sensors Article

Design, Fabrication, Simulation and Characterization of a Novel Dual-Sided Microelectrode Array for Deep Brain Recording and Stimulation Zongya Zhao 1,2 , Ruxue Gong 1,2 , Hongen Huang 1,2 and Jue Wang 1,2, * 1

2

*

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.) 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 deep brain stimulation (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 electron 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 signals 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 neurons 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 steel 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 silicon-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 hippocampus 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 880 (EIS) 2016, and 16, other

3 of 16 electrochemical methods. In addition, the stimulating effects of the fabricated 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. Materials 2. Materials and and Methods Methods 2.1. Microelectrode Microelectrode Arrays Arrays Design Design and andFabrication Fabrication 2.1. The microelectrode microelectrode arrays arrays were were designed designed according according to to the the size size of of rat rat brain brain and and relative relative The positional relationship relationship of of the the STN, STN, GPi GPi and and striatum striatum of of BGS BGS (Figure (Figure 1), 1), and and our our purpose purpose was was to to positional simultaneously record electrophysiological information from the striatum, GPi and STN of BGS. simultaneously record electrophysiological information from the striatum, GPi and STN of BGS. Figure22shows showsthe thedetailed detaileddesign designparameters. parameters.The Thedesigned designed microelectrode array with dimensions Figure microelectrode array with dimensions of 9 mm × µm 400 ˆμm 200(l μm w × t) employed 14 round-shaped gold microelectrode sites 9ofmm ˆ 400 200×µm ˆ w (l ˆ ×t) employed 14 round-shaped gold microelectrode sites distributed distributed each side. These sites could be divided intostimulating three types: three stimulating sites on each side. on These sites could be divided into three types: three sites (diameter of 100 µm), (diameter of 100 μm), seven electrophysiological recording sites 20 μm) and four seven electrophysiological recording sites (diameter of 20 µm) and(diameter four sites of with diameter of 50sites µm with for diameter of 50 μm used for other experimental the 50 future. For could example, these 50 used other experimental purposes in the future. Forpurposes example, in these µm sites be modified μm sites could be modified with theforion-exchange Nafion for detection of [49], neurotransmitter with the ion-exchange resin Nafion detection of resin neurotransmitter dopamine or modified dopamine [49], or modified with aL-glutamate protein matrix layer containing L-glutamate oxidase as with a protein matrix layer containing oxidase as glutamate sensors [50]. At the front of glutamate sensors [50]. At the front of the designed microelectrode, three stimulating sites and three the designed microelectrode, three stimulating sites and three recording sites were mutually spaced recording were mutually spaced equal center-to-center distance of 250 The distance with equalsites center-to-center distance ofwith 250 µm. The distance between the tip and μm. the center of the between the tip and the center of the first recording site was 300 μm, by with the fabrication first recording site was 300 µm, defined by the fabrication process. Thesedefined four sites diameter process. fourrecording sites with sites diameter 50 μm and four recording sites were also mutually spaced of 50 µmThese and four wereofalso mutually spaced with equal center-to-center distance with center-to-center distance of 250 μm.connected Each recording site waspad electrically connected of 250equal µm. Each recording site was electrically to a bonding (400 µm ˆ 400 µm) to viaa pad (400 μm × 400 paths μm) via 5 μm-wide gold conductive 5 μm-wide gaps between 5bonding µm-wide gold conductive with 5 µm-wide gaps between paths them, with and each stimulating site was them, and connected each stimulating site was electrically connected a bondingpaths pad with via 10 μm-wide gaps gold electrically to a bonding pad via 10 µm-wide gold to conductive 5 µm-wide conductive paths with 5 μm-wide gaps between them. between them.

Figure 1.1. Schematic Schematic diagram diagram of of median median sagittal sagittal plane plane of of Sprague-Dawley Sprague-Dawley rat rat brains brains showing showing the the Figure relative positional relationship of the STN, GPi and striatum (coordinate unit: mm). relative positional relationship of the STN, GPi and striatum (coordinate unit: mm).

Sensors 2016, 16, 880

4 of 16

Sensors 2016, 16, 880 Sensors 2016, 16, 880

4 of 16

4 of 16

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) Figure3.3.Schematic Schematicdiagram diagram of of the the process process flow: (a) oxidation; (b)(b) spinning photoresist and Figure flow:layer; (a)thermal thermal oxidation; spinning photoresist spinning photoresist for patterning passivation (h) exposing electrode sites and bonding pads; and patterning by lithography; (c) deposition of adhesion layer Cr; (d) deposition of Au layer; (e) lifting patterning by lithography; (c) DRIE; deposition of adhesion Cr; (d) deposition of Au layer; (e) lifting off (i) spinning photoresist for (j) DRIE and devicelayer release.

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 for passivation layer; (h) exposing electrode sites and bonding pads;The Figurephotoresist 3 showed thepatterning main fabrication forelectrode a silicon-based array. photoresist for patterning passivation layer; (h)process exposing sites andmicroelectrode bonding pads; (i) spinning (i) spinning photoresist for DRIE; (j) DRIE and device release. substrate was of about 100 mm diameter, double-side polished, p-doped (1 0 0) photoresist fortypically DRIE; (j)comprised DRIE and device release. silicon wafers with a thickness of 200 μm. In the first step, the substrate was thermally oxidized at Figure showed theμm main fabrication process for a silicon-based microelectrode array. The 1150 °C to3 yield a 1.5 silicon dioxide (SiO 2) on both sides as an insulating layer (Figure 3a). Figure 3 showed the main fabrication process for adouble-side silicon-based microelectrode substrate wasa typically comprised of about 100 mm diameter, 0array. 0) Secondly, 5 μm-thick negative tone photoresist (nLOF-2035, AZ Electronicpolished, Materials)p-doped was spun(1on The substrate was typically comprised of about 100 mm diameter, double-side polished, p-doped silicon with thickness of 200layer μm. comprising In the first the step, the substrate was thermally wires oxidized and wafers patterned to adefine the metal electrode sites, interconnecting and(1at0 0) silicon wafers with thickness of 200 µm. the first step,sides the was thermally oxidized 1150 °C to yield a a1.5 μm silicon dioxide (SiO 2) on both as an insulating layer chromium (Figure 3a). at bonding pads by lithography (Figure 3b). In This was followed by substrate sputtering 20 nm-thick ˝ C to yield 1150 a 1.5 µmlayer silicon dioxide (SiO ) on both sides as nm-thick an (Figure 3a). Secondly, Secondly, μm-thick negative tone3c) photoresist (nLOF-2035, AZinsulating Electronic Materials) was spun (Cr) as aan5 adhesion (Figure and 2evaporating a 300 gold layer (Au) layer (Figure 3d)on patterned toThe define the metal layer comprising the electrode sites, metal interconnecting wires and a 5and µm-thick negative tone photoresist (nLOF-2035, AZoff Electronic Materials) wasand spun on and patterned sequentially. lift-off process was used to lift the unpatterned photoresist by pads by lithography (Figure 3b). This was followed by sputtering 20 nm-thick chromium dissolving the photoresist in an amine-solvent mixture (Figure 3e). Thirdly, the subsequent 2 by tobonding define the metal layer comprising the electrode sites, interconnecting wires and bonding pads SiO2 passivation layer was made from plasma-enhanced chemical deposition (Cr)μm-thick as an adhesion layer (Figure 3c) and evaporating a 300 nm-thick chromium gold (Au)vapor layer (Figure 3d) lithography (Figure 3b). This was followed by sputtering 20 nm-thick (Cr) as an adhesion (PECVD) (Figure 3f). This passivation layer was patterned reactive through sequentially. The process used to lift off(Au) the using unpatterned metal and (RIE) photoresist by layer (Figure 3c) andlift-off evaporating awas 300 nm-thick gold layer (Figureion 3d)etching sequentially. The lift-off

dissolving the photoresist in unpatterned an amine-solvent (Figure 3e). thethe subsequent 2 in process was used to lift off the metalmixture and photoresist by Thirdly, dissolving photoresist μm-thick SiO 2 passivation layer was made from plasma-enhanced chemical vapor deposition an amine-solvent mixture (Figure 3e). Thirdly, the subsequent 2 µm-thick SiO2 passivation layer was (PECVD) (Figure 3f). This passivation layer was patterned using reactive ion etching (RIE) through made from plasma-enhanced chemical vapor deposition (PECVD) (Figure 3f). This passivation layer

Sensors 2016, 16, 880

5 of 16

Sensors 2016, 16, 880 as the photoresist

16 a hard mask (Figure 3g) to open electrode sites and bonding pads (Figure5 of 3h). After the process on the front side, the wafer was turned over to repeat the above steps in the back. Afterpatterned that, additional twice lithography, left-off and RIE definedas the dimensions of probes to was using reactive ion etching (RIE) through thesteps photoresist a hard mask 5(Figure 3g) to Sensors 2016, 16, 880 of 16 realize the outline from the front and back surface of wafer by removing SiO 2 outside the probe. open electrode sites and bonding pads (Figure 3h). After the process on the front side, the wafer was Finally,over a the 10tophotoresist μm-thick photoresist (AZ-9260, AZ Electronic Materials) was pads applied to3h). serve asand the as a hard mask (Figure to open electrode sites and bonding (Figure turned repeat the above steps in the3g) back. After that, additional twice lithography, left-off After the(Figure process on theand frontdeep side, the wafer was turned over (DRIE) to repeat the above steps in the back.the probes etch mask layer 3i) reactive ion etching was used to release RIE steps defined the dimensions of probes to realize the outline from the front and back surface of After that, additional twice lithography, left-off and RIE steps defined the dimensions of probes to from the wafer (Figure 3j). Thethe overview of an entire silicon wafer with the probes and frames is wafer by removing probe. Finally, a 10 µm-thick photoresist (AZ-9260, AZ Electronic realize the SiO outline from the front and back surface of wafer by removing SiO2 outside the probe. 2 outside shown in Figure Finally, a4.10 μm-thick photoresist (AZ-9260, Electronic Materials) applied to serve the Materials) was applied to serve as the etch maskAZ layer (Figure 3i) andwas deep reactive ion as etching (DRIE)

etch mask layer (Figure from 3i) andthe deep reactive ion etching (DRIE) was used probeswafer with was used to release the probes wafer (Figure 3j). The overview of to anrelease entirethe silicon from the wafer (Figure 3j). The overview of an entire silicon wafer with the probes and frames is the probesshown and frames is shown in Figure 4. in Figure 4.

Figure 4. Picture of the entire wafer with 120 microelectrode array in holder frames.

Figure 4. 4. Picture Picture of of the the entire entire wafer Figure wafer with with 120 120 microelectrode microelectrode array array in in holder holder frames. frames. 2.2. Microelectrode Arrays Package

2.2. Microelectrode Package 2.2. Microelectrode Arrays Package AfterArrays fabrication 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 ofpurpose the array, package was begun by combining Afterdesigned fabrication the microelectrode microelectrode array, package begun combining gold gold wire wire for thisof was used for packaging (Figure 5a). On was the back side ofby the microelectrode bonding and flip-chip bonding technology, and aa printed circuit board specifically array, bonding pads between bonding the microelectrode array and PCB were closely integrated by(PCB) flip-chip bonding technology technology and flip-chip technology, and printed circuit board (PCB) specifically technology, and the bonds formed via an anisotropic conductive (ACF, designed this was used for (Figure 5a). the backadhesive side of of film the microelectrode microelectrode designed for for this purpose purpose was usedwere for packaging packaging (Figure 5a). On On the back side the AC-7206U-18, HITACH): first, solder bumps were formed on inner bonding pads ofclosely PCB by reflow array, bonding pads between the microelectrode array and PCB were integrated by array, bonding pads between the microelectrode array and PCB were closely integrated by flip-chip 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 anforanisotropic conductive adhesive film (ACF, (ACF, technology, theby bonds were formed adhesive film padsand of PCB a temporary bonding at 80via °C, 1an Mpaanisotropic 5 s, and a conductive final bonding was performed AC-7206U-18, HITACH): first, solder bumps were formed on inner bonding pads of PCB by reflow between the bonding padssolder of microelectrode array formed and ACF at °C, 3 bonding Mpa for 20pads s. Oneof thePCB front by reflow AC-7206U-18, HITACH): first, bumps were on170 inner diameter gold with wires a connected traces on the PCB using goldinner wire bonding soldering, then piece of 1.5 ˆ mm completely covered every soldering,side, then33aaμm piece of ACF ACF with a size size of ofthe 1.5bond × 55 pads mm with completely covered every inner bonding ˝ bonding technology (Figure 5b,c). Additionally, a biocompatible adhesive was employed to pads of PCB by a temporary bonding at 80 C, 1 Mpa for 5 s, and a final bonding was performed pads of PCB by a temporary bonding at 80 °C, 1 Mpa for 5 s, and a final bonding was performed strengthen the bond and seal the contact pads. ˝ C, 3 Mpa for 20 s. One the front between between the the bonding bonding pads pads of of microelectrode microelectrode array array and and ACF ACF at at 170 170 °C, 3 Mpa for 20 s. One the front side, side, 33 33 µm μm diameter diameter gold gold wires wires connected connected the the bond bond pads pads with with traces traces on on the the PCB PCB using using gold gold wire wire bonding technology (Figure 5b,c). Additionally, a biocompatible adhesive was employed to strengthen bonding technology (Figure 5b,c). Additionally, a biocompatible adhesive was employed to the bond and theand contact strengthen theseal bond 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.

Sensors 2016, 16, 880

6 of 16

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 H2 SO4 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 [Fe(CN)6 ]3´/4´ (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 Figure 6a, 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 (Figure 6c), 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. Figure 6b 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 Sensors 2016, 16, 880

7 of 16

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 Sensors 2016, 16, 880 7 of 16 AuNPs (Figure 6d), lots of gold nanoparticles were densely and uniformly distributed on the surface were densely and uniformly distributed on the surface of the stimulating site, which greatly reduced of theand stimulating site, whichSiO greatly reduced impedance, increased surface roughness and enhanced thermally oxidized , which illustrated that the layer capability was well removed by impedance, increased surface 2roughness and enhanced thepassivation charge injection of stimulating the charge injection capability of stimulating sites [56]. Figure 7 showed an SEM image of the tip reactive ion etching (RIE). an After electrodeposition of of AuNPs (Figure 6d), lots of gold sites [56]. Figure 7 showed SEM image of the tip a microelectrode array and ananoparticles recording site of alocated microelectrode and adistributed recording atthe the tip of the electrode shank. It could were densely and uniformly onsite thelocated surface of stimulating site, whichtip greatly reduced at the tip ofarray the electrode shank. It could be clearly observed that a sharp geometry withbe impedance, surface and enhanced the charge capability of released stimulating clearly observed that sharp tiproughness geometry with accurate shape injection was presented, indicating that accurate shapeincreased was apresented, indicating that the microelectrode arrays were well fromthe sites [56]. Figure 7 were showed anreleased SEM image of silicon the tip of a microelectrode array ion and etching a recording site microelectrode arrays well from wafer by deep reactive (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) electrodeposition of AuNPs. Figure 6. SEM images of stimulating sites (b,d) and recording sites (a,c) before (a,b) and after (c,d) electrodeposition of AuNPs. electrodeposition of AuNPs.

Figure of microelectrode microelectrodeshank. shank. Figure7.7.SEM SEMimages images of tip of Figure 7. SEM images of tip of microelectrode shank.

Sensors 2016, 16, 880

8 of 16

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 Thegenerated aim of this was to demonstrate spread electrical in STN byFEM DBS simulation [57]. The extracellular potentialthe (V)efficient distributions inof STN duringstimulation monopole in STN generated by DBS [57]. The extracellular potential (V) distributions in STN monopole stimulation on double-side site #0 were studied. The extracellular potential during (V) distributions stimulationfrom on double-side site #0 were studied. Thecurrent extracellular potential (V) are distributions generated monopole stimulation at different intensities in STN shown in generated Figures 8 from monopole stimulation at different current intensities in STN are shown in Figures 9. and 9. Figure 8 shows the one-dimensional distributions of the extracellular potential (V) 8inand three Figure 8 shows the one-dimensional distributions of the extracellular potential (V) in three directions at directions at various stimulus intensities. Figure 9 shows the three-dimensional extracellular various stimulus intensities. Figure 9 shows the three-dimensional extracellular distribution potential distribution isosurface of −0.5 V (the extracellular excitation threshold)potential generated by DBS at isosurface of ´0.5 V (the extracellular excitation threshold) generated by DBS at different currents. different currents. When applying current intensities of 100, 150, 250 and 350 μA, the voltage values When applying current intensities of 100, 150, 250 and 350 µA, the voltage values at the surface of at the surface of electrode site #0 were 2.3096, 3.4644, 5.7739 and 8.0835 V, respectively. The potential electrode siteincreased #0 were as 2.3096, 3.4644, stimulus 5.7739 and 8.0835 V, respectively. potential distribution distribution the current was increased. In order toThe qualify the effects of the increased as the current stimulus was increased. In order to qualify the effects of the stimulation, VTA stimulation, VTA was calculated as the vital standard to measure the stimulus effects [58]. As shown was calculated as theapplying vital standard to measure theofstimulus [58]. AsμA shown in Figure 10a,values when in Figure 10a, when current intensities 100, 150,effects 250 and 350 to STN, the VTA applying current intensities of 100, 150, 250 and 350 µA to STN, the VTA values were 0.6321, 0.8346, 3 were 0.6321, 0.8346, 2.5067 and 4.2805 mm , which indicated that the VTA values increased with the 2.5067 and 4.2805 mm3 , which indicatedwe that the VTA values increased with of thedouble-side stimulation monopole intensities. stimulation intensities. Furthermore, compared the stimulus effects Furthermore, we that compared the stimulus effectsstimulation. of double-side with that of stimulation with of single-side monopole For monopole single-sidestimulation monopole stimulation, single-side monopole stimulation. For single-side monopole stimulation, when applying when applying current intensities of 100, 150, 250 and 350 μA, the VTA values were 0.1669, current 0.3233, intensities 100, 150, 250 and 350 10b), µA, the VTA values 0.1669, 0.3233, and 1.2404 mm3 3 (Figure 0.7055 andof1.2404 mm showing that were the VTA value of 0.7055 single-side monopole (Figure 10b),was showing that the VTA value single-side monopolemonopole stimulation was significantly stimulation significantly lower thanofthat of double-side stimulation (Figurelower 10a) than that of double-side monopole stimulation (Figure 10a) when applying the same current intensity. when applying the same current intensity.

Figure Figure 8. 8. One-dimensional One-dimensional distributions distributions of of the the extracellular extracellular potential potential (V) (V) in in three three directions directions of of X-axis X-axis (a); Y-axis (b) and Z-axis (c) generated by monopole stimulation in STN at various stimulus (a); Y-axis (b) and Z-axis (c) generated by monopole stimulation in STN at various stimulus intensities intensities (100, 150, and 350 μA). (100, 150, 250 and 350250 µA).

Sensors 2016, 16, 880

9 of 16

Sensors 2016, 16, 880

9 of 16

Sensors 2016, 16, 880

9 of 16

Figure 9. Three-dimensional extracellular potential distribution isosurface basedononthe the extracellular Figure 9. Three-dimensionalextracellular extracellularpotential potential distribution distribution isosurface Figure 9. Three-dimensional isosurfacebased based on theextracellular extracellular excitation threshold of −0.5 V generated by monopole stimulation in STN at various stimulus excitation threshold of −0.5 V generated by monopole stimulation in STN at various stimulus excitation threshold of ´0.5 V generated by monopole stimulation in STN at various stimulus currents currents (−100, −150, −250 and −350 μA). currents (−100, −150, −350 μA). (´100, ´150, ´250 and−250 ´350and µA).

Figure 10.10. Effects ofof the during (a) (a)double-side double-sidemonopole monopole stimulation; Figure Effects thestimulus stimulusintensity intensityon on the the VTA VTA during stimulation; Figure 10. Effects of the stimulus intensity on the VTA during (a) double-side monopole stimulation; (b) single-side monopole stimulation. (b) single-side monopole stimulation. (b) single-side monopole stimulation.

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, 10 of 16 sites in H216, SO880 4 possessed the same properties as those of recording sites. The stimulating and recording sites were further electrochemically characterized by electrochemical impedance (EIS)Array to measure the electrode impedances in 0.1 M PBS 3.3. Electrochemical Behavior ofspectroscopy the Microelectrode using electrochemical analyzer and the results were presented in Figure 11b. For a typical recording Figure 11a shows theμm cyclic voltammetric a recording site with a diameter of about 20 µm 4.1 in site with diameter of 20 (curve a of Figureresponse 11b) , theofaverage measured impedance was 0.5 M H SO . The typical potential peak appearing at about 900 mV corresponded to the reduction 4 and the average impedance was reduced to about 0.9 MΩ after electrodeposition of MΩ at 12 kHz of Au oxide species thatwas the in cyclic voltammogram of the Au microelectrode site in AuNPs (curve b of [59,60], Figure implying 11b), which rough agreement with previously published results H SO matched the classic electrochemical response of bulk Au in H SO . These results indicated 2 4 2 4 [35,37]. The average impedance of a typical stimulating site with diameter of 100 μm reduced from that thin film microelectrode sites possessed electrochemical properties as bulk Auwere and about 200 KΩAu(curve a of Figure 11c) to aboutthe 80same KΩ (curve a of Figure 11c) after AuNPs could be reasonably expected to possess the samethat recording as traditional Au microwires deposited on its surface. These results implied AuNPscapabilities could greatly increase electron transfer used for neural recording. In addition, the cyclic voltammetric responses of stimulating sites in H2 SO 4 rates and surface roughness of electrode site, reduce interfacial impedances and enhance the possessed the same properties as those of recording sites. performance of stimulating/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 H2 SO4 ; 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 were furthercontaining electrochemically characterized by 6]3−/4− and the measuring cyclic voltammograms in 0.1sites M KCl solution 2 mM [Fe(CN) electrochemical impedance spectroscopy (EIS) to in measure impedances M PBS using results were shown in Figure 12. As shown Figurethe 12,electrode the absolute valuesinof0.1reduction and electrochemical analyzer and the results were presented in Figure 11b. For a typical recording site with oxidation peak currents were almost equal, and the peak potentials were definite values and diameter of 20ofµm a of Figure , the average impedance wasof about 4.1 MΩ at independent the(curve scan rates, which 11b) indicated that the measured electrochemical processes microelectrode 1sites kHzin and impedance was reduced to about6]0.9 after electrodeposition of AuNPs 3−/4−MΩ 0.1the M average KCl solution containing 2 mM [Fe(CN) were reversible. For stimulating site (curve b of Figure 11b), which was in rough agreement with previously published results (Figure 12a), the values of the peak currents increased with scan rates varying from 20 mV/s[35,37]. to 200 The average impedance a typical peak stimulating site diameter ofsites 100 were µm reduced from about mV/s, and reduction andofoxidation currents of with the stimulating proportional to the 200 KΩ (curve Figure to about KΩ (curve a ofmV/s Figurethrough 11c) afteranalysis, AuNPs were deposited square root ofa of the scan11c) rates in the80range 20–200 suggesting the on its surface. These resultswere implied that AuNPs could greatly process increase[61]. electron rates and electrochemical responses a typical surface-controlled For transfer the recoding site surface of electrode site, reducea interfacial impedances andsigmoidal enhance the performance of (Figure roughness 12b), the cyclic voltammograms typical showed a typical pattern due to the stimulating/recording sites. small area of the recording sites. The increase of peak currents was quite small with the increase of and sites were further characterized by scanThe ratesstimulating in Figure 12b duerecording to the small sizemodified and edgewith effectAuNPs of the recording sites [62]. These results 3 ´ /4 ´ measuring cyclic voltammograms in 0.1 M KCl solution containinggood 2 mM [Fe(CN)6 ] and the demonstrated that the fabricated microelectrode array possessed electrochemical properties results were shown in Figure 12. As shown in Figure 12, the absolute values of reduction and oxidation and stability. In addition, inset of Figure 12a was the cyclic voltammogram of the bare stimulating peak currents almost equal, and the peak potentials were definite values and independent of site in 2 mM were [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 solution containing 2 mM [Fe(CN)6 ]3´/4´ 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

possessed cyclic Sensors 2016,good 16, 880electrochemical properties and stability. In addition, inset of Figure 12a was the 11 of 16 voltammogram of the bare stimulating site in 2 mM [Fe(CN)6 ]3´/4´ at scan rate of 100 mV/s, and the oxidationvalues peak currents of stimulating site modified with AuNPs (curve csite of Figure 12a,with scanAuNPs rate of absolute of reduction and oxidation peak currents of stimulating modified 100 mV/s) much higher than those of thewere baremuch stimulating 12a) under the (curve c of were Figure 12a, scan rate of 100 mV/s) highersite than(inset thoseofofFigure the bare stimulating same experimental conditions, showing that AuNPs conditions, could greatly increase rates site (inset of Figure 12a) under the same experimental showing thatelectron AuNPs transfer could greatly and surface roughness the and electrode The same conclusion reached from the inset in increase electron transferofrates surfacesite. roughness of the electrodecan site.beThe same conclusion can be Figure 12b. reached from the inset in Figure 12b.

Figure 12. 12. Cyclic Cyclic voltammograms voltammograms of of the the stimulating stimulating site site modified modified with with AuNPs AuNPs (a) (a) and and recording recording site site Figure 3−/4− 3´/4´ 6 ] . Scan rate: 20, 50, 100, modified with AuNPs (b) in 0.1 M KCl solution containing 2 mM [Fe(CN) modified with AuNPs (b) in 0.1 M KCl solution containing 2 mM [Fe(CN)6 ] . Scan rate: 20, 50, 150 and 200 mV/s (from(from a to e). of Figure 12a: 12a: cycliccyclic voltammogram of bare stimulating site in 100, 150 and 200 mV/s a toInset e). Inset of Figure voltammogram of bare stimulating site2 3−/4− 3´/4´ 6] at scan rate rate of 100 mV/s. Inset of of Figure 12b: bare mM [Fe(CN) in 2 mM [Fe(CN) at scan of 100 mV/s. Inset Figure 12b:cyclic cyclicvoltammogram voltammogram of of bare 6] 3−/4− at scan rate of 100 mV/s. recording site site in in 22 mM mM [Fe(CN) [Fe(CN)66]3´/4´ recording at scan rate of 100 mV/s.

3.4. In Vivo Neural Recordings

confirm thethe ability to In vivo recordings in the the brain brain of ofthe theanesthetized anesthetizedrats ratswere wereperformed performedtoto confirm ability record spontaneous discharge signals from multiple nuclei at the fabricated microelectrode array. to record spontaneous discharge signals from multiple nuclei at the fabricated microelectrode array. (Laboratory Animal Center at Xi’an Xi’an Jiaotong Jiaotong Male Sprague-Dawley (SD) rats weighing 250–300 g (Laboratory of Medicine) Medicine) were were used used in in the theelectrophysiological electrophysiological experiments. experiments. All animal animal University School of 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 12 ofthe 16 natricum (3 mg/100 g). After craniotomy and resection of the dura mater, cortical surface was exposed. The microelectrode array was obliquely inserted into the rat brain at mm lateralofto72Bregma subdural) by a micromanipulator with a constant speed 10 μm/s an angle degreeand with0 mm the horizontal plane from the pia mater (coordinates: 0 mmofposterior and an mm insertion depth of 8 mm. signals from the brain were recorded using a Cerebus and 2.5 lateral to Bregma and 0Neural mm subdural) by a micromanipulator with a constant speed of multi-channel data acquisition system (Blackrock Microsystems, Salt Lake city, UT, USA). The 10 µm/s and an insertion depth of 8 mm. Neural signals from the brain were recorded using a Cerebus original neural signals were amplified, filtered by a bandpass ranging from 1 Hz to 7.5 kHz and multi-channel data acquisition system (Blackrock Microsystems, Salt Lake city, UT, USA). The original digitized (sampling 30 kHz)filtered for offline neural signals wererate: amplified, by aanalysis. bandpass ranging from 1 Hz to 7.5 kHz and digitized The obtained original neural signals were filtered by a bandpass from 500 Hz to 5 kHz to get the (sampling rate: 30 kHz) for offline analysis. multiple unit activity (MUA) of signals nervouswere nuclei. Figure showedfrom the 500 simultaneously The obtained original neural filtered by a13 bandpass Hz to 5 kHz recorded to get the MUA from striatum, GPi and STN, which substantiated the ability of the fabricated microelectrode multiple unit activity (MUA) of nervous nuclei. Figure 13 showed the simultaneously recorded MUA array to simultaneously record spontaneous discharge signals multiple microelectrode nuclei. In addition, from striatum, GPi and STN, which substantiated the ability of from the fabricated arraythe to average signal-to-noise ratio (SNR) ofdischarge the neuralsignals signalsfrom wasmultiple calculated. The SNR of the neural signal simultaneously record spontaneous nuclei. In addition, the average was defined asratio the (SNR) average amplitude of spikes The to the root mean square of was the signal-to-noise of peak-to-peak the neural signals was calculated. SNR of the neural signal background noise, and the SNR of neural signals from the striatum, GPi and STN was 3.6, 6.3 and defined as the average peak-to-peak amplitude of spikes to the root mean square of the background 4.7, respectively. noise, and the SNR of neural signals from the striatum, GPi and STN was 3.6, 6.3 and 4.7, respectively. Sensors 2016, 16, 880 1% pelltobarbitalum

Figure 13. 13. Simultaneously Simultaneouslyrecorded recorded MUA MUA from from striatum striatum (a); (a); GPi GPi (b) (b) and STN (c). Figure

4. 4. Conclusions Conclusions In In this this paper, the design, simulation, fabrication fabrication and and characterization characterization of of aa novel dual-sided dual-sided microelectrode microelectrode array array have have been reported. The The microelectrode microelectrode array array was was specially specially designed designed and and fabricated fora rat a Parkinson’s rat Parkinson’s model to the stimulate the subthalamic nucleus and fabricated for diseasedisease model to stimulate subthalamic nucleus and simultaneously simultaneously record electrophysiological signalsnuclei from multiple nuclei of the basal ganglia system. record electrophysiological signals from multiple of the basal ganglia system. The package of The package ofmicroelectrode the dual-sidedarray microelectrode arraywire combined gold wire bonding technology and the dual-sided combined gold bonding technology and flip-chip bonding flip-chip bonding fororder the first time. Inthe order to enhance the stimulating/recording technology for thetechnology first time. In to enhance 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. 2.

3.

4.

5.

6. 7. 8.

9.

10.

11. 12.

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] 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] 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. 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] 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] 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. 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. 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] 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. 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] Montgomery, E.B. Effects of GPi stimulation on human thalamic neuronal activity. Clin. Neurophysiol. 2006, 117, 2691–2702. [CrossRef] [PubMed] 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

13.

14. 15.

16. 17. 18. 19. 20. 21.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32.

33. 34. 35.

14 of 16

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. 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. 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] 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] Delong, M.; Wichmann, T. Circuits and circuit disorders of the basal ganglia. Arch. Neurol. 2007, 64, 20–24. [CrossRef] [PubMed] 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] 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. Mcintyre, C.C.; Hahn, P.J. Network perspectives on the mechanisms of deep brain stimulation. Neurobiol. Dis. 2009, 38, 329–337. [CrossRef] [PubMed] 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] 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] Cheung, K.C. Implantable microscale neural interfaces. Biomed. Microdevices 2008, 9, 923–938. [CrossRef] [PubMed] 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] 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] Wise, K.D. Silicon microsystems for neuroscience and neural prostheses. IEEE Eng. Med. Biol. Mag. 2005, 24, 22–29. [CrossRef] [PubMed] 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] 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] Yao, Y.; Gulari, M.N.; Wiler, J.A.; Wise, K.D. A Microassembled Low-Profile Three-Dimensional Microelectrode Array for Neural Prosthesis Applications. J. Microelectromech. Syst. 2007, 16, 977–988. [CrossRef] 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] 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] 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] Fekete, Z. Recent advances in silicon-based neural microelectrodes and microsystems: A review. Sens. Actuators B Chem. 2015, 215, 300–315. [CrossRef] 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] 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

36.

37. 38. 39.

40. 41. 42.

43.

44.

45. 46.

47.

48. 49.

50.

51.

52. 53. 54. 55.

15 of 16

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. Du, J.; Blanche, T.J.; Harrison, R.R.; Lester, H.A.; Masmanidis, S.C. Multiplexed, high density electrophysiology with nanofabricated neural probes. PLoS ONE 2011, 6, e26204. [CrossRef] [PubMed] Tanghe, S.J.; Wise, K.D. A 16-channel CMOS neural stimulating array. IEEE J. Solid-State Circuits 1992, 27, 1819–1825. [CrossRef] 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. 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] 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] 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] 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] 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. Neuron 2015, 88, 1136–1148. [CrossRef] [PubMed] 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] 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. 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] 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] 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] 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] 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] 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] 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] 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] 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

56.

57. 58. 59.

60.

61. 62. 63.

16 of 16

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. 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] 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] 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] 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] Fang, B.; Wang, G.; Zhang, W.; Li, M.; Kan, X. Fabrication of Fe3 O4 Nanoparticles Modified Electrode and Its Application for Voltammetric Sensing of Dopamine. Electroanal 2005, 17, 744–748. [CrossRef] 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] Suzuki, I.; Mao, F.; Shirakawa, K.; Jiko, H.; Gotoh, M. Carbon nanotube 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/).