exposing electrode sites and bonding pads; photoresistFigure for3 showed patterning the passivation main fabrication layer; (h process) exposing for electrode a silicon-based sites and microelectrode bonding pads; (array.i) spinning The (i) spinning photoresist for DRIE; (j) DRIE and device release. photoresistsubstrate was for typically DRIE; (j) comprised DRIE and deviceof about release. 100 mm diameter, double-side polished, p-doped (1 0 0) silicon wafers with a thickness of 200 μm. In the first step, the substrate was thermally oxidized at Figure 3 showed the main fabrication process for a silicon-based microelectrode array. The 1150 °C to yield a 1.5 μm silicon dioxide (SiO2) on both sides as an insulating layer (Figure 3a). Figure3 showed the main fabrication process for a silicon-based microelectrode array. substrateSecondly, was a typically5 μm-thick comprised negative tone of about photoresist 100 mm (nLOF-2035, diameter, AZ double-side Electronic polished, Materials) p-doped was spun (1 on 0 0) Thesilicon substrateand waferspatterned was with typicallyto adefine thickness comprisedthe metal of 200 layer ofμm. about comprisingIn the 100 first mm thestep, diameter, electrode the substrate double-side sites, interconnecting was polished,thermally p-dopedwiresoxidized and(1 at 0 0) silicon1150bonding °C wafers to yieldpads with bya a 1.5lithography thickness μm silicon of(Figure 200dioxideµ 3b).m. In(SiOThis the2 )wa on firsts followedboth step, sides the by substrateassputtering an insulating was20 nm-thick thermally layer chromium(Figure oxidized 3a). at ˝ 1150Secondly,(Cr)C to as yield aan 5 adhesionμ am-thick 1.5 µm layer siliconnegative (Figure dioxide tone 3c) photoresist (SiOand 2evaporating) on both(nLOF-2035, sides a 300 as nm-thick anAZ insulating Electronic gold layer(Au)Materials) (Figurelayer (Figurewas3a). spun Secondly, 3d) on a 5andµm-thicksequentially. patterned negative toThe define tonelift-off the photoresist process metal layerwas (nLOF-2035, used comprising to lift AZ off the Electronic the electrode unpatterned Materials) sites, metalinterconnecting was and spun photoresist onand wires patterned byand tobonding definedissolving the pads metal theby lithographyphotoresist layer comprising in(Figure an amine-solvent the 3b). electrode This wa mixtures sites, followed interconnecting (Figure by sputtering 3e). Thirdly, wires 20 nm-thick andthe subsequent bonding chromium pads 2 by lithography(Cr)μ m-thickas an adhesion (Figure SiO2 passivation3b). layer This (Figure was layer followed 3c) was and made byevaporatingsputtering from plasma-enhanced a 20300 nm-thick nm-thick chromium chemicalgold (Au) vapor (Cr)layer asdeposition (Figure an adhesion 3d) layersequentially.(PECVD) (Figure3 (Figurec) The and lift-off evaporating3f). This process passivation a was 300 nm-thick usedlayer wasto li goldpaft tternedoff (Au) the using layerunpatterned reactive (Figure 3ionmetald) etching sequentially. and (RIE)photoresist through The lift-off by processdissolving was usedthe photoresist to lift off thein unpatternedan amine-solvent metal mixture and photoresist (Figure 3e). by Thirdly, dissolving the the subsequent photoresist 2 in μm-thick SiO2 passivation layer was made from plasma-enhanced chemical vapor deposition an amine-solvent mixture (Figure3e). Thirdly, the subsequent 2 µm-thick SiO2 passivation layer was made(PECVD) from (Figure plasma-enhanced 3f). This passivation chemical layer vapor was deposition patterned (PECVD) using reactive (Figure ion3f). etching This passivation (RIE) through layer

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Sensorsthe photoresist2016, 16, 880 as a hard mask (Figure 3g) to open electrode sites and bonding pads (Figure5 of3h). 16 After the process on the front side, the wafer was turned over to repeat the above steps in the back. After that, additional twice lithography, left-off and RIE steps defined the dimensions of probes to was patternedSensors using2016, 16, 880 reactive ion etching (RIE) through the photoresist as a hard mask5 (Figure of 16 3g) to openrealize electrode the outline sites from and bondingthe front pads and (Figureback surface3h). After of wafer the process by removing on the front SiO2 side, outside the waferthe probe. was turnedFinally, over a the10 to photoresistμ repeatm-thick the asphotoresist abovea hard mask steps (AZ-9260, (Figure in the 3g) back. toAZ op AfterElectronicen electrode that, additional sitesMaterials) and bonding twice was pads lithography,applied (Figure to 3h).serve left-off as andthe After the process on the front side, the wafer was turned over to repeat the above steps in the back. RIEetch stepsmask defined layer (Figure the dimensions 3i) and deep of probes reactive to realize ion etching the outline (DRIE) from was the used front to and release back the surface probes of from the waferAfter that, (Figure additional 3j). twiceThe lithography,overview left-offof an andentire RIE siliconsteps defined wafer the with dimensions the probes of probes and to frames is wafer by removingrealize the SiOoutline2 outside from the the front probe. and back Finally, surface a 10of waferµm-thick by removing photoresist SiO2 outside (AZ-9260, the probe. AZ Electronic Materials)shown in FigureFinally, was applied a4. 10 μm-thick to serve photoresist as the etch (AZ-9260, mask AZ layer Electronic (Figure Materials)3i) and was deep applied reactive to serve ion etchingas the (DRIE) was used toetch release mask layer the probes(Figure from3i) and the deep wafer reactive (Figure ion etching3j). The (DRIE) overview was used of anto release entire the silicon probes wafer with from the wafer (Figure 3j). The overview of an entire silicon wafer with the probes and frames is the probesshown and frames in Figure is 4. shown in Figure4.

Figure 4. Picture of the entire wafer with 120 microelectrode array in holder frames. Figure 4. Picture of the entire wafer with 120 microelectrode array in holderholder frames.frames. 2.2. Microelectrode Arrays Package 2.2. Microelectrode After Arraysfabrication Package of the microelectrode array, package was begun by combining gold wire bonding technology and flip-chip bonding technology, and a printed circuit board (PCB) specifically After fabrication of the microelectrode array, package was begun by combining gold wire After designedfabrication for this of purpose the microelectrode was used for packaging array, (Figure package 5a). On wasthe back begun side of by the microelectrodecombining gold wire bonding technologyarray, bonding and pads flip-chipflip-chip between bondingthe microelectrode technology, array and and PCB a printedwere closely circuit integrated board by (PCB) flip-chip specificallyspecifically designed fortechnology, this purpose and the was bonds used were for packagingformed via an (Figure(F igureanisotropic5 5a).a). On Onconductive thethe backback adhesive sideside ofof thefilmthe microelectrodemicroelectrode(ACF, AC-7206U-18, HITACH): first, solder bumps were formed on inner bonding pads of PCB by reflow array, bonding bonding pads pads between between the themicroelectrode microelectrode array array and PCB and were PCB closely were closelyintegrated integrated by flip-chip by soldering, then a piece of ACF with a size of 1.5 × 5 mm completely covered every inner bonding flip-chip technology, and the bonds were formed via an anisotropic conductive adhesive film (ACF, technology,pads and of PCB the by bonds a temporary were bonding formed at 80 via °C, 1an Mpa anisotropic for 5 s, and a conductivefinal bonding wasadhesive performed film (ACF, AC-7206U-18,between HITACH): the bonding first,first, pads solder of microelectrode bumps were array formedand ACF at on 170 inner °C, 3 bondingMpa for 20 padss. One of the PCB front by reflowreflow soldering, side, then 33 a μ piecem diameter ofof ACFACF gold withwithwires aconnecteda sizesize ofof the 1.51.5 bond ˆ× 5 pads mm with completelycompletely traces on the covered PCB using every gold innerinner wire bondingbonding bonding technology (Figure 5b,c). Additionally,˝ a biocompatible adhesive was employed to pads of PCBPCB byby aa temporarytemporary bondingbonding at 80 °C,C, 1 Mpa for 5 s,s, andand aa finalfinal bondingbonding waswas performedperformed strengthen the bond and seal the contact pads. ˝ between the bonding pads of microelectrodemicroelectrode arrayarray andand ACFACF atat 170170 °C,C, 33 MpaMpa forfor 2020 s.s. OneOne thethe front side, 33 µμm diameterdiameter gold wires connected the bond pads with traces on the PCB using gold wire bonding technologytechnology (Figure (Figure5b,c). 5b,c). Additionally, Additionally, a biocompatible a biocompatible adhesive adhesive was employed was toemployed strengthen to thestrengthen bond and the seal bond the and contact seal pads.the contact pads.

Figure 5. (a) Schematic diagram of the specifically designed PCB; (b) assembly scheme for connecting the double-sided microelectrode array with PCB; (c) optical image of schemed microelectrode array.

Figure 5. (a) Schematic diagram of the specifically designed PCB; (b) assembly scheme for connecting Figure 5. (a) Schematic diagram of the specifically designed PCB; (b) assembly scheme for connecting the double-sided microelectrode array with PCB; (c) optical image of schemed microelectrode array. the double-sided microelectrode array with PCB; (c) optical image of schemed microelectrode array.

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2.3. FEM of STN Stimulation It would be difficult to perform experiments to directly measure the electric field distribution in brain tissue generated by DBS, but FEM provides a solution to quantitatively evaluate the neural response to DBS in a simulated environment. Three-dimensional models of STN stimulation were built in order to demonstrate the effects of extracellular stimulation using self-designed microelectrode array. The experiments could be mainly described in four steps: (1) a symmetric FEM of a neural probe surrounded by a homogeneous and isotropic volume conductor representing brain tissue was created using the FEM software COMSOL Multiphysics [51]; (2) the stimulus current was applied to the electrodes using a monopole stimulation mode at single or double side (reference electrode was at infinity for monopole stimulation mode); (3) the potential distribution in the model was calculated using a FEM solver; (4) and the volume of tissue activity (VTA) was estimated using MATLAB based on the extracellular excitation threshold of ´0.5 V [52]. The electrical conductivity of electrode contacts and the substrate of the microelectrode array were 5.99 ˆ 107 and 1 ˆ 10´19 S/m respectively [53], and an electrical conductivity of 0.2 S/m was applied in brain tissue [54]. Besides, we named the three stimulating sites #0, #1, #2, respectively, from the bottom to the top of the microelectrode array. The X, Y and Z axes were defined as follows: the Z axis was along the shaft of microelectrode probe (longitudinal axis of probe), the Y axis was vertical to the side surface of the probe, and the X axis was vertical to the X–Y plane.

2.4. Electrochemical Characterization of the Microelectrode Array Electrochemical characterization of the microelectrode arrays were analyzed with the CHI 650E electrochemical analyzer (Shanghai CH Instruments, Shanghai, China) at room temperature in a conventional three-electrode electrochemical cell, which was composed of a fabricated microelectrode array as the working electrode, a platinum wire as the auxiliary electrode, and an Ag/AgCl as the reference electrode. The electrochemical impedance spectroscopy (EIS) was performed in 0.1 M phosphate buffer solution (PBS) at an amplitude of 50 mV and frequencies between 1 Hz and 100 kHz. Here, electrochemical characterization of stimulating sites (diameter of 100 µm) and recording sites (diameter of 20 µm) were mainly studied. Before all the experiments, the microelectrode sites were pretreated in 0.5 M H2SO4 solution for cleaning surface of Au films until the cyclic voltammograms of Au microelectrode sites showed stable electrochemical behavior. In addition, in order to reduce the impedances of microelectrode sites, gold nanoparticles (AuNPs) were electrodeposited on the surface of microelectrode sites by immersing the microelectrode array into 0.1 M PBS (pH = 5.0) containing 1 mM chloroauric acid and applying a constant potential of ´0.8 V for 200 s [55]. And then, the cyclic voltammograms of stimulating and recording sites at different scan rate (20, 50, 100, 150 and 200 mV/s) were obtained in 0.1 M KCl solution containing 2 mM 3´/4´ [Fe(CN)6] (1 mM K3[Fe(CN)6] + 1 mM K4[Fe(CN)6]).

3. Results and Discussion

3.1. Morphological Characterization The surface morphologies of the fabricated microelectrode array were examined by scanning electron microscopy (SEM), and here we mainly studied the morphological characterization of stimulating sites (diameter of 100 µm) and recording sites (diameter of 20 µm). As shown in Figure6a, the recording site with a diameter of 20 µm possessed a round shape and smooth surface. Moreover, 5 µm-wide interconnecting wire connected with the sites was also visible. After electrodeposition of AuNPs (Figure6c), dendritic nanoparticles with a 3D structure grown on the surface of the recording site, which could greatly increase the surface roughness, reduced the impedance of the electrode site and facilitated neural recordings. Figure6b shows that the stimulating site with a diameter of 100 µm also had a round shape and smooth surface, and the inset figure with higher magnification indicates that there was a clear boundary between the gold layer and thermally oxidized SiO2, which illustrated Sensors 2016, 16, 880 7 of 16 Sensors 2016, 16, 880 7 of 16

and thermally oxidized SiO2, which illustrated that the passivation layer was well removed by that the passivation layer was well removed by reactive ion etching (RIE). After electrodeposition of reactive ion etching (RIE). After electrodeposition of AuNPs (Figure 6d), lots of gold nanoparticles AuNPsSensors (Figure 2016, 166d),, 880 lots of gold nanoparticles were densely and uniformly distributed on the7 of surface 16 were densely and uniformly distributed on the surface of the stimulating site, which greatly reduced of the stimulating site, which greatly reduced impedance, increased surface roughness and enhanced impedance,and thermally increased oxidized surface SiO 2roughness, which illustrated and enhanced that the the passivation charge injection layer capabilitywas well removedof stimulating by the charge injection capability of stimulating sites [56]. Figure7 showed an SEM image of the tip sitesreactive [56]. Figureion etching 7 showed (RIE). anAfter SEM electrodeposition image of the tip of ofAuNPs a microelectrode (Figure 6d), lots array of goldand ananoparticles recording site of alocated microelectrodewere denselyat the tip and of array uniformlythe electrode and adistributed recording shank. Iton sitecould the locatedsurf beace clearly of at the the observed stimulating tip of the that site, electrode a sharpwhich tip greatly shank. geometry reduced It could with be clearlyaccurateimpedance, observed shape increased thatwas apresented, sharp surface tip roughness indicating geometry and that with enhanced th accuratee microelectrode the charge shape injection was arrays presented, capabilitywere well indicating of releasedstimulating thatfrom the sites [56]. Figure 7 showed an SEM image of the tip of a microelectrode array and a recording site microelectrodesilicon wafer arraysby deep were reactive well ion released etching from (DRIE). silicon wafer by deep reactive ion etching (DRIE). located at the tip of the electrode shank. It could be clearly observed that a sharp tip geometry with accurate shape was presented, indicating that the microelectrode arrays were well released from silicon wafer by deep reactive ion etching (DRIE).

Figure 6. SEM images of stimulating sites (b,d) and recording sites (a,c) before (a,b) and after (c,d) Figure 6. SEM images of stimulating sites (b,d) and recording sites (a,c) before (a,b) and after (c,d) electrodepositionFigure 6. SEM images of AuNPs. of stimulating sites (b,d) and recording sites (a,c) before (a,b) and after (c,d) electrodeposition of AuNPs. electrodeposition of AuNPs.

FigureFigure 7. 7. SEM SEM images images ofof tip ofof microelectrodemicroelectrode shank. shank. Figure 7. SEM images of tip of microelectrode shank.

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3.2. Effective Areas of STN Stimulation Based on FEM 3.2. Effective Areas of STN Stimulation Based on FEM The aim of this FEM simulation was to demonstrate the efficient spread of electrical stimulation in STNThe generated aim of this by FEM DBS simulation[57]. The extracellular was to demonstrate potential the(V) efficientdistributions spread inof STN electrical during stimulation monopole stimulationin STN generated on double-side by DBS [57 ].site The #0 extracellular were studie potentiald. The (V)extracellular distributions potential in STN during(V) distributions monopole generatedstimulation from on double-side monopole stimulation site #0 were studied.at different The current extracellular intensities potential in STN (V) are distributions shown in generatedFigures 8 andfrom 9. monopole Figure 8 shows stimulation the one-dimensional at different current distributions intensities of inthe STN extracellular are shown potential in Figures (V)8 in and three9. directionsFigure8 shows at various the one-dimensional stimulus intensities. distributions Figure of the 9 extracellularshows the potentialthree-dimensional (V) in three extracellular directions at potentialvarious stimulus distribution intensities. isosurface Figure of −90.5 shows V (the the extracellular three-dimensional excitation extracellular threshold) potential generated distribution by DBS at differentisosurface currents. of ´0.5 When V (the applying extracellular current excitation intensities threshold) of 100, 150, generated 250 and by 350 DBS μA, at the different currents. values Whenat the surface applying of electrode current intensities site #0 were of 100,2.3096, 150, 3.4644, 250 and 5.7739 350 andµA, 8.0835 the voltage V, respectively. values at theThe surface potential of distributionelectrode site increased #0 were as 2.3096, the curr 3.4644,ent stimulus 5.7739 and was 8.0835 increased. V, respectively. In order to The qualify potential the effects distribution of the stimulation,increased as VTA the current was calculated stimulus wasas the increased. vital standard In order to measure to qualify the the stimulus effects of effects the stimulation, [58]. As shown VTA wasin Figure calculated 10a, when as the applying vital standard curren tot measureintensities the of stimulus 100, 150, effects 250 and [58 350]. As μ shownA to STN, in Figure the VTA 10a, values when wereapplying 0.6321, current 0.8346, intensities 2.5067 and of 100,4.2805 150, mm 2503, andwhich 350 indicatedµA to STN, that the the VTA VTA values values were increased 0.6321, with 0.8346, the 3 stimulation2.5067 and 4.2805 intensities. mm , whichFurthermore, indicated we that compared the VTA valuesthe stimulus increased effects with of the double-side stimulation monopole intensities. stimulationFurthermore, with we that compared of single-side the stimulus monopole effects st ofimulation. double-side For monopolesingle-side stimulation monopole stimulation, with that of whensingle-side applying monopole current stimulation. intensities of For 100, single-side 150, 250 and monopole 350 μA, stimulation,the VTA values when were applying 0.1669, current0.3233, 3 0.7055intensities and of 1.2404 100, 150, mm 2503 (Figure and 350 10b),µA,the showing VTA values that werethe VTA 0.1669, value 0.3233, of 0.7055single-side and 1.2404 monopole mm stimulation(Figure 10b), was showing significantly that the VTAlower value than of that single-side of double-side monopole monopole stimulation stimulation was significantly (Figure lower 10a) whenthan that applying of double-side the same monopole current intensity. stimulation (Figure 10a) when applying the same current intensity.

Figure 8. One-dimensional distributions of the extrace extracellularllular potential (V) in three directions of X-axis (a); YY-axis-axis ( b()b and) andZ-axis Z-axis (c) generated(c) generated by monopole by monopole stimulation stimulation in STN atin variousSTN at stimulus various intensitiesstimulus intensities(100, 150, 250 (100, and 150, 350 250µA). and 350 μA).

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Figure 9. Three-dimensional extracellular potential distribution isosurface based on the extracellular FigureFigure 9. Three-dimensional9. Three-dimensional extracellular extracellular potentialpotential distribution isosurface isosurface based based on on the the extracellular extracellular excitation threshold of −0.5 V generated by monopole stimulation in STN at various stimulus excitationexcitation threshold threshold of ´of0.5 −0.5 V generated V generated by monopoleby monopole stimulation stimulation in STN in atSTN various at various stimulus stimulus currents currents (−100, −150, −250 and −350 μA). (´100,currents´150, (−100,´250 −150, and −´250350 andµA). −350 μA).

FigureFigure 10. 10.Effects Effects of of the the stimulus stimulus intensity intensity onon thethe VTA during ( (aa)) double-side double-side monopole monopole stimulation; stimulation; Figure 10. Effects of the stimulus intensity on the VTA during (a) double-side monopole stimulation; (b)( single-sideb) single-side monopole monopole stimulation. stimulation. (b) single-side monopole stimulation.

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3.3. Electrochemical Behavior of the Microelectrode Array Figure 11a shows the cyclic voltammetric response of a recording site with a diameter of 20 μm in 0.5 M H2SO4. The typical potential peak appearing at about 900 mV corresponded to the reduction of Au oxide species [59,60], implying that the cyclic voltammogram of the Au microelectrode site in H2SO4 matched the classic electrochemical response of bulk Au in H2SO4. These results indicated that thin film Au microelectrode sites possessed the same electrochemical properties as bulk Au and could be reasonably expected to possess the same recording capabilities as traditional Au microwires used for neural recording. In addition, the cyclic voltammetric responses of stimulating Sensors 2016, 16, 880 10 of 16 sites in H2SO4 possessed the same properties as those of recording sites. The stimulating and recording sites were further electrochemically characterized by 3.3.electrochemical Electrochemical impedance Behavior of spectroscopy the Microelectrode (EIS) Array to measure the electrode impedances in 0.1 M PBS using electrochemical analyzer and the results were presented in Figure 11b. For a typical recording site withFigure diameter 11a shows of 20 the μ cyclicm (curve voltammetric a of Figure response 11b) , the of average a recording measured site with impedance a diameter was of about 20 µm 4.1 in 0.5MΩ M at H 12 SOkHz4. Theand typicalthe average potential impedance peak appearing was reduced at about to about 900 mV 0.9 correspondedMΩ after electrodeposition to the reduction of ofAuNPs Au oxide (curve species b of [Figure59,60], implying11b), which that was the in cyclic rough voltammogram agreement with of thepreviously Au microelectrode published results site in H[35,37].2SO4 matched The average the classicimpedance electrochemical of a typical responsestimulating of bulksite with Au indiameter H2SO4. of These 100 μ resultsm reduced indicated from thatabout thin 200 film KΩ Au (curve microelectrode a of Figure sites 11c) possessed to about the 80 sameKΩ (curve electrochemical a of Figure properties 11c) after as bulkAuNPs Au were and coulddeposited be reasonably on its surface. expected These to possessresults implied the same that recording AuNPs capabilities could greatly astraditional increase electron Au microwires transfer usedrates forand neural surface recording. roughness In addition, of electrode the cyclic site, voltammetric reduce interfacial responses impedances of stimulating and sites enhance in H2SO the4 possessedperformance the of same stimulat propertiesing/recording as those sites. of recording sites.

Figure 11. (a) Representative cyclic voltammogram of a recording site immersed in 0.5 M H2SO4; (b) Figure 11. (a) Representative cyclic voltammogram of a recording site immersed in 0.5 M H2SO4; impedances of a recording site (curve a, b) and a stimulating site (curve c, d). (b) impedances of a recording site (curve a, b) and a stimulating site (curve c, d).

The stimulating and recording sites modified with AuNPs were further characterized by The stimulating and recording sites were further electrochemically characterized by measuring cyclic voltammograms in 0.1 M KCl solution containing 2 mM [Fe(CN)6]3−/4− and the electrochemicalresults were shown impedance in Figure spectroscopy 12. As shown (EIS) to in measure Figure the 12, electrode the absolute impedances values inof 0.1 reduction M PBS using and electrochemicaloxidation peak analyzer currents and were the resultsalmost wereequal, presented and the in peak Figure potentials 11b. For awere typical definite recording values site withand µ diameterindependent of 20 of them (curve scan rates, a of Figurewhich 11indicatedb) , the averagethat the measuredelectrochemical impedance processes was of about microelectrode 4.1 MΩ at 1 kHz and the average impedance was reduced to about 0.9 MΩ after electrodeposition of AuNPs sites in 0.1 M KCl solution containing 2 mM [Fe(CN)6]3−/4− were reversible. For stimulating site (curve(Figure b 12a), of Figure the values 11b), of which the peak was currents in rough increa agreementsed with with scan previously rates varying published from 20 results mV/s [ 35to, 37200]. µ ThemV/s, average and reduction impedance and of oxidation a typical peak stimulating currents site of withthe stimulating diameter of sites 100 werem reduced proportional from aboutto the 200square KΩ (curveroot of a ofthe Figure scan 11ratesc) to aboutin the 80 range KΩ (curve 20–200 a of mV/s Figure through 11c) after analysis, AuNPs weresuggesting deposited the onelectrochemical its surface. These responses results were implied a typical that AuNPs surface- couldcontrolled greatly process increase [61]. electron For transferthe recoding rates andsite surface(Figure roughness12b), the cyclic of electrode voltammograms site, reduce a interfacialtypical showed impedances a typical and sigmoidal enhance thepattern performance due to the of stimulating/recordingsmall area of the recording sites. sites. The increase of peak currents was quite small with the increase of scanThe rates stimulating in Figure 12b and due recording to the small sites size modified and edge with effect AuNPs of the recording were further sites [62]. characterized These results by 3´/4´ measuringdemonstrated cyclic that voltammograms the fabricated microelectrode in 0.1 M KCl solution array possessed containing good 2 mM electrochemical [Fe(CN)6] propertiesand the resultsand stability. were shown In addition, in Figure inset 12. Asof Figure shown 12a in Figure was the12, thecyclic absolute voltammogram values of reduction of the bare and stimulating oxidation peak currents were almost equal, and the peak potentials were definite values and independent of site in 2 mM [Fe(CN)6]3−/4− at scan rate of 100 mV/s, and the absolute values of reduction and the scan rates, which indicated that the electrochemical processes of microelectrode sites in 0.1 M KCl 3´/4´ solution containing 2 mM [Fe(CN)6] were reversible. For stimulating site (Figure 12a), the values of the peak currents increased with scan rates varying from 20 mV/s to 200 mV/s, and reduction and oxidation peak currents of the stimulating sites were proportional to the square root of the scan rates in the range 20–200 mV/s through analysis, suggesting the electrochemical responses were a typical surface-controlled process [61]. For the recoding site (Figure 12b), the cyclic voltammograms a typical showed a typical sigmoidal pattern due to the small area of the recording sites. The increase of peak currents was quite small with the increase of scan rates in Figure 12b due to the small size and edge effect of the recording sites [62]. These results demonstrated that the fabricated microelectrode array Sensors 2016, 16, 880 11 of 16

possessedSensors 2016, good16, 880 electrochemical properties and stability. In addition, inset of Figure 12a was the11 cyclic of 16 3´/4´ voltammogram of the bare stimulating site in 2 mM [Fe(CN)6] at scan rate of 100 mV/s, and the absoluteoxidation values peak currents of reduction of stimulating and oxidation site modified peak currents with AuNPs of stimulating (curve c site of Figure modified 12a, with scan AuNPs rate of (curve100 mV/s) c of were Figure much 12a, scanhigher rate than of 100those mV/s) of the were bare muchstimulating higher site than (inset those of of Figure the bare 12a) stimulating under the sitesame (inset experimental of Figure 12conditions,a) under the showing same experimental that AuNPs conditions,could greatly showing increase that electron AuNPs transfer could greatly rates increaseand surface electron roughness transfer of rates the andelectrode surface site. roughness The same of theconclusion electrode can site. be The reached same conclusionfrom the inset can bein reachedFigure 12b. from the inset in Figure 12b.

Figure 12. Cyclic voltammograms of the stimulating site modifiedmodified with AuNPs (a) and recording sitesite 6 3−3/4´− /4´ modifiedmodified with AuNPs ( b) in in 0.1 0.1 M M KCl KCl solution solution containing containing 2 2 mM mM [Fe(CN) [Fe(CN)]6] . Scan. Scan rate: rate: 20, 50, 20, 100, 50, 100,150 and 150 and200 mV/s 200 mV/s (from (from a to e). a to Inset e). Insetof Figure of Figure 12a: 12cya:clic cyclic voltammogram voltammogram of bare of barestimulating stimulating site in site 2 3−/4− 3´/4´ inmM 2 mM[Fe(CN) [Fe(CN)6] 6] at scanat scanrate rateof 100 of 100mV/s. mV/s. Inset Inset of ofFigure Figure 12b: 12 b:cyclic cyclic voltammogram voltammogram of of bare 3−´/4/4− ´ recordingrecording sitesite inin 22 mMmM [Fe(CN)[Fe(CN)66] at scanat scan rate rate of 100 of 100 mV/s. mV/s.

3.4. In Vivo Neural Recordings InIn vivovivo recordings in the the brain brain of of the the anesthetized anesthetized ra ratsts were were performed performed to toconfirm confirm the the ability ability to torecord record spontaneous spontaneous discharge discharge signals signals from from multiple multiple nuclei nuclei at at the the fabricated fabricated microelectrode microelectrode array. array. Male Sprague-Dawley (SD) rats weighing 250–300 g (Laboratory(Laboratory Animal Center at Xi’anXi’an JiaotongJiaotong University School ofof Medicine)Medicine) werewere usedused inin thethe electrophysiologicalelectrophysiological experiments.experiments. All animalanimal experiments were conducted in accordance with protocols approved by the animal ethics committee of Xi’an Jiaotong University. The SD rats were anesthetized using an intraperitoneal injection of 1% pelltobarbitalum natricum (3 mg/100 g). After craniotomy and resection of the dura mater, the cortical surface was exposed. The microelectrode array was obliquely inserted into the rat brain at an angle of 72 degree with the horizontal plane from the pia mater (coordinates: 0 mm posterior and 2.5

Sensors 2016, 16, 880 12 of 16 of Xi’an Jiaotong University. The SD rats were anesthetized using an intraperitoneal injection of Sensors1% pelltobarbitalum 2016, 16, 880 natricum (3 mg/100 g). After craniotomy and resection of the dura mater,12 of the 16 cortical surface was exposed. The microelectrode array was obliquely inserted into the rat brain at mman angle lateral of to 72 Bregma degree and with 0 mm the horizontalsubdural) by plane a micromanipulator from the pia mater with (coordinates: a constant speed 0 mm of posterior 10 μm/s andand 2.5an mminsertion lateral depth to Bregma of 8 mm. and 0Neural mm subdural) signals from by a micromanipulatorthe brain were recorded with a constantusing a Cerebus speed of multi-channel10 µm/s and an data insertion acquisition depth ofsystem 8 mm. (Blackrock Neural signals Microsystems, from the brain Salt were Lake recorded city, UT, using USA). a Cerebus The originalmulti-channel neural data signals acquisition were amplified, system (Blackrock filtered by Microsystems, a bandpass Saltranging Lake from city, UT,1 Hz USA). to 7.5 The kHz original and digitizedneural signals (sampling were rate: amplified, 30 kHz) filtered for offline by aanalysis. bandpass ranging from 1 Hz to 7.5 kHz and digitized (samplingThe obtained rate: 30 original kHz) for neural offline signals analysis. were filtered by a bandpass from 500 Hz to 5 kHz to get the multipleThe obtainedunit activity original (MUA) neural of signalsnervous were nucl filteredei. Figure by a13 bandpass showed from the 500simultaneously Hz to 5 kHz recorded to get the MUAmultiple from unit striatum, activity GPi (MUA) and of STN, nervous which nuclei. substantia Figureted 13 theshowed ability the of simultaneously the fabricated microelectrode recorded MUA arrayfrom striatum,to simultaneously GPi and STN,record which spontaneous substantiated discharge the ability signals of from the fabricated multiple microelectrodenuclei. In addition, array the to averagesimultaneously -to-noise record spontaneousratio (SNR) of discharge the neural signals signals from was multiplecalculated. nuclei. The SNR In addition, of the neural the average signal wassignal-to-noise defined as ratiothe (SNR)average of peak-to-peak the neural signals amplitude was calculated. of spikes Theto the SNR root of themean neural square signal of wasthe backgrounddefined as the noise, average and peak-to-peakthe SNR of neural amplitude signals of from spikes the to striatum, the root mean GPi and square STN of was the background3.6, 6.3 and 4.7,noise, respectively. and the SNR of neural signals from the striatum, GPi and STN was 3.6, 6.3 and 4.7, respectively.

FigureFigure 13. 13. SimultaneouslySimultaneously recorded recorded MUA MUA from from striatum striatum ( (aa);); GPi GPi ( (b)) and and STN STN ( (c).).

4.4. Conclusions Conclusions InIn this this paper, the design, simulation, fabrication fabrication and and characterization characterization of of a a novel dual-sided microelectrodemicroelectrode array array have been reported. The The mi microelectrodecroelectrode array array was was specially specially designed designed and fabricatedfabricated forfor a rata Parkinson’srat Parkinson’s disease disease model tomodel stimulate to thestimulate subthalamic the subthalamic nucleus and simultaneouslynucleus and simultaneouslyrecord electrophysiological record electrophysiological signals from multiple signals nuclei from multiple of the basal nuclei ganglia of the system. basal ganglia The package system. of Thethe dual-sidedpackage of microelectrodethe dual-sided arraymicroelectrode combined goldarray wire combined bonding gold technology wire bonding and flip-chip technology bonding and flip-chiptechnology bonding for the technology first time. Infor orderthe first to enhance time. In the order stimulating/recording to enhance the stimulating/recording functionality of the functionality of the microelectrode, the impedances of the stimulating/recording sites were reduced by electroplating gold nanoparticles on the thin film gold electrode sites. SEM revealed that the stimulating/recording sites with accurate shapes and clear boundaries were well etched by reactive ion etching technology. In vivo neural recording experiments indicated that the designed and fabricated microelectrode array could successfully produce multiple-unit electrical activity from the

Sensors 2016, 16, 880 13 of 16 microelectrode, the impedances of the stimulating/recording sites were reduced by electroplating gold nanoparticles on the thin film gold electrode sites. SEM revealed that the stimulating/recording sites with accurate shapes and clear boundaries were well etched by reactive ion etching technology. In vivo neural recording experiments indicated that the designed and fabricated microelectrode array could successfully produce multiple-unit electrical activity from the striatum, GPi and STN simultaneously, demonstrating its potential application for studying the mechanisms of deep brain stimulation in rat models. Future work should concentrate first and foremost on integration of additional functionalities beyond stimulation and recording, such as integration of biosensors on the microelectrode array shaft [63], which can measure changes in the concentration of neurotransmitters when stimulating brain nucleus.

Acknowledgments: This work was financially supported by the National Natural Science Foundation of China (Grant No. 61271088, 61431012). Author Contributions: Jue Wang guided the experiments. Hongen Huang and Ruxue Gong performed the in vivo neural recording experiments. Zongya Zhao carried out the majority of the experiments, including the fabrication and electrochemical measurements of the obtained microelectrode array, and paper writing. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Hagai Bergman, M.D.; Günther, M.D. Deuschl Pathophysiology of Parkinson’s disease: From clinical neurology to basic neuroscience and back. Mov. Disord. 2002, 17, S28–S40. [CrossRef][PubMed] 2. Narabayashi, H. The neural mechanisms and progressive nature of symptoms of Parkinson’s disease—Based on clinical, neurophysiological and morphological studies. J. Neural Transm. Parkinson’s Dis. Dement. Sect. 1995, 10, 63–75. [CrossRef] 3. Adrian, W.; Steven, G.; Thelekat, V.; Crispin, J.; Niall, Q.; Rosalind, M.; Richard, S.; Natalie, I.; Caroline, R. Jane Deep brain stimulation plus best medical therapy versus best medical therapy alone for advanced Parkinson’s disease (PD SURG trial): A randomised, open-label trial. Lancet Neurol. 2010, 9, 581–591. 4. Okun, M.S.; Gallo, B.V.; Mandybur, G.; Jagid, J.; Foote, K.D.; Revilla, F.J.; Alterman, R.; Jankovic, J.; Simpson, R. Junn Subthalamic deep brain stimulation with a constant-current device in Parkinson’s disease: An open-label randomised controlled trial. Lancet Neurol. 2012, 11, 140–149. [CrossRef] 5. Limousin-Dowsey, P.; Pollak, P.; Blercom, N.V.; Krack, P.; Benazzouz, A.; Benabid, A.L. Thalamic, subthalamic nucleus and internal pallidum stimulation in Parkinson’s disease. J. Neurol. 1999, 246, II42–II45. [CrossRef] [PubMed] 6. Myrandele, D.; Davis, T.L.; Konrad, P.E.; Roberts, A.G.; Pfister, A.A.; David, C.P. Deep brain stimulation: A new treatment for Parkinson’s disease. Tenn. Med. 2003, 96, 33–35. 7. Deep-Brain Stimulation for Parkinson’s Disease Study Group. Deep-brain stimulation of the subthalamic nucleus or the pars interna of the globus pallidus in Parkinson’s disease. N. Engl. J. Med. 2001, 345, 956–963. 8. Koop, M.M.; Andrzejewski, A.; Hill, B.C.; Heit, G.; Bronte-Stewart, H.M. Improvement in a quantitative measure of bradykinesia after microelectrode recording in patients with Parkinson’s disease during deep brain stimulation surgery. Mov. Disord. 2006, 21, 673–678. [CrossRef][PubMed] 9. Etienne, P.; Damien, D.; Malin, M.; Francois, V.; Josephs, G.; Jean-Guy, V. Effect of deep brain stimulation of GPI on neuronal activity of the thalamic nucleus ventralis oralis in a dystonic patient. Neurophysiol. Clin. 2003, 33, 169–173. 10. Galati, S.; Mazzone, P.; Fedele, E.; Pisani, A.; Peppe, A.; Pierantozzi, M.; Brusa, L.; Tropepi, D.; Moschella, V.; Raiteri, M. Biochemical and electrophysiological changes of substantia nigra pars reticulata driven by subthalamic stimulation in patients with Parkinson’s disease. Eur. J. Neurosci. 2006, 23, 2923–2928. [CrossRef] [PubMed] 11. Montgomery, E.B. Effects of GPi stimulation on human thalamic neuronal activity. Clin. Neurophysiol. 2006, 117, 2691–2702. [CrossRef][PubMed] 12. Takao, H.; Elder, C.M.; Okun, M.S.; Patrick, S.K.; Vitek, J.L. Stimulation of the subthalamic nucleus changes the firing pattern of pallidal neurons. J. Neurosci. 2003, 23, 1916–1923. Sensors 2016, 16, 880 14 of 16

13. Hitoshi, K.; Yoshihisa, T.; Atsushi, N.; Satomi, C. Balance of monosynaptic excitatory and disynaptic inhibitory responses of the globus pallidus induced after stimulation of the subthalamic nucleus in the monkey. J. Neurosci. 2005, 25, 585–594. 14. Weidong, X.; Russo, G.S.; Takao, H.; Jianyu, Z.; Vitek, J.L. Subthalamic nucleus stimulation modulates thalamic neuronal activity. J. Neurosci. 2008, 28, 11916–11924. 15. Johnson, M.D.; Vitek, J.L.; Mcintyre, C.C. Pallidal stimulation that improves parkinsonian motor symptoms also modulates neuronal firing patterns in primary motor cortex in the MPTP-treated monkey. Exp. Neurol. 2009, 219, 359–362. [CrossRef][PubMed] 16. Kopell, B.H.; Rezai, A.R.; Jin, W.C.; Vitek, J.L. Anatomy and physiology of the basal ganglia: Implications for deep brain stimulation for Parkinson’s disease. Mov. Disord. 2006, 21, S238–S246. [CrossRef][PubMed] 17. Delong, M.; Wichmann, T. Circuits and circuit disorders of the basal ganglia. Arch. Neurol. 2007, 64, 20–24. [CrossRef][PubMed] 18. Parent, A.; Hazrati, L.N. Functional anatomy of the basal ganglia. I. The cortico-basal ganglia-thalamo-cortical loop. Brain Res. Rev. 1995, 20, 91–127. [CrossRef] 19. André, P.; Lili-Naz, H. Functional anatomy of the basal ganglia. II. The place of subthalamic nucleus and external pallidium in basal ganglia circuitry. Brain Res. Rev. 1995, 20, 128–154. 20. Mcintyre, C.C.; Hahn, P.J. Network perspectives on the mechanisms of deep brain stimulation. Neurobiol. Dis. 2009, 38, 329–337. [CrossRef][PubMed] 21. Shi, L.H.; Luo, F.; Woodward, D.J.; Chang, J.Y. Basal ganglia neural responses during behaviorally effective deep brain stimulation of the subthalamic nucleus in rats performing a treadmill locomotion test. Synapse 2006, 59, 445–457. [CrossRef][PubMed] 22. Fiáth, R.; Grand, L.; Kerekes, B.; Pongrácz, A.; Vázsonyi, É.; Márton, G.; Battistig, G.; Ulbert, I. A novel multisite silicon probe for laminar neural recordings. Procedia Comput. Sci. 2011, 7, 310–311. [CrossRef] 23. Cheung, K.C. Implantable microscale neural interfaces. Biomed. Microdevices 2008, 9, 923–938. [CrossRef] [PubMed] 24. Norlin, P.; Kindlundh, M.; Mouroux, A.; Yoshida, K.; Hofmann, U.G. A 32-site neural recording probe fabricated by DRIE of SOI substrates. J. Micromech. Microeng. 2002, 12, 414–419. [CrossRef] 25. Ruther, P.; Herwik, S.; Kisban, S.; Seidl, K.; Paul, O. Recent Progress in Neural Probes Using Silicon MEMS Technology. IEEJ Trans. Electr. Electron. 2010, 5, 505–515. [CrossRef] 26. Wise, K.D. Silicon microsystems for neuroscience and neural prostheses. IEEE Eng. Med. Biol. Mag. 2005, 24, 22–29. [CrossRef][PubMed] 27. Wise, K.D.; Angell, J.B.; Starr, A. An Integrated-Circuit Approach to Extracellular Microelectrodes. IEEE Trans. Biomed. Eng. 1970, BME-17, 238–247. [CrossRef] 28. Bai, Q.; Wise, K.D.; Anderson, D.J. A high-yield microassembly structure for three-dimensional microelectrode arrays. IEEE Trans. Biomed. Eng. 2000, 47, 281–289. [PubMed] 29. Yao, Y.; Gulari, M.N.; Wiler, J.A.; Wise, K.D. A Microassembled Low-Profile Three-Dimensional Microelectrode Array for Neural Applications. J. Microelectromech. Syst. 2007, 16, 977–988. [CrossRef] 30. Rousche, P.J.; Normann, R.A. Chronic recording capability of the Utah intracortical electrode array in cat sensory cortex. J. Neurosci. Methods 1998, 82, 1–15. [CrossRef] 31. Maynard, E.M.; Nordhausen, C.T.; Normann, R.A. The Utah Intracortical Electrode Array: A recording structure for potential brain-computer interfaces. Electroencephalogr. Clin. Neurophysiol. 1997, 102, 228–239. [CrossRef] 32. Campbell, P.K.; Jones, K.E.; Huber, R.J.; Horch, K.W.; Normann, R.A. A silicon-based, three-dimensional neural interface: Manufacturing processes for an intracortical electrode array. IEEE Trans. Biomed. Eng. 1991, 38, 758–768. [CrossRef][PubMed] 33. Fekete, Z. Recent advances in silicon-based neural microelectrodes and microsystems: A review. Sens. Actuators B Chem. 2015, 215, 300–315. [CrossRef] 34. Aarts, A.A.A.; Neves, H.P.; Puers, R.P.; Hoof, C.V. An interconnect for out-of-plane assembled biomedical probe arrays. J. Micromech. Microeng. 2008, 18, 777–786. [CrossRef] 35. Du, J.; Roukes, M.L.; Masmanidis, S.C. Dual-side and three-dimensional microelectrode arrays fabricated from ultra-thin silicon substrates. J. Micromech. Microeng. 2009, 19, 1403–1407. [CrossRef] Sensors 2016, 16, 880 15 of 16

36. Lee, Y.T.; Moser, D.; Holzhammer, T.; Fang, W.; Paul, O.; Ruther, P. Ultrathin, dual-sided silicon neural microprobes realized using BCB bonding and aluminum sacrificial etching. In Proceedings of the IEEE 26th International Conference on Micro Electro Mechanical Systems (MEMS), Taipei, Taiwan, 20–24 January 2013. 37. Du, J.; Blanche, T.J.; Harrison, R.R.; Lester, H.A.; Masmanidis, S.C. Multiplexed, high density with nanofabricated neural probes. PLoS ONE 2011, 6, e26204. [CrossRef][PubMed] 38. Tanghe, S.J.; Wise, K.D. A 16-channel CMOS neural stimulating array. IEEE J. Solid-State Circuits 1992, 27, 1819–1825. [CrossRef] 39. Seidl, K.; Lemke, B.; Ramirez, H.; Herwik, S. CMOS-based high-density silicon microprobe for stress mapping in intracortical applications. In Proceedings of the 23rd IEEE International Conference on Micro Electro Mechanical Systems, Hong Kong, China, 24–28 January 2010; Voulme 33, pp. 35–38. 40. Chen, J.; Wise, K.D.; Hetke, J.F.; Bledsoe, S.C. A multichannel neural probe for selective chemical delivery at the cellular level. IEEE Trans. Biomed. Eng. 1997, 44, 760–769. [CrossRef][PubMed] 41. Seidl, K.; Spieth, S.; Herwik, S.; Steigert, J.; Zengerle, R.; Paul, O.; Ruther, P. In-plane silicon probes for simultaneous neural recording and drug delivery. J. Micromech. Microeng. 2010, 20, 105006–105011. [CrossRef] 42. Pongrácz, A.; Fekete, Z.; Márton, G.; Bérces, Z.; Ulbert, I.; Fürjes, P. Deep-brain silicon multielectrodes for simultaneous in vivo neural recording and drug delivery. Sens. Actuators B Chem. 2013, 189, 97–105. [CrossRef] 43. Fekete, Z.; Pálfi, E.; Márton, G.; Handbauer, M.; Bérces, Z.; Ulbert, I.; Pongrácz, A.; Négyessy, L. Combined in vivo recording of neural signals and iontophoretic injection of pathway tracers using a hollow silicon microelectrode. Sens. Actuators B Chem. 2016.[CrossRef] 44. Wu, F.; Stark, E.; Ku, P.C.; Wise, K.D.; Buzsáki, G.; Yoon, E. Monolithically Integrated µLEDs on Silicon Neural Probes for High-Resolution Optogenetic Studies in Behaving Animals. 2015, 88, 1136–1148. [CrossRef][PubMed] 45. Kiss, M.; Földesy, P.; Fekete, Z. Optimization of a Michigan-type silicon microprobe for infrared neural stimulation. Sens. Actuators B Chem. 2015, 224, 676–682. [CrossRef] 46. Xu, H.; Weltman, A.; Hsiao, M.C.; Scholten, K.; Meng, E.; Berger, T.W.; Song, D. Design of a flexible parylene-based multi-electrode array for multi-region recording from the rat hippocampus. IEEE Eng. Med. Biol. 2015, 2015, 7139–7142. 47. Márton, G.; Baracskay, P.; Cseri, B.; Plósz, B.; Juhász, G.; Fekete, Z.; Pongrácz, A. A silicon-based microelectrode array with a microdrive for monitoring brainstem regions of freely moving rats. J. Neural Eng. 2016, 13, 026025. [CrossRef][PubMed] 48. Lozano, A.M.; Dostrovsky, J.; Chen, R.; Ashby, P. Deep brain stimulation for Parkinson’s disease: Disrupting the disruption. Lancet Neurol. 2002, 1, 225–231. [CrossRef] 49. Johnson, M.D.; Franklin, R.K.; Gibson, M.D.; Brown, R.B.; Kipke, D.R. Implantable microelectrode arrays for simultaneous electrophysiological and neurochemical recordings. J. Neurosci. Methods 2008, 174, 62–70. [CrossRef][PubMed] 50. Frey, O.; Holtzman, T.; Mcnamara, R.M.; Theobald, D.E.H.; van der Wal, P.D.; de Rooij, N.F.; Dalley, J.W.; Koudelka-Hep, M. Simultaneous neurochemical stimulation and recording using an assembly of biosensor silicon microprobes and SU-8 microinjectors. Sens. Actuators B Chem. 2011, 154, 96–105. [CrossRef] 51. Mcintyre, C.C.; Mori, S.; Sherman, D.L.; Thakor, N.V.; Vitek, J.L. Electric field and stimulating influence generated by deep brain stimulation of the subthalamic nucleus. Clin. Neurophysiol. 2004, 115, 589–595. [CrossRef][PubMed] 52. Butson, C.R.; Mcintyre, C.C. Role of electrode design on the volume of tissue activated during deep brain stimulation. J. Neural Eng. 2006, 3, 1–8. [CrossRef][PubMed] 53. Yousif, N.; Bayford, R.; Liu, X. The influence of reactivity of the electrode-brain interface on the crossing electric current in therapeutic deep brain stimulation. Neuroscience 2008, 156, 597–606. [CrossRef][PubMed] 54. Geddes, L.A.; Baker, L.E. The specific resistance of biological material—A compendium of data for the biomedical engineer and physiologist. Med. Biol. Eng. Comput. 1967, 5, 271–293. [CrossRef] 55. Li, S.J.; Deng, D.H.; Shi, Q.; Liu, S.R. Electrochemical synthesis of a graphene sheet and gold nanoparticle-based nanocomposite, and its application to amperometric sensing of dopamine. Microchim. Acta 2012, 177, 325–331. [CrossRef] Sensors 2016, 16, 880 16 of 16

56. Yong, H.K.; Kim, A.Y.; Kim, G.H.; Han, Y.H.; Chung, M.A.; Jung, S.D. Electrochemical and in vitro neuronal recording characteristics of multi-electrode arrays surface-modified with electro-co-deposited gold-platinum nanoparticles. Biomed. Microdevices 2016, 18, 1–7. 57. Butson, C.R.; Maks, C.B.; Mcintyre, C.C. Sources and effects of electrode impedance during deep brain stimulation. Clin. Neurophysiol. 2006, 117, 447–454. [CrossRef][PubMed] 58. Butson, C.R.; Cooper, S.E.; Henderson, J.M.; Mcintyre, C.C. Patient-specific analysis of the volume of tissue activated during deep brain stimulation. Neuroimage 2007, 34, 661–670. [CrossRef][PubMed] 59. Shahrokhian, S.; Rastgar, S. Construction of an electrochemical sensor based on the electrodeposition of Au-Pt nanoparticles mixtures on multi-walled carbon nanotubes film for voltammetric determination of cefotaxime. Analyst 2012, 137, 2706–2715. [CrossRef][PubMed] 60. Zhao, Z.; Zhang, M.; Chen, X.; Li, Y.; Wang, J. Electrochemical Co-Reduction Synthesis of AuPt Bimetallic Nanoparticles-Graphene Nanocomposites for Selective Detection of Dopamine in the Presence of Ascorbic Acid and Uric Acid. Sensors 2015, 15, 16614–16631. [CrossRef][PubMed]

61. Fang, B.; Wang, G.; Zhang, W.; Li, M.; Kan, X. Fabrication of Fe3O4 Nanoparticles Modified Electrode and Its Application for Voltammetric Sensing of Dopamine. Electroanal 2005, 17, 744–748. [CrossRef] 62. Qin, G.; Liu, Y.; Liu, H.; Ding, Y.; Qi, X.; Du, R. Fabrication of bio-microelectrodes for deep-brain stimulation using microfabrication and electroplating process. Microsyst. Technol. 2009, 15, 933–939. [CrossRef] 63. Suzuki, I.; Mao, F.; Shirakawa, K.; Jiko, H.; Gotoh, M. multi-electrode array chips for noninvasive real-time measurement of dopamine, action potentials, and postsynaptic potentials. Biosens. Bioelectron. 2013, 49, 270–275. [CrossRef][PubMed]

© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).