Microsyst Technol (2015) 21:139–145 DOI 10.1007/s00542-013-2017-3
Implantable electrode array with platinum black coating for brain stimulation in fish Chuan Zhang · Jing‑Quan Liu · Hong‑Chang Tian · Xiao‑Yang Kang · Jing‑Cheng Du · Yue‑Feng Rui · Bin Yang · Chun‑Sheng Yang
Received: 30 August 2013 / Accepted: 28 November 2013 / Published online: 12 December 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract Electrical stimulation of certain part of the neural tissue could evoke specific functional response. The interface plays an important role in stimulation process. Tungsten wire electrodes have long been used as an ideal interface for neural signal recording and stimulation. In this work, an electrode array with good electrochemical property and biocompatibility was successfully fabricated in a novel and convenient way. A PDMS electrode holder was made to fix the electrodes with certain distance. To achieve a better stimulation effect, a platinum black coating was electroplated, and the electrochemical properties of the electrode were tested. Tests results of the impedance and charge injection capacity showed great improvement in electrochemical property. To test the durability of the platinum black coating, the electrode was dealt with ultrasound vibration for 10 min and then re-tested for charge storage capacity lost. The array was then implanted into the midbrain of crucian carp by a surgical procedure. A stimulation pulse was applied in order to elicit a change in locomotion. By stimulating the midbrain part of the fish brain, both side turning movements were observed. Also, this work provides the fundamental experiment for the development of bio-robotic fish.
C. Zhang · J.-Q. Liu (*) · H.-C. Tian · X.-Y. Kang · J.-C. Du · Y.-F. Rui · B. Yang · C.-S. Yang National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Shanghai, China e-mail: [email protected]
C. Zhang · J.-Q. Liu · H.-C. Tian · X.-Y. Kang · J.-C. Du · Y.-F. Rui · B. Yang · C.-S. Yang Institute of Micro and Nano Science and Technology, Shanghai Jiao Tong University, Shanghai, China
Abbreviations PDMS Polydimethylsiloxane CSC Charge storage capacity Qinj Charge injection capacity SCE Saturated calomel electrode Nflm Nucleus of the medial longitudinal fasciculus CSF Cerebrospinal fluid
1 Introduction Stimulation of certain group of neurons could evoke specific response. Implantable prostheses used for treatment in muscle neural injury and brain diseases have been widely reported (Peckham 2005; Perlmutter and Mink 2006). The interface between neural tissue and implanted prostheses plays an important role, because the contact interface determines the charge delivery ability, which is the main consideration in neural recording and stimulation process. Also characteristics like biocompatibility for implantation, stability during stimulation process and ability to deliver charge in the stimulation process require careful consideration. Tungsten micro wire electrode is widely used in neurophysiologic experiment for its excellent characteristics. It has been frequently used as common and convenient prostheses for both neural recording and stimulation (Prasad et al. 2012; Williams et al. 1999). Although problems may be met during long term implantation considering the inflammatory response and other effects (Prasad et al. 2012; Patrick et al. 2011), it is still an ideal interface for short term experiment. Also, wire electrodes have tiny damage to the tissue and enough mechanical strength during implantation procedure. Because of these properties, here we choose tungsten wire for later stimulation experiment.
Here we introduce a convenient method to build a tungsten electrode array. The electrodes were settled in a fabricated PDMS holder. Then the array were insulated by parylene C thin film with only the tip of the tungsten wire electrodes exposed as stimulation sites. To enhance the electrochemical properties, a platinum black coating was electroplated onto the electrode sites. Increase in charge storage and injection capacity could give rise to the electrochemical surface area and improve the ability to deliver electron to the nerve tissue. To confirm the durability of platinum black coating, the electrode was tested after being dealt with an ultrasound vibration for 10 min. The CSCC (Cathodic Charge Storage Capacity) change after ultrasound was tested. To prove its applicability, this electrode array was implanted into the midbrain part of the fish by a surgical procedure. A positive rectangular pulse train was applied to induce a locomotion response in a free swimming fish. Activities of the fish were recorded by a camera fixed above the tank. In addition, applying stimulation pulse in animals or insects to build an artificial controlled “robot” is getting popular recently (Sanjiv et al. 2002; Sato and Maharbiz 2010; Sato et al. 2009). These kinds of robot are called cyborgs or bio-robots. Our research strongly provides a fundamental research for future development in bio-robotic fish at the same time.
2 Experiment A mould was fabricated to make a holder for tungsten micro wire. The fabrication process is shown in Fig. 1. First, a silicon wafer was cleaned by acetone bath with ultrasound for 3 min and then dried at a temperature of 180 centigrade for 3 h. When the wafer cooled down, a 150 μm thick SU-8 was spin-coated onto the wafer. After lithography, PDMS was casted onto the SU-8 mould with a thickness of 1 mm and heated at a temperature of 85 centigrade for 2 h. After solidification, the PDMS holder was peeled off the wafer. This PDMS electrodes holder is built with parallel channels of 75 μm width and 100 μm deep. The distance between two neighbor channels is 1 mm. The whole size of the PDMS holder is 6 × 6 mm, and thickness 1 mm. Tungsten electrodes (TGW0325, WPI, USA) were cleaned by soaked in acetone bath with ultrasound for 3 min. Then the electrodes were flushed with de-ionized water and dried in the drying oven for 1 h. After this preparation, the electrodes were placed on the PDMS holder into the channels to form a parallel electrode array. To form a multi-row electrode array, we can bond single layers of PDMS holder together. Because PDMS has a relatively low surface energy, it is difficult to keep layers stick together. The surface of both side of the holder was treated with O2 ion plasma for 30 s, thus a hydrophilic property
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Fig. 1 Fabrication process of PDMS electrode holder. a A 150 μm SU-8 was spun onto a silicon wafer. b SU-8 patterned. c PDMS was poured onto the SU-8 mould and solidified. d PDMS holder was peeled off the mould, and tungsten micro wires were stuck into the channels to form an array
could be induced (Chen et al. 2012). As a result, single layer array could be bonded to each other to form a multi-layer tungsten wire array, as shown in Fig. 2b. The coin used for comparison has a diameter of 22.5 mm. Figure 2a shows an example of 2 × 4 tungsten wire electrode array fabricated using this process. When the array was made, it was covered with a 5 μm thick parylene C thin film by chemical vapor deposition (CVD) using a deposition machine (PDS 2010, SCS, USA). The parylene C film stands as an insulating layer. In addition, parylene C has an excellent biocompatibility with FDA approval, which has long been used as an excellent coating for implantable device (Schmidt et al. 1988). After this insulating process, the tips of the insulated tungsten wires are carefully cut to expose a circular round section as the electrode site. The length of all the tungsten wires for implantation was kept 4 mm. To improve the electro-chemical properties, a platinum black coating was then electroplated onto the electrode sites. The whole process was carried on using an electrochemical workstation (CHI660B, Chenhua, China). Considering the lack of durability of platinum black coating using DC electroplating, here we adopted a method of electroplating in chloroplatinic acid solution with ultrasonic bath agitator. This method could improve the electrochemical property significantly as DC electroplating and the stability at the same time (Desai et al. 2010; Rui et al. 2012). The electroplating process was applied a repeating pulse
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Fig. 2 Schematic view of the bonding process and results. a A 2 × 4 tungsten wire electrode array made using the methods mentioned above. A top PDMS block layer was also bounded as cover for under electrode layer. b Schematic view of bonding process between layers of electrode array
Fig. 3 SEM image of tungsten electrode fabricated using these methods
of 5 ms:500 ms duty ratios. The peak current density is 2.5 A/cm2, which 100 times greater than the overall current density 2.5 A/dm2. The short duty cycle could give a break for the re-balance of ion concentration near the electrode site. The whole process lasts 500 cycles. After electroplating, the array was carefully kept with electrode sites soaking into normal saline for later usage. Normal DC plating methods of platinum black would result problem in durability. To confirm the durability of the platinum black coating, the electrodes were soaked in de-ionized water under an ultrasound vibration for 10 min. Then the electrode was re-tested for electrochemical property, especially of CSCC lost, which is due to the falling out of platinum black coating.
The electrochemical properties were then tested by an electrochemical workstation. During the test, saturated calomel electrode (SCE) was used as the reference electrode. The impedance at different frequency, ranging from 0.1 to 105 Hz, was measured, as shown in Fig. 4a. Lower impedance requires lower stimulation voltage and results higher signal to noise ratio. The impedance at a frequency of 1 kHz is an important parameter (Cogan 2008). As we can see from the figure, the impedance was greatly reduced, from 16.6 to 3.5 kΩ, at 1 kHz. Another important parameter is impedance at 50 Hz, which is the frequency at stimulation procedure. The impedance was decreased from 150 to 13.3 kΩ. Figure 4b shows the phase angle of the electrode before and after coating. The relationship between impedance and phase angle was determined by equation:
3 Results and discussion
Z(w) = |Z|eiθ
3.1 Surface property and electrochemical characteristics The surface of the electrode site was observed by SEM. Figure 3 shows the SEM picture of the cross section of one single tungsten wire electrode. The electrode is insulated by parylene C thin film. The surface of the electrode site is densely covered by a porous structure of platinum black, which significantly increases the effective surface area. Greater surface area means lower impedance and higher charge injection ability (Cogan 2008; Long et al. 2011).
According to the equation, at lower frequency, the modified electrode shows a slight capacitive character. This means platinum black coating is responsible for the decrease in both resistive and capacitive resistance. The cyclic voltammograms of the modified wire electrode before and after coated with platinum black are shown in Fig. 5. The current polarity was chose to be cathode positive. The anodic peak is observed partly due to the residual chloroplatinic acid solution in the layers of platinum black coating, which would be flatter after repetitive test and usage. The Cathodal Charge Storage Capacity
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Fig. 5 Cyclic voltammograms of electrode before and after platinum black electro-plating
Fig. 4 Electrochemical properties tested using a workstation before and after electro-plating at different frequency. a Electrochemical impedance spectroscopy; b phase angle
(CSCC) means the amount of available electron for charge delivery in cathode, which could be identified by the CV area above zero. To further confirm the improvement in properties for later stimulation process, the charge-injection capacity (Qinj) is tested, as shown in Fig. 6. The stimulation pulse train was generated by a pulse stimulator (A.M.P.I., Master 8, Israel). The charge injection capacity is tested in normal saline. Voltage responses for the given pulse train are shown in Fig. 6. Charge injection limit is the biggest cathodic charge when the voltage is greater than 0.6 V compared with the reference electrodes (Luo et al. 2011). The charge injection limit increases greatly, from 0.286 to 1.906 mC/cm2 on average. It reflects the obvious improvement by platinum black coating in the ability to deliver enough charge, because the electrode would carry out
Fig. 6 Voltage response of the coated and uncoated electrodes regarding to the same stimulation pulse. The coated electrode shows a lower change in voltage
lower voltage generation for a given stimulation current (Venkatraman et al. 2011). After 10 min ultrasound, the CV scan was processed again to confirm the CSC lost. As we can find out in Fig. 7, the CSCC only decreased for 21.3 %. Also, the impedance only suffered from a slight reduction at 1 kHz after ultrasound vibration. Even after long term usage, the platinum black coating would significantly lower the impedance with only a very thin layer of coating. This proved the excellent mechanical durability of platinum black coating for stimulation in a relatively long term experiment. Also, former
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Fig. 7 Comparison of cyclic voltammograms of electrode before and after ultrasound vibration
researchers also proved that platinum black is non-toxic and has a good biocompatibility for implantation (Dymond et al. 1970; Rui et al. 2012). All these provide solid evidences that platinum black coating would have an excellent performance in later stimulation experiment. 3.2 Stimulation results Crucian carp, kept in a fish tank for more than 1 week to adapt to the environment, was anesthetized using 150– 200 mg/L tricainemethanesulfonate (MS-222) solution for more than 10 min before the surgery. The dosage of MS-222 differs between individuals. The surgery began when the body balances of the fish were lost and only gill movement was observed. The fish was covered with a wet towel in case of damage to its body, and then fixed onto a surgical holding apparatus. The skin above the head was carefully removed using scalpel in order to expose the skull and enhance the binding force of dental cement in later process. Then a circle hole with a diameter about 7 mm was open using an electrical drill above the midbrain part of the fish skull. The cerebrospinal fluid (CSF) was removed to expose the midbrain. Normal saline (0.9 %) was used to keep the brain ion concentration. During the whole process, a 20–50 mg/L MS-222 was continued pumping into the buccal cavity to irrigate the fish gill. Then a 2 × 4 electrodes array was implanted into the midbrain part using a micro-manipulator (World Precision Instrument Inc., Kite-L) in a 10 μm step. The array was place to keep the two parallel rows of electrodes implanted into left and right part of the midbrain respectively, as the
Fig. 8 The schematic view of the whole stimulation process. a The stimulation system. b The view of implantation sites and stimulation wave form. The red line in the fish brain shows the approximate position of two rows of electrodes. Teo optic tectum, CC corpus cerebella
red lines shown in Fig. 8b. The electrode site was chosen to locate near the nucleus of the medial longitudinal fasciculus (Nflm), which is proved to be responsible for the initialization in the center pattern generator (Kobayashi et al. 2009; Kashin et al. 1974; Uematsu and Todo 1997) in spinal cord. Then the brain was covered and fixed by dental cement. After the procedure, the fish was taken back to the tank with clean water for recovery from anaesthesia. After recovery, a positive rectangular pulse train was applied to the midbrain using a TDT system (RZ5D, USA, FL), the induced locomotion of the fish is observed by a camera fixed above the fish tank. The schematic view of the whole stimulation system for this experiment was shown in Fig. 8a. Electrical stimulation near one side of the Nflm would induce a turning movement towards the stimulation side, with a tail flaps to the other side (Kobayashi et al. 2009). The stimulation frequency was set to 50 Hz with a wave width of 2.5 ms. The current amplitude was increased from 10 to 100 μA with a 10 μA step to confirm the appropriate current to evoke locomotion change in fish. To figure out the best stimulation point, every electrode of the electrode array was tested in this procedure. It is reported that as the current intensity increases, the amplitude and frequency of the fin movement will increase when the latency decrease (Kashin et al. 1974). The preferred stimulation point, however, would require lower current intensity to evoke a
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was electroplated onto the surface in an ultrasound bath to improve the durability of the coating. The electrochemical tests showed the property of the wire electrode was greatly enhanced including a drop in impedance and an increase in charge injection capacity. To further prove the feasibility of the array, a stimulation experiment in fish brain was carried out using the array fabricated in this method. By stimulating the midbrain part of the fish brain, both side turning movements were observed. This work provides the fundamental experiment for the development of biorobotic fish. Acknowledgments The authors thank to financial support from the National Natural Science Foundation of China (No. 51035005, 61076107), 973 Program (2013CB329401). Shanghai Municipal Science and Technology Commission (No. 11JC1405700, 13511500200). Minhang District Project (2011MH084). WUXI-SJTU project (2011JDZX017), NDFC Funding (9140A26060313JW3385). The authors are also grateful to the colleagues for their essential contribution to this work.
References Fig. 9 Stimulation results. a–c Right turning movements induced by stimulation in the midbrain. d–f Left turning movements
noticeable movement, and stimulation at this point would induce a more rhythmic swimming locomotion. The turning movement was successfully induced with the lowest current of 10 μA. Both left and right turning movements were induced, although the left turning movement is not in a balance and rhythmic way, Fig. 9. This is probably because the stimulation points on the left side of the brain were too far from the Nflm. The swimming activity stops almost when the stimulation stops. When the current amplitude increased, the swimming locomotion became fiercer with greater tail flip and faster body movement. The lowest initial current amplitude was bigger when it located farther from the position of Nflm. In addition, the wave width also affected the intensity of the locomotion of the fish. But when the pulse width is greater than 8 ms, the fish only showed a sudden rush and stopped quickly. This result provides basic research for future research on the accurate control of fish movement.
4 Conclusions In this paper, we provide a simple method to build a tungsten wire electrode array, whose size could be easily changed. The whole array was insulated by parylene C and only the stimulation sites were exposed. To enhance the electrochemical property, a platinum black coating
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