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All-carbon-nanotube flexible multi-electrode array for neuronal recording and stimulation

All-carbon-nanotube flexible multi-electrode array for neuronal recording and stimulation Biomed Microdevices (2014) 16:43–53 DOI 10.1007/s10544-013-9804-6 All-carbon-nanotube flexible multi-electrode array for neuronal recording and stimulation Moshe David-Pur & Lilach Bareket-Keren & Giora Beit-Yaakov & Dorit Raz-Prag & Yael Hanein Published online: 24 August 2013 The Author(s) 2013. This article is published with open access at Springerlink.com . . Abstract Neuro-prosthetic devices aim to restore impaired Keywords Carbon nanotubes Multi electrode array . . . function through artificial stimulation of the nervous system. Neuronal recording Neuronal stimulation Flexible A lingering technological bottleneck in this field is the reali- Prosthesis zation of soft, micron sized electrodes capable of injecting enough charge to evoke localized neuronal activity without causing neither electrode nor tissue damage. Direct stimula- 1 Introduction tion with micro electrodes will offer the high efficacy needed in applications such as cochlear and retinal implants. Here we Flexible neuronal micro electrode technology progressed ex- present a new flexible neuronal micro electrode device, tensively over the past several decades hand in hand with the based entirely on carbon nanotube technology, where both overall development in the field of neuro-prosthetics. Several the conducting traces and the stimulating electrodes consist novel fabrication approaches suited for micro electrode appli- of conducting carbon nanotube films embedded in a poly- cations were devised. These schemes attempt to achieve flex- meric support. The use of carbon nanotubes bestows the ible electronic technology integration with high surface rough- electrodes flexibility and excellent electrochemical proper- ness while maintaining bio-compatibility and durability in ties. As opposed to contemporary flexible neuronal elec- physiological conditions. Commonly, these devices use metal trodes, the technology presented here is both robust and electrodes such as gold (Sandison et al. 2002;Chen etal. the resulting stimulating electrodes are nearly purely ca- 2009, 2011a; Wester et al. 2009;Lacour etal. 2010;Wei pacitive. Recording and stimulation tests with chick retinas et al. 2011), titanium (Takeuchi et al. 2004), electroplated were used to validate the advantageous properties of the platinum black (Adams et al. 2005;Rodger etal. 2008; electrodes and demonstrate their suitability for high-efficacy Graudejus et al. 2009, 2012; Rui et al. 2011), tungsten (Wei neuronal stimulation applications. et al. 2011), platinum (Cheung et al. 2007; Mercanzini et al. 2008; Myllymaa et al. 2009; Viventi et al. 2011) and iridium (Rodger et al. 2008; Fomani and Mansour 2011)deposited on Moshe David-Pur and Lilach Bareket-Keren contributed equally to this various flexible supports such as polyimide (Sandison et al. work. 2002; Takeuchi et al. 2004; Cheung et al. 2007; Viventi et al. Electronic supplementary material The online version of this article 2011), parylene C (Rodger et al. 2008; Wester et al. 2009)or (doi:10.1007/s10544-013-9804-6) contains supplementary material, poly(dimethylsiloxane) (PDMS) (Graudejus et al. 2009, 2012; which is available to authorized users. Lacour et al. 2010; Wei et al. 2011). These metal electrodes : : : M. David-Pur L. Bareket-Keren G. Beit-Yaakov Y. Hanein achieve neural stimulation by Faradaic current injection School of Electrical Engineering, Tel-Aviv University, through the electrode-electrolyte interface. Electron transfer, Tel-Aviv 6997801, Israel associated with the Faradaic charge stimulation, can induce : : : : M. David-Pur L. Bareket-Keren G. Beit-Yaakov D. Raz-Prag irreversible reduction and oxidation reactions that can damage Y. Hanein (*) both the electrode and the tissue (Merrill et al. 2005; Cogan Tel-Aviv University Center for Nanoscience and Nanotechnology, 2008). Storage and injection of charge can also occur from Tel-Aviv University, Tel-Aviv 6997801, Israel valence changes in multivalent electrode coatings such as e-mail: [email protected] 44 Biomed Microdevices (2014) 16:43–53 Iridium oxide (Robblee et al. 1983; Klein et al. 1989)that 2Methods undergo reversible reduction-oxidation reactions (Merrill et al. 2005;Cogan 2008). Consequently, capacitive charge 2.1 Flexible CNT MEA fabrication stimulation is preferable for neuronal stimulation, as it involves only a displacement current associated with Flexible CNT MEAs were fabricated as follows. First, standard charging and discharging of the electrode-electrolyte dou- lithography (AZ1518 photoresist; Clariant) was used to form ble layer (Merrill et al. 2005). Common capacitive elec- the desired circuit pattern on a Silicon/Silicon dioxide (Si/ trode materials include titanium nitride (TiN), tantalum- SiO ) support. A 2.5 nm Ni catalyst layer was deposited using tantalum oxide and the more recently investigated carbon an e-beam evaporator (VST). A resist lift-off process was then nanotubes (CNTs) (Rose et al. 1985; Gabay et al. 2007; performed, followed by an oxygen plasma treatment to remove Cogan 2008). Conducting polymers, such as polypyrrole all photoresist residues. Next, MWCNTs were grown by chem- (PPy) and poly(ethylenedioxythiophene) (PEDOT) are mixed ical vapor deposition (CVD) (Lindberg Blue) with ethylene conductors, exhibiting both electron and ion transport within (20 sccm) and hydrogen (1,000 sccm) at 900 °C. A flexible the polymer film (Ludwig et al. 2006; Abidian et al. 2010; substrate, medical adhesive tape, parylene C, polyimide or Blau et al. 2011). poly(dimethylsiloxane) (PDMS), was applied and peeled off A related key requirement in neuronal electrode technology with the CNT pattern. Medical adhesive tape (Steri-Drape, is large specific capacitance (C ). Large specific capacitance 3 M) was attached to the CNT pattern and pressed lightly. reduces the electrode impedance, without increasing its geo- Parylene C was applied by on the CNT pattern by vapor metric area. The reduction in impedance is essential for effi- deposition. Polyimide, prepared from a poly(pyromellitic cient, high resolution neuronal recording and stimulation dianhydride-co-4,4′-oxydianiline) 15 wt.% solution in N- (Robinson 1968;Loebet al. 1995; Merrill et al. 2005; methyl-2-Pyrrolidone (Sigma-Aldrich) was spin coated and Cogan 2008). One of the best materials to exhibit both large cured at 350 °C under nitrogen atmosphere. Uncured PDMS specific capacitance as well as non-Faradaic behavior is po- (Sylgard 184, Dow Corning), mixed in a 10:1 ratio by weight, −2 rous TiN with C in the range of 2 mFcm (Gabay et al. was casted or spin coated and cured at 60 °C. Peeling-off of 2007). It was recently demonstrated that pristine CNTs exhibit very thin PDMS films (~100 μm) required the deposition of a similar performances to those of TiN with C values in the thin Cr layer (2 nm) followed by Au layer (6 nm) using an e- −2 range of 3–10 mFcm (Gabay et al. 2007). Accordingly, beam evaporator prior to PDMS application to reduce the CNTs have been suggested by several studies as a future adhesion between the SiO and the PDMS. To guarantee the material for neuronal stimulation applications and several final cleanliness of the CNT film, half cured PDMS films fabrication schemes have been studied. Primarily, direct (60 °Cfor 5min) wereused inastateofa viscouspolymer growth of CNT electrodes (Wang et al. 2006; Gabay et al. and were applied as an adhesive film onto the CNTs. The 2007;Su et al. 2010) as well as CNT coatings of metal use of partially cured films substantially reduced wetting of electrodes by electro-polymerization (Keefer et al. 2008), drop the CNTs. Finally, a passivation PDMS membrane with coating from a solution (Gabriel et al. 2009) and micro-contact predefined holes and the CNT flexible circuit were bonded. printing (Fuchsberger et al. 2011) on a rigid support were The PDMS passivation layer was prepared using a SU8- described. To accommodate flexibility, CNT transfer onto 3050 (MICRO-CHEM) patterned mold (see Supplementary a polymeric support (Su, Lin et al. 2009;Tsaietal. 2009; Fig. 1). PDMS passivation was bonded using a custom made Carnahan et al. 2010;Chang-Jianetal. 2010) was recently holder mounted on a microscope stage. PDMS-PDMS bonding presented. However, the lack of a simple platform to was promoted by oxygen plasma treatment to both films. allow the realization of fully functional devices consisting of Oxidation of PDMS surface exposes silanol groups (Si-OH) pristine CNT surfaces has left this technology so far largely so when the two films are brought together they form covalent unused. siloxane bonds (Si-O-Si) which provide excellent sealing (Duffy Here, we present a novel flexible neuronal micro elec- et al. 1998). Bonding with polyimide and parylene C substrates trode device, based solely on multi-walled CNT (MWCNT) was achieved by means of an intermediate thin layer of liquid films embedded in a flexible polymeric support. We dem- PDMS followed by curing at 60 °C. Finally, the medical tape onstrate a new simple and robust fabrication technique to was bonded with the passivation by exploiting the adhesiveness realize the seamless CNT circuit on the flexible substrate. of the tape. These processes yielded 30–65 % clean and capac- Next, the electrical and electrochemical properties of the itive electrodes utilizing an entirely manual preparation. We CNT electrodes and of the CNT conducting traces were expect that mechanizing the process can dramatically improve studied, using a scheme of specially designed electrode the yield. For electrophysiological experiments the flexible arrays. Finally, the flexible CNT MEA was applied for CNT MEA was mounted on a PCB (49×49 mm )with 60 extracellular neuronal recording and stimulation of chick Au traces and contact pads. A glass chamber was mounted retinas. on top of the PCB using uncured PDMS. Biomed Microdevices (2014) 16:43–53 45 2.2 Electrical resistance measurements of CNT films (MultiChannel Systems MC_Card, Reutlingen, Germany) and recorded (MultiChannel Systems MC_Rack, Reutlingen, Sets of CNT bars with different lengths and constant width and Germany). All additional signal analysis was performed using height, were fabricated between TiN pads as follows. A 100 nm Matlab software (MathWorks). Electrically stimulated neuro- TiN layer was sputtered (MRC RF sputter) on a Si/SiO support nal activity was digitized at 20 kHz and spikes were detected followed by lithography and reactive ion etching (Nextral 860) to by setting a threshold of signal to noise ratio (SNR) SNR>4 pattern the TiN pads. Due to a marked difference between the (related to the pre-stimulation noise level). Due to amplifier diffusion rate of Ni through SiO and TiN at the CNT growth saturation artifact, the period of 20 ms post stimulation was temperature, two layers of Ni were deposited by an e-beam ignored. The response of the retinal site to electrical stimula- evaporator (VST). The first layer (8 nm) was deposited on the tion was defined as the detected spikes count. inner half of the TiN pads and the second layer (2.5 nm) between the TiN pads on the SiO substrate. Finally a CNT film was 2.6 Electrical stimulation grown by CVD (for detailed illustration see Supplementary Fig. 2). Current versus voltage screen of the different length Chick retinas were electrically stimulated using a dedicated TiN-CNTs-TiN bars was recorded and their electrical resistance stimulator (STG-1008, Multi-Channel Systems, Reutlingen, was calculated (for details see Supplementary Fig. 3). Germany) through one of the MEA electrodes each time (versus an external reference) with charge-balanced bi- 2.3 Electrochemical analysis phasic (cathodic first) current stimulation (pulse width: 1 ms and pulse amplitude: 1–10 μA). Each stimulation session The electrochemical properties of the CNT electrodes were char- included stimulations at the entire intensity range (increased acterized by performing cyclic voltammetry (CV) and electro- by 1 μA every 10 s) and was repeated five times. To validate chemical impedance spectroscopy (EIS) in PBS. An Ag/AgCl that the electrical stimulation resulted from synaptic process- electrode served as a reference electrode and a platinum wire as a es, synaptic blockers CNQX (Sigma) and APV (Sigma) were counter electrode. CV measurements were conducted using a applied (75 μM and 400 μMrespectively). potentiostat (263A Princeton Applied Research) under ambient conditions and recorded using the PowerCV software (Princeton Applied Research). The DC capacitance was derived from the 3Results oxidation current versus the scan rate data according to the relation:i=C·dV/dt in which i is the charging current, C is the 3.1 All-CNT flexible MEA fabrication DC capacitance and dV/dt is the scan rate. EIS measurements We investigated a new fabrication technique utilizing a combi- were conducted under equilibrium conditions by applying small (10 mV) AC signals over the frequency range of 1 Hz to 10 kHz nation of micro and nano schemes to realize non-Faradaic CNT using a lock-in amplifier (SR830, Stanford Research Systems) basedelectrodeswithveryhighspecificcapacitance usinga and a potentiostst (263A, Princeton Applied Research). simple fabrication process. To support a simple and robust fabrication process, the electrodes are made exclusively of 2.4 Retina preparation and handling CNTs so no complex fabrication integration was required. The general fabrication process, described in Fig. 1a, is based on Embryonic chick retinas (day 14) were isolated and trans- loosely-bound MWCNT films grown using CVD process from ferred to the experimental chamber, placed RGC layer down athinNilayer (Fig. 1a-2). The Ni layer is deposited on a onto the flexible MEAs. Better coupling between the tissue support Si/SiO substrate (Fig. 1a-1). An uncured polymer and the electrodes was achieved by placing a small piece of (e.g. PDMS or polyimide) is then casted on the substrate polyestermembranefilter(5 μm pores; Sterlitech, Kent, WA, with the CNT film. After curing, the CNTs are integrated USA) on the retina followed by a ring weight which served as with the polymer. The polymer and the CNT films can then a slice anchor holder. Retinas were kept at physiological be peeled-off from the surface (Fig. 1a-3). Similar results conditions according to a previously reported protocol can be obtained by applying an adhesive tape against the (Hammerle et al. 1994) with temperature of 34 °C and perfuse CNT pattern or by using vapor deposition of Parylene C. The (2–5 ml/min) with oxygenated artificial cerebro-spinal fluid. CNT carrying film and a second layer of holey PDMS membrane are then bonded together (Fig. 1a-4) to form a 2.5 Electrical recording flexible circuit containing passivated CNT conducting tracks and exposed CNT electrodes. The biocompatibility of PDMS, Neuronal electrical signals were amplified (gain ×1,200, parylene C and polyimide is well established. Polyimide and MultiChannel Systems MEA1060-Inv, Reutlingen, Germany), parylene C have comparable elastic moduli of ~2–4 GPa (two digitized using a 128-channel analogue to digital converter to three orders of magnitude lower than that of metal and 46 Biomed Microdevices (2014) 16:43–53 Fig. 1 All-CNT flexible multi- electrode arrays. a Electrode fabrication scheme. (1) The process is based on a single photolithographically defined Ni catalyst layer. (2) The CNT film is then grown using a CVD process. (3) Next, the film is transferred to a polymeric support (e.g. medical adhesive tape, PDMS, Parylene C, polyimide). (4) Finally, a second polymeric layer (PDMS) with predefined holes is bonded with the CNT carrying film for passivation. b Different patterns of flexible CNT electrode arrays on different support layers: (1) PDMS, (2) medical adhesive tape, (3) Parylene C and (4) polyimide silicon), while PDMS elasticity (depending on preparation and most importantly, at no stage of the process, the surfaces of conditions) can be further reduced down to ~0.05 MPa the CNTelectrodes are exposed to any solvents, photo-resists, or (Rousche et al. 2001; Brown et al. 2005;Rodger etal. 2008; electro-plating baths rendering the entire process very clean, and Meacham et al. 2011). Polyimide can be patterned using therefore ensuring the non-Faradaic nature of the electrodes. standard microfabrication such as photolithography and reac- While the process described above appears to be straight tive ion etching (Cheung et al. 2007;Mercanzini et al. 2008) forward, two critical properties must be carefully maintained to and parylene C has superior resistance to moisture. Finally, the guarantee proper function of the end device. Foremost, is the adhesive medical tape enables quick and simple fabrication high effective surface area of the electrodes. Clean CNTs have with well exposed CNT films. Such films may be well suited outstanding electrochemical properties, however, impurities for skin-applied electrode arrays. and polymeric residues can dramatically hamper the proper The process is general enough to include additional layers operation of the electrodes. Indeed, we have noticed that the for multi-layer stacking, as well as to incorporate additional cleanliness of the electrode surface can be compromised if the elements such as photodiodes. Photodiodes integration with polymer (e.g. PDMS) penetrates the CNT film. The second CNT electrode array would enable neuronal stimulation using critical requirement is the electrical conductivity of the CNT light, a desirable feature in retinal implants aimed at substitut- interconnects. ing degenerated photoreceptors. This scheme has several notable advantages over previously 3.2 Characterization of flexible CNT devices proposed concepts. Foremost, it is simple for implementation, requiring only two independent lithographic steps. Unlike dis- We begin by discussing the cleanliness of the CNT films and persion methods, the use of standard lithography allows high their electrochemical properties. We found that different poly- resolution patterning of the CNT film and a simple integrating of mers and deposition methods (e.g. spin coating, applying adhe- the CNT pattern with the polymer substrate. Moreover, the sive tape, and vapor deposition) dramatically affect the extent of entire device it based only on very few elementary fabrication the polymer penetration into the film. Accordingly, careful steps. Additionally, the device benefits from strong overall validation of the morphological and electrochemical properties stability against peeling and degradation due to seamless inte- of the electrodes is important. Validation was achieved by using electrode arrays with different electrode diameter (100, 150, gration between the electrodes and conducting traces. Finally Biomed Microdevices (2014) 16:43–53 47 200, 250, 300, 350, 400 and 450 μm). Electrode arrays were intertwined MWCNTs were observed on the medical tape and realized following the scheme depicted in Fig. 1a and were then on the Parylene C surfaces (Fig. 2a-1 and a-2). Cross section systematically tested. The CNT film cleanliness was first vali- imageofthe CNTfilmonamedicaladhesivetape(Fig. 2a-1, dated qualitatively using environmental scanning electron mi- inset) demonstrates a CNT film on top of the flexible medical croscopy (ESEM). Figure 2a shows ESEM images of a typical tape substrate. Under proper preparation conditions clean CNT CNTsurface on a medical adhesive tape (Fig. 2a-1), on Parylene films were reliably transferred to all different flexible substrates C(Fig. 2a-2) and on PDMS (Fig. 2a-3). While part of the CNT described above (see Section 2). film is embedded in the cured PDMS, the top surface of the The ESEM imaging was followed by electrochemical CNTs is clearly exposed (Fig. 2a-3). Apparently clean, highly characterization using CV that records current resulting from Fig. 2 Electrochemical and transport properties of CNT devices. a An ESEM image of MWCNTs on a medical adhesive tape; Inset: a zoom out ESEM cross section image of a MWCNT film on a medical adhesive tape (marked with arrow) (1), Parylene C (2), and PDMS (3), scale bar: 2 μm; Inset scale bar: 100 μm. b CV scans of a CNT electrode (100 μm in diameter) at different scan rates with blue, red and black lines corresponding to scan rates of 15, 50 and 150 mV/s respectively. c Charging current versus scan rate of a CNT electrode (100 μm in diameter), solid line is a linear fit. d CNT electrode capacitance versus electrode surface area, solid line is a linear fit. Inset: Microscope image of CNT electrodes (100, 150, 200 and 250 μmin diameter). Measurements shown are for a single representative set of devices. e CNT electrode (100 μm in diameter) impedance versus frequency. All electrochemical measurements were performed in PBS with an Ag/AgCl reference electrode. f Raman spectrum of a MWCNT film. g CNT film electrical resistance versus number of squares. Inset: Microscope image of different length TiN-CNTs-TiN bars used to derive film electrical resistance. Measurements shown are for a single representative set of devices 48 Biomed Microdevices (2014) 16:43–53 scanning the applied voltage, and EIS, which measures film resistivity was identified, during or after these manipula- frequency-dependent changes in the impedance. CV and EIS tions. A major concern when considering the biocompatibility measurements were performed with a three-electrode cell con- of the CNTelectrodes is Ni traces and we have tested our CNT figuration using phosphate buffered saline (PBS) and Ag/AgCl electrodes for Ni traces and performed biocompatibility tests reference electrode. The CV data (Fig. 2b) is markedly flat, by culturing rat cortical cells on the CNT films (according to a showing no signs of reactivity, as expected from clean CNT previously reported protocol (Shein et al. 2009)). We have electrodes (Gabay et al. 2007). Current versus scan-rate plots conducted energy-dispersive x-ray spectroscopy (EDS) tests show clear linear dependence (Fig. 2c) in accordance with a that revealed very small residues of Ni. Apparently Ni is double layer capacitor model. Finally, the capacitance of differ- effectively embedded in the CNTs and has no adverse effects. ent size electrodes was calculated and plotted and the specific Finally, to reliably measure the electrical resistance of the capacitance value was derived, yielding values as high as 2 MWCNT traces, a special testing scheme was implemented. −2 mFcm (Fig. 2d). Variation of the impedance with frequency Sets of different length MWCNT bars (width and height (1 Hz to 10 kHz) is presented in Fig. 2e. The impedance of a remained constant) were fabricated (Supplementary Fig. 2)with 100 μm diameter CNT electrode (including its long conducting TiN contacts (TiN-CNTs-TiN). The TiN pads are instrumental to trace) at biologically relevant frequency for neural recording of achieve reliable Ohmic contacts to the CNT films, guarantying 1kHz is 55 kΩ. The electrochemical measurements were also consistent measurements. It should be noted that while the used as a tool to directly quantify the extent of the clean surface. contact resistance of TiN is substantial, TiN is a conducting Sensitive surface analytical methods such as X-ray photoelec- material most suitable for CNT growth under the high temper- tron spectroscopy (XPS) could also be used as complementary ature of the CVD process and therefore is a very convenient tool to electrochemical measurements. material to perform the film resistance validation discussed here. Since the CNT films also constitute the circuit lines of our Current versus voltage trace for each TiN-CNTs-TiN bar was devices, their electrical resistance is consequential (Agrawal recorded and the electrical resistance was calculated. All sam- et al. 2007). The electrical performances of MWCNTs depend ples exhibited an Ohmic behavior with values ranging between on many factors such as average length, diameter, wall num- 2 and 15 kΩ. To derive their sheet resistance, electrical resistance ber, structural defects, film thickness, and the amount of values were plotted versus the number of squares in each bar amorphous carbon (Ferrari and Robertson 2000). While some (Fig. 2g; for explanation on sheet resistance calculations see of these parameters can be controlled in the growth process to Supplementary Fig. 3). Values ranging between 160 and 1,850 optimize the conductivity of the films, CNT films generally Ω/□ for different CVD growth conditions of the MWCNT film suffer from poor conductivity compared with typical metals. were obtained. Owing to the high electrode-electrolyte imped- We note that for our device needs, owing to the large ance values, we conclude that the CVD grown MWCNT films electrode-solution impedance, exceptionally high trace con- are conducting well enough to be readily used as effective ductivity is not critically important and values in the order of conducting traces for our application. several kilo ohms are acceptable. To validate the CNT film quality and to quantify the 3.3 Extracellular neuronal recording and stimulation electrical conductivity values, CNT films were characterized using the flexible CNT MEA using Raman spectroscopy (RS) and direct electrical measure- ments respectively. RS was performed to characterize the Having established the electrical as well as the electrochemical nature and the quality of the MWCNT films (Fig. 2f). properties of the CNT films, we now turn to describe the electro- Raman spectrum of the CNT films show two distinct peaks physiological performances of the flexible electrodes. An elec- at 1,360 (D-band) and 1,580 cm−1(G-band)(Thomsen and trode array compatible with a standard multi-electrode array Reich 2007). We used the ratio between the D and the G band recording and stimulation setup was realized on a printed circuit (I /I ) as a crude characterization of the defect density and board (PCB) support (Fig. 3a). The array consists of 16 elec- D G each CNT film was measured at 20 different sites. The I /I trodes on a medical tape support each connected to an external D G for all films was higher than one, indicating fairly poor film pad. A top PDMS passivation layer, 150 μm thick and with quality associated with the highly entangled CNTs. However, 50 μm diameter holes, was used to define the effective size of we have extensively used similar films in the past to perform the electrodes (Fig. 3a, inset). The flexible array was then recording from dissociated neurons (Gabay et al. 2007; Shein mounted onto the PCB carrier to accommodate the link between et al. 2009) and from mouse retina (Shoval et al. 2009)with the electrodes and external amplifiers. A glass cylinder was excellent results. The obtained films are thus very well suited glued to the PCB support to serve as a well for the physiological for neuronal stimulation. To validate the durability of the CNT medium. films upon mechanical stress we have tested the electrical Embryonic chick retina (day 14) was used as a neuronal properties of the CNT films following repeated cycles (up to model. The retina was extracted and transferred to the medium chamber under physiological conditions. The retina was then 30 cycles) of folding and winding. No significant change in Biomed Microdevices (2014) 16:43–53 49 Fig. 3 Flexible CNT MEA for extracellular neuronal recording and stimulation. a The flexible CNT electrode array mounted on a PCB support linking the electrodes to external amplifiers (scale bar: 5 mm). The array consists of 16 electrodes and a top passivation layer with 50 μm-diameter holes which define the electrode effective size. An embryonic chick retina (day 14) was flattened on the electrode array. The edge of the retina is marked with a dashed line. Inset: enlargement of the electrodes area (area marked with a solid line; scale bar: 200 μm). b Acircuit model for extracellular recording and stimulation from a neural tissue using the flexible electrode array. The model demonstrates the electrochemical interface resistance and capacitance of the CNT electrode and the solution derived shunt capacitance as well as the point of stimulation flattened on the electrode array (Fig. 3a), with the retinal 4 Discussion and conclusions ganglion cell (RGC) layer facing down (as in an epi-retinal implant) and was anchored with a weight. Figure 3b illustrates We can now turn to look at how our new CNT electrodes rank a circuit model for extracellular recording and stimulation of compared with previously reported technologies. Table 1 neuronal tissue using the micro electrode array, depicting the summarizes specific DC capacitance, stimulation threshold electrochemical interface resistance and the capacitance of the and SNR values obtained with other CNT and flexible elec- CNT electrode as well as the solution derived shunt capaci- trode technologies. The table refers to studies that demonstrat- tance and the stimulation point. ed either recording or stimulation of neuronal activity. DC −2 At day 14 the embryonic retina is still at an early develop- capacitance values of 1–10 mFcm were measured from mental stage and clear spontaneous activity waves were most CNT electrodes on both rigid and flexible substrates. recorded demonstrating the overall functionality of the device The all CNT flexible MEA presented in this study is well −2 and the setup. We next tested the CNT electrodes suitability to within this range with 2 mFcm , exceeding both CNT elec- −2 evoke electrical activity in the retina tissue. Stimulation was trodes grown directly on flexible polyimide with 0.1 mFcm achieved at currents as low as 4 μA(Fig. 4a and b)and (Hsu et al. 2010;Chenetal. 2011b) aswellasPtelectrodes stimulation pulse width of 1 ms. With nearly perfectly capac- coated with SWCNT (drop coating) on a rigid Pyrex substrate −6 −2 itive electrodes, these values are well within the limits of safe with 4.5·10 mFcm (Gabriel et al. 2009). It should be noted stimulation. The observed electrical response is typical for that SNR and stimulation threshold values depend on the pre-synaptic cells activation. Validation of the synaptic pro- examined tissue as well as on the size and shape of the cesses was achieved with the use of the synaptic blockers 6- electrode. Therefore they cannot be used as a direct measure cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5- of MEA devices. The SNR in particular, provides only a phosphonovaleric acid (APV). 400 s after the introduction of validation for the acceptable performance of the electrodes. the synaptic blockers no retinal ganglion cell activation was A stimulation threshold of 4 nC measured by our flexible measured (Fig. 4c). CNT MEA is lower than that reported by other CNT MEA 50 Biomed Microdevices (2014) 16:43–53 rigid and flexible technologies, our device benefits from the advantages of a very clean and simple fabrication scheme and most importantly a seamless integration between the electrode and the circuit, ultimately supporting a reliable and scalable fabrication of state of the art flexible MEAs. To conclude this discussion, our novel flexible CNT electrodes, as other clean carbon basedelectrodes, aredistinguishedbyhavinga clear capacitive nature. Being produced by a simple, clean and robust process, these electrodes properties surpass previously de- scribed technologies. Notwithstanding these promising results, some improve- ments in the fabrication scheme are desirable. For exam- ple, the 150 μm insulation layer locates our electrodes at a significant distance from the tissue, limiting the spatial reso- lution of the device as reflected in the relative low amplitude of the recorded signals (Fig. 4a). Reducing the thickness of the insulation layer will also improve the adhesion of the tissue to the electrodes, further promoting the spatial resolution. To summarize, a new scheme based on CNTs was presented and demonstrated as an advantageous approach to form high performance neuronal electrode array devices. The electrodes gain their performances from the combination of several dif- ferent CNT properties. Foremost, CNTs films have extremely large surface area making them very effective electrochemical electrode with capacitive charge injection mechanism. CNTs are also inert and strong, making the electrodes stable in biological conditions. As CNT films are suitable to withhold bending, they are very well suited for flexible electronic appli- cations. In the realm of multi-electrode arrays, this feature is particularly important as flexible MEA devices are of great interest for implantable applications. Unlike other coatings Fig. 4 Electrical recording and stimulation of chick retina with that may tend to crack and disconnect from the flexible flexible CNT MEA. a Evoked activity using a biphasic cathodic first substrate during bending, CNT films are durable owing to pulse (arrowhead). The large signal at t=0 is an artifact of the stimulation. their remarkable mechanical properties and the unique struc- b Firing rate of evoked activity at different stimulation intensities (3–10 ture of the MWCNTs film. The entangled bundles of tubes, nC). c Firing rate of evoked activity after synaptic blockers CNQX and APV application (stimulation was applied every 10 s). After 400 s no forming a dense and continuous yet porous film, make these retinal ganglion cells activation is observed films particularly optimal for neuronal applications. Additio- nally, the adhesion between the CNTs and the polymeric substrate is strong, making the CNT film an integrated part technologies (both rigid and flexible) and is similar to values of the substrate. Since CNTs are chemically inert they are obtained with TiN commercial devices, further demonstrating also durable against corrosion, a very common challenge in the overall high quality of our devices. conventional metal technology in biological applications. The We have shown that our new flexible all CNT MEA perfor- circuit structure is seamless and all elements, connecting pads, mances are equivalent to rigid CNT technologies with the conducting traces and electrodes are made of CNT. This is an obvious major advantage of being flexible. Two flexible CNT enormous advantage for both in vivo and in vitro long term use technologies used for neuronal recording and stimulation were since it eliminates delamination of the coatings and the for- reported before. Lin and co-workers fabricated a vertically mation of cracks. These cracks result with leakage currents aligned CNT (VACNT) MEA embedded in Parylene-C film and failure of the device as often occurs with layering and (Lin et al. 2009) while Hsu and co-workers used low temper- connection of different materials. All these properties are ature CVD (i.e. 400 °C) to directly grow CNT MEA on added to the relatively simple and robust fabrication process polyimide (Hsu et al. 2010;Chenet al. 2011b). The specific discussed above. This fabrication process can be easily ex- capacitance of our CNT MEA is significantly higher than that tended to include elements such as photodiodes and allows for stacking of different functionality layers, make the all-carbon- of the directly grown CNTs on polyimide. Compared with both Biomed Microdevices (2014) 16:43–53 51 Table 1 Neuronal recording and stimulation multi-electrode technologies Reference Electrode description In vitro testing scheme Area (μm ) Specific DC Stimulation SNR −2 capacitance (mFcm ) threshold Rigid CNT Wang et al. (2006) Vertically aligned MWCNT (CVD) MEA on Embryonic rat hippocampal cells 2,500–10,000 1.6 10 nC NA MEAs a quartz substrate with PEGPL coating Gabay et al. (2007) MWCNT (CVD) MEA on a Si substrate Rat cortical cultures 5,024 10 NA 135 Commercial TiN MEA; not coated with CNT Rat cortical cultures 706 2.5 NA 4 Keefer et al. (2008) ITO MEAcoatedwithMWCNT-Au Mice frontal cortex cultures 314 3.24 195 mV Recording (electrochemical deposition) Gabrieletal. (2009) Pt MEA coated with SWCNT (drop coating) on Isolated rabbit retinas 1,256 0.000045 NA 21 aPyrex substrate Su et al. (2010) Cone-shaped Si MEA coated with MWCNT Crayfish giant neurons 10–2,000 2.5 NA 42.3 (CVD) after O plasma Fuchsberger et al. (2011) TiN MEA coated with MWCNT (micro-contact Rat postnatal hippocampal 5,024 2.5 NA Recording printing). cultures Flexible CNT Lin et al. (2009) Vertically aligned CNT electrodes embedded in Crayfish nerve cord 1,962 Not available NA 257 MEAs Parylene C film Hsu et al. (2010) CNT MEA (CVD) on polyimide after Crayfish giant neurons 3,600–40,000 0.1 NA 150 UV-ozone modification Chen et al. (2011b) CNT MEA (CVD) on polyimide after UV-ozone Crayfish caudal photoreceptor 7,850–125,600 0.21 NA 6.2 modification In vivo, EcoG of rat motor cortex NA 8.68 This paper CNTs on a medical tape Embryonic chick retina 1,962–125,664 2 4 nC 20 Other flexible Blau et al. (2011) PEDOT:PSS+5 % ethylene glycol (v/v) Embryonic rat and mice hearts 11,304 Not available NA 100 MEAs electrodes on PDMS Rat cortico-hippocampl cultures 5 Chen et al. (2011a) Au MEA on Parylene C reinforced with Crayfish lateral giant nerve 2,500 Not available NA 32 SU8 and PEG filled micro channels NA-stimulation/recording were not applied using the MEA Calculated from EIS at 1 Hz Calculated from data in the article 52 Biomed Microdevices (2014) 16:43–53 A.C. Ferrari, J. Robertson, Interpretation of Raman spectra of disordered nanotube flexible neural electrodes, presented here, a promis- and amorphous carbon. Phys. Rev. B 61(20), 14095–14107 (2000) ing element in future neuro-prosthetic devices. A.A. Fomani, R.R. Mansour, Fabrication and characterization of the flexible neural microprobes with improved structural design. Sen- Acknowledgments The authors thank Nurit Atar for providing the sors Actuators B Phys. 168(2), 233–241 (2011) polyimide substrates. Micro and nano fabrication and characterization were K. Fuchsberger, A. Le Goff et al., Multiwalled carbon-nanotube- performed at Tel Aviv University Center for Nanoscience and Nanotech- functionalized microelectrode arrays fabricated by microcontact nology. We also acknowledge the support of a grant from the Israel printing: platform for studying chemical and electrical neuronal Ministry of Science and Technology, the Israel Science Foundation and signaling. Small 7(4), 524–530 (2011) the European Research Council funding under the European Community’s T. Gabay, M. Ben-David et al., Electro-chemical and biological properties Seventh Framework Program (FP7/2007– 2013)/ERC grant agreement of carbon nanotube based multi-electrode arrays. Nanotechnology FUNMANIA-306707. 18(3) (2007) G. Gabriel, R. Gomez et al., Easily made single-walled carbon nanotube Open Access This article is distributed under the terms of the Creative surface microelectrodes for neuronal applications. Biosens. 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All-carbon-nanotube flexible multi-electrode array for neuronal recording and stimulation

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Biomed Microdevices (2014) 16:43–53 DOI 10.1007/s10544-013-9804-6 All-carbon-nanotube flexible multi-electrode array for neuronal recording and stimulation Moshe David-Pur & Lilach Bareket-Keren & Giora Beit-Yaakov & Dorit Raz-Prag & Yael Hanein Published online: 24 August 2013 The Author(s) 2013. This article is published with open access at Springerlink.com . . Abstract Neuro-prosthetic devices aim to restore impaired Keywords Carbon nanotubes Multi electrode array . . . function through artificial stimulation of the nervous system. Neuronal recording Neuronal stimulation Flexible A lingering technological bottleneck in this field is the reali- Prosthesis zation of soft, micron sized electrodes capable of injecting enough charge to evoke localized neuronal activity without causing neither electrode nor tissue damage. Direct stimula- 1 Introduction tion with micro electrodes will offer the high efficacy needed in applications such as cochlear and retinal implants. Here we Flexible neuronal micro electrode technology progressed ex- present a new flexible neuronal micro electrode device, tensively over the past several decades hand in hand with the based entirely on carbon nanotube technology, where both overall development in the field of neuro-prosthetics. Several the conducting traces and the stimulating electrodes consist novel fabrication approaches suited for micro electrode appli- of conducting carbon nanotube films embedded in a poly- cations were devised. These schemes attempt to achieve flex- meric support. The use of carbon nanotubes bestows the ible electronic technology integration with high surface rough- electrodes flexibility and excellent electrochemical proper- ness while maintaining bio-compatibility and durability in ties. As opposed to contemporary flexible neuronal elec- physiological conditions. Commonly, these devices use metal trodes, the technology presented here is both robust and electrodes such as gold (Sandison et al. 2002;Chen etal. the resulting stimulating electrodes are nearly purely ca- 2009, 2011a; Wester et al. 2009;Lacour etal. 2010;Wei pacitive. Recording and stimulation tests with chick retinas et al. 2011), titanium (Takeuchi et al. 2004), electroplated were used to validate the advantageous properties of the platinum black (Adams et al. 2005;Rodger etal. 2008; electrodes and demonstrate their suitability for high-efficacy Graudejus et al. 2009, 2012; Rui et al. 2011), tungsten (Wei neuronal stimulation applications. et al. 2011), platinum (Cheung et al. 2007; Mercanzini et al. 2008; Myllymaa et al. 2009; Viventi et al. 2011) and iridium (Rodger et al. 2008; Fomani and Mansour 2011)deposited on Moshe David-Pur and Lilach Bareket-Keren contributed equally to this various flexible supports such as polyimide (Sandison et al. work. 2002; Takeuchi et al. 2004; Cheung et al. 2007; Viventi et al. Electronic supplementary material The online version of this article 2011), parylene C (Rodger et al. 2008; Wester et al. 2009)or (doi:10.1007/s10544-013-9804-6) contains supplementary material, poly(dimethylsiloxane) (PDMS) (Graudejus et al. 2009, 2012; which is available to authorized users. Lacour et al. 2010; Wei et al. 2011). These metal electrodes : : : M. David-Pur L. Bareket-Keren G. Beit-Yaakov Y. Hanein achieve neural stimulation by Faradaic current injection School of Electrical Engineering, Tel-Aviv University, through the electrode-electrolyte interface. Electron transfer, Tel-Aviv 6997801, Israel associated with the Faradaic charge stimulation, can induce : : : : M. David-Pur L. Bareket-Keren G. Beit-Yaakov D. Raz-Prag irreversible reduction and oxidation reactions that can damage Y. Hanein (*) both the electrode and the tissue (Merrill et al. 2005; Cogan Tel-Aviv University Center for Nanoscience and Nanotechnology, 2008). Storage and injection of charge can also occur from Tel-Aviv University, Tel-Aviv 6997801, Israel valence changes in multivalent electrode coatings such as e-mail: [email protected] 44 Biomed Microdevices (2014) 16:43–53 Iridium oxide (Robblee et al. 1983; Klein et al. 1989)that 2Methods undergo reversible reduction-oxidation reactions (Merrill et al. 2005;Cogan 2008). Consequently, capacitive charge 2.1 Flexible CNT MEA fabrication stimulation is preferable for neuronal stimulation, as it involves only a displacement current associated with Flexible CNT MEAs were fabricated as follows. First, standard charging and discharging of the electrode-electrolyte dou- lithography (AZ1518 photoresist; Clariant) was used to form ble layer (Merrill et al. 2005). Common capacitive elec- the desired circuit pattern on a Silicon/Silicon dioxide (Si/ trode materials include titanium nitride (TiN), tantalum- SiO ) support. A 2.5 nm Ni catalyst layer was deposited using tantalum oxide and the more recently investigated carbon an e-beam evaporator (VST). A resist lift-off process was then nanotubes (CNTs) (Rose et al. 1985; Gabay et al. 2007; performed, followed by an oxygen plasma treatment to remove Cogan 2008). Conducting polymers, such as polypyrrole all photoresist residues. Next, MWCNTs were grown by chem- (PPy) and poly(ethylenedioxythiophene) (PEDOT) are mixed ical vapor deposition (CVD) (Lindberg Blue) with ethylene conductors, exhibiting both electron and ion transport within (20 sccm) and hydrogen (1,000 sccm) at 900 °C. A flexible the polymer film (Ludwig et al. 2006; Abidian et al. 2010; substrate, medical adhesive tape, parylene C, polyimide or Blau et al. 2011). poly(dimethylsiloxane) (PDMS), was applied and peeled off A related key requirement in neuronal electrode technology with the CNT pattern. Medical adhesive tape (Steri-Drape, is large specific capacitance (C ). Large specific capacitance 3 M) was attached to the CNT pattern and pressed lightly. reduces the electrode impedance, without increasing its geo- Parylene C was applied by on the CNT pattern by vapor metric area. The reduction in impedance is essential for effi- deposition. Polyimide, prepared from a poly(pyromellitic cient, high resolution neuronal recording and stimulation dianhydride-co-4,4′-oxydianiline) 15 wt.% solution in N- (Robinson 1968;Loebet al. 1995; Merrill et al. 2005; methyl-2-Pyrrolidone (Sigma-Aldrich) was spin coated and Cogan 2008). One of the best materials to exhibit both large cured at 350 °C under nitrogen atmosphere. Uncured PDMS specific capacitance as well as non-Faradaic behavior is po- (Sylgard 184, Dow Corning), mixed in a 10:1 ratio by weight, −2 rous TiN with C in the range of 2 mFcm (Gabay et al. was casted or spin coated and cured at 60 °C. Peeling-off of 2007). It was recently demonstrated that pristine CNTs exhibit very thin PDMS films (~100 μm) required the deposition of a similar performances to those of TiN with C values in the thin Cr layer (2 nm) followed by Au layer (6 nm) using an e- −2 range of 3–10 mFcm (Gabay et al. 2007). Accordingly, beam evaporator prior to PDMS application to reduce the CNTs have been suggested by several studies as a future adhesion between the SiO and the PDMS. To guarantee the material for neuronal stimulation applications and several final cleanliness of the CNT film, half cured PDMS films fabrication schemes have been studied. Primarily, direct (60 °Cfor 5min) wereused inastateofa viscouspolymer growth of CNT electrodes (Wang et al. 2006; Gabay et al. and were applied as an adhesive film onto the CNTs. The 2007;Su et al. 2010) as well as CNT coatings of metal use of partially cured films substantially reduced wetting of electrodes by electro-polymerization (Keefer et al. 2008), drop the CNTs. Finally, a passivation PDMS membrane with coating from a solution (Gabriel et al. 2009) and micro-contact predefined holes and the CNT flexible circuit were bonded. printing (Fuchsberger et al. 2011) on a rigid support were The PDMS passivation layer was prepared using a SU8- described. To accommodate flexibility, CNT transfer onto 3050 (MICRO-CHEM) patterned mold (see Supplementary a polymeric support (Su, Lin et al. 2009;Tsaietal. 2009; Fig. 1). PDMS passivation was bonded using a custom made Carnahan et al. 2010;Chang-Jianetal. 2010) was recently holder mounted on a microscope stage. PDMS-PDMS bonding presented. However, the lack of a simple platform to was promoted by oxygen plasma treatment to both films. allow the realization of fully functional devices consisting of Oxidation of PDMS surface exposes silanol groups (Si-OH) pristine CNT surfaces has left this technology so far largely so when the two films are brought together they form covalent unused. siloxane bonds (Si-O-Si) which provide excellent sealing (Duffy Here, we present a novel flexible neuronal micro elec- et al. 1998). Bonding with polyimide and parylene C substrates trode device, based solely on multi-walled CNT (MWCNT) was achieved by means of an intermediate thin layer of liquid films embedded in a flexible polymeric support. We dem- PDMS followed by curing at 60 °C. Finally, the medical tape onstrate a new simple and robust fabrication technique to was bonded with the passivation by exploiting the adhesiveness realize the seamless CNT circuit on the flexible substrate. of the tape. These processes yielded 30–65 % clean and capac- Next, the electrical and electrochemical properties of the itive electrodes utilizing an entirely manual preparation. We CNT electrodes and of the CNT conducting traces were expect that mechanizing the process can dramatically improve studied, using a scheme of specially designed electrode the yield. For electrophysiological experiments the flexible arrays. Finally, the flexible CNT MEA was applied for CNT MEA was mounted on a PCB (49×49 mm )with 60 extracellular neuronal recording and stimulation of chick Au traces and contact pads. A glass chamber was mounted retinas. on top of the PCB using uncured PDMS. Biomed Microdevices (2014) 16:43–53 45 2.2 Electrical resistance measurements of CNT films (MultiChannel Systems MC_Card, Reutlingen, Germany) and recorded (MultiChannel Systems MC_Rack, Reutlingen, Sets of CNT bars with different lengths and constant width and Germany). All additional signal analysis was performed using height, were fabricated between TiN pads as follows. A 100 nm Matlab software (MathWorks). Electrically stimulated neuro- TiN layer was sputtered (MRC RF sputter) on a Si/SiO support nal activity was digitized at 20 kHz and spikes were detected followed by lithography and reactive ion etching (Nextral 860) to by setting a threshold of signal to noise ratio (SNR) SNR>4 pattern the TiN pads. Due to a marked difference between the (related to the pre-stimulation noise level). Due to amplifier diffusion rate of Ni through SiO and TiN at the CNT growth saturation artifact, the period of 20 ms post stimulation was temperature, two layers of Ni were deposited by an e-beam ignored. The response of the retinal site to electrical stimula- evaporator (VST). The first layer (8 nm) was deposited on the tion was defined as the detected spikes count. inner half of the TiN pads and the second layer (2.5 nm) between the TiN pads on the SiO substrate. Finally a CNT film was 2.6 Electrical stimulation grown by CVD (for detailed illustration see Supplementary Fig. 2). Current versus voltage screen of the different length Chick retinas were electrically stimulated using a dedicated TiN-CNTs-TiN bars was recorded and their electrical resistance stimulator (STG-1008, Multi-Channel Systems, Reutlingen, was calculated (for details see Supplementary Fig. 3). Germany) through one of the MEA electrodes each time (versus an external reference) with charge-balanced bi- 2.3 Electrochemical analysis phasic (cathodic first) current stimulation (pulse width: 1 ms and pulse amplitude: 1–10 μA). Each stimulation session The electrochemical properties of the CNT electrodes were char- included stimulations at the entire intensity range (increased acterized by performing cyclic voltammetry (CV) and electro- by 1 μA every 10 s) and was repeated five times. To validate chemical impedance spectroscopy (EIS) in PBS. An Ag/AgCl that the electrical stimulation resulted from synaptic process- electrode served as a reference electrode and a platinum wire as a es, synaptic blockers CNQX (Sigma) and APV (Sigma) were counter electrode. CV measurements were conducted using a applied (75 μM and 400 μMrespectively). potentiostat (263A Princeton Applied Research) under ambient conditions and recorded using the PowerCV software (Princeton Applied Research). The DC capacitance was derived from the 3Results oxidation current versus the scan rate data according to the relation:i=C·dV/dt in which i is the charging current, C is the 3.1 All-CNT flexible MEA fabrication DC capacitance and dV/dt is the scan rate. EIS measurements We investigated a new fabrication technique utilizing a combi- were conducted under equilibrium conditions by applying small (10 mV) AC signals over the frequency range of 1 Hz to 10 kHz nation of micro and nano schemes to realize non-Faradaic CNT using a lock-in amplifier (SR830, Stanford Research Systems) basedelectrodeswithveryhighspecificcapacitance usinga and a potentiostst (263A, Princeton Applied Research). simple fabrication process. To support a simple and robust fabrication process, the electrodes are made exclusively of 2.4 Retina preparation and handling CNTs so no complex fabrication integration was required. The general fabrication process, described in Fig. 1a, is based on Embryonic chick retinas (day 14) were isolated and trans- loosely-bound MWCNT films grown using CVD process from ferred to the experimental chamber, placed RGC layer down athinNilayer (Fig. 1a-2). The Ni layer is deposited on a onto the flexible MEAs. Better coupling between the tissue support Si/SiO substrate (Fig. 1a-1). An uncured polymer and the electrodes was achieved by placing a small piece of (e.g. PDMS or polyimide) is then casted on the substrate polyestermembranefilter(5 μm pores; Sterlitech, Kent, WA, with the CNT film. After curing, the CNTs are integrated USA) on the retina followed by a ring weight which served as with the polymer. The polymer and the CNT films can then a slice anchor holder. Retinas were kept at physiological be peeled-off from the surface (Fig. 1a-3). Similar results conditions according to a previously reported protocol can be obtained by applying an adhesive tape against the (Hammerle et al. 1994) with temperature of 34 °C and perfuse CNT pattern or by using vapor deposition of Parylene C. The (2–5 ml/min) with oxygenated artificial cerebro-spinal fluid. CNT carrying film and a second layer of holey PDMS membrane are then bonded together (Fig. 1a-4) to form a 2.5 Electrical recording flexible circuit containing passivated CNT conducting tracks and exposed CNT electrodes. The biocompatibility of PDMS, Neuronal electrical signals were amplified (gain ×1,200, parylene C and polyimide is well established. Polyimide and MultiChannel Systems MEA1060-Inv, Reutlingen, Germany), parylene C have comparable elastic moduli of ~2–4 GPa (two digitized using a 128-channel analogue to digital converter to three orders of magnitude lower than that of metal and 46 Biomed Microdevices (2014) 16:43–53 Fig. 1 All-CNT flexible multi- electrode arrays. a Electrode fabrication scheme. (1) The process is based on a single photolithographically defined Ni catalyst layer. (2) The CNT film is then grown using a CVD process. (3) Next, the film is transferred to a polymeric support (e.g. medical adhesive tape, PDMS, Parylene C, polyimide). (4) Finally, a second polymeric layer (PDMS) with predefined holes is bonded with the CNT carrying film for passivation. b Different patterns of flexible CNT electrode arrays on different support layers: (1) PDMS, (2) medical adhesive tape, (3) Parylene C and (4) polyimide silicon), while PDMS elasticity (depending on preparation and most importantly, at no stage of the process, the surfaces of conditions) can be further reduced down to ~0.05 MPa the CNTelectrodes are exposed to any solvents, photo-resists, or (Rousche et al. 2001; Brown et al. 2005;Rodger etal. 2008; electro-plating baths rendering the entire process very clean, and Meacham et al. 2011). Polyimide can be patterned using therefore ensuring the non-Faradaic nature of the electrodes. standard microfabrication such as photolithography and reac- While the process described above appears to be straight tive ion etching (Cheung et al. 2007;Mercanzini et al. 2008) forward, two critical properties must be carefully maintained to and parylene C has superior resistance to moisture. Finally, the guarantee proper function of the end device. Foremost, is the adhesive medical tape enables quick and simple fabrication high effective surface area of the electrodes. Clean CNTs have with well exposed CNT films. Such films may be well suited outstanding electrochemical properties, however, impurities for skin-applied electrode arrays. and polymeric residues can dramatically hamper the proper The process is general enough to include additional layers operation of the electrodes. Indeed, we have noticed that the for multi-layer stacking, as well as to incorporate additional cleanliness of the electrode surface can be compromised if the elements such as photodiodes. Photodiodes integration with polymer (e.g. PDMS) penetrates the CNT film. The second CNT electrode array would enable neuronal stimulation using critical requirement is the electrical conductivity of the CNT light, a desirable feature in retinal implants aimed at substitut- interconnects. ing degenerated photoreceptors. This scheme has several notable advantages over previously 3.2 Characterization of flexible CNT devices proposed concepts. Foremost, it is simple for implementation, requiring only two independent lithographic steps. Unlike dis- We begin by discussing the cleanliness of the CNT films and persion methods, the use of standard lithography allows high their electrochemical properties. We found that different poly- resolution patterning of the CNT film and a simple integrating of mers and deposition methods (e.g. spin coating, applying adhe- the CNT pattern with the polymer substrate. Moreover, the sive tape, and vapor deposition) dramatically affect the extent of entire device it based only on very few elementary fabrication the polymer penetration into the film. Accordingly, careful steps. Additionally, the device benefits from strong overall validation of the morphological and electrochemical properties stability against peeling and degradation due to seamless inte- of the electrodes is important. Validation was achieved by using electrode arrays with different electrode diameter (100, 150, gration between the electrodes and conducting traces. Finally Biomed Microdevices (2014) 16:43–53 47 200, 250, 300, 350, 400 and 450 μm). Electrode arrays were intertwined MWCNTs were observed on the medical tape and realized following the scheme depicted in Fig. 1a and were then on the Parylene C surfaces (Fig. 2a-1 and a-2). Cross section systematically tested. The CNT film cleanliness was first vali- imageofthe CNTfilmonamedicaladhesivetape(Fig. 2a-1, dated qualitatively using environmental scanning electron mi- inset) demonstrates a CNT film on top of the flexible medical croscopy (ESEM). Figure 2a shows ESEM images of a typical tape substrate. Under proper preparation conditions clean CNT CNTsurface on a medical adhesive tape (Fig. 2a-1), on Parylene films were reliably transferred to all different flexible substrates C(Fig. 2a-2) and on PDMS (Fig. 2a-3). While part of the CNT described above (see Section 2). film is embedded in the cured PDMS, the top surface of the The ESEM imaging was followed by electrochemical CNTs is clearly exposed (Fig. 2a-3). Apparently clean, highly characterization using CV that records current resulting from Fig. 2 Electrochemical and transport properties of CNT devices. a An ESEM image of MWCNTs on a medical adhesive tape; Inset: a zoom out ESEM cross section image of a MWCNT film on a medical adhesive tape (marked with arrow) (1), Parylene C (2), and PDMS (3), scale bar: 2 μm; Inset scale bar: 100 μm. b CV scans of a CNT electrode (100 μm in diameter) at different scan rates with blue, red and black lines corresponding to scan rates of 15, 50 and 150 mV/s respectively. c Charging current versus scan rate of a CNT electrode (100 μm in diameter), solid line is a linear fit. d CNT electrode capacitance versus electrode surface area, solid line is a linear fit. Inset: Microscope image of CNT electrodes (100, 150, 200 and 250 μmin diameter). Measurements shown are for a single representative set of devices. e CNT electrode (100 μm in diameter) impedance versus frequency. All electrochemical measurements were performed in PBS with an Ag/AgCl reference electrode. f Raman spectrum of a MWCNT film. g CNT film electrical resistance versus number of squares. Inset: Microscope image of different length TiN-CNTs-TiN bars used to derive film electrical resistance. Measurements shown are for a single representative set of devices 48 Biomed Microdevices (2014) 16:43–53 scanning the applied voltage, and EIS, which measures film resistivity was identified, during or after these manipula- frequency-dependent changes in the impedance. CV and EIS tions. A major concern when considering the biocompatibility measurements were performed with a three-electrode cell con- of the CNTelectrodes is Ni traces and we have tested our CNT figuration using phosphate buffered saline (PBS) and Ag/AgCl electrodes for Ni traces and performed biocompatibility tests reference electrode. The CV data (Fig. 2b) is markedly flat, by culturing rat cortical cells on the CNT films (according to a showing no signs of reactivity, as expected from clean CNT previously reported protocol (Shein et al. 2009)). We have electrodes (Gabay et al. 2007). Current versus scan-rate plots conducted energy-dispersive x-ray spectroscopy (EDS) tests show clear linear dependence (Fig. 2c) in accordance with a that revealed very small residues of Ni. Apparently Ni is double layer capacitor model. Finally, the capacitance of differ- effectively embedded in the CNTs and has no adverse effects. ent size electrodes was calculated and plotted and the specific Finally, to reliably measure the electrical resistance of the capacitance value was derived, yielding values as high as 2 MWCNT traces, a special testing scheme was implemented. −2 mFcm (Fig. 2d). Variation of the impedance with frequency Sets of different length MWCNT bars (width and height (1 Hz to 10 kHz) is presented in Fig. 2e. The impedance of a remained constant) were fabricated (Supplementary Fig. 2)with 100 μm diameter CNT electrode (including its long conducting TiN contacts (TiN-CNTs-TiN). The TiN pads are instrumental to trace) at biologically relevant frequency for neural recording of achieve reliable Ohmic contacts to the CNT films, guarantying 1kHz is 55 kΩ. The electrochemical measurements were also consistent measurements. It should be noted that while the used as a tool to directly quantify the extent of the clean surface. contact resistance of TiN is substantial, TiN is a conducting Sensitive surface analytical methods such as X-ray photoelec- material most suitable for CNT growth under the high temper- tron spectroscopy (XPS) could also be used as complementary ature of the CVD process and therefore is a very convenient tool to electrochemical measurements. material to perform the film resistance validation discussed here. Since the CNT films also constitute the circuit lines of our Current versus voltage trace for each TiN-CNTs-TiN bar was devices, their electrical resistance is consequential (Agrawal recorded and the electrical resistance was calculated. All sam- et al. 2007). The electrical performances of MWCNTs depend ples exhibited an Ohmic behavior with values ranging between on many factors such as average length, diameter, wall num- 2 and 15 kΩ. To derive their sheet resistance, electrical resistance ber, structural defects, film thickness, and the amount of values were plotted versus the number of squares in each bar amorphous carbon (Ferrari and Robertson 2000). While some (Fig. 2g; for explanation on sheet resistance calculations see of these parameters can be controlled in the growth process to Supplementary Fig. 3). Values ranging between 160 and 1,850 optimize the conductivity of the films, CNT films generally Ω/□ for different CVD growth conditions of the MWCNT film suffer from poor conductivity compared with typical metals. were obtained. Owing to the high electrode-electrolyte imped- We note that for our device needs, owing to the large ance values, we conclude that the CVD grown MWCNT films electrode-solution impedance, exceptionally high trace con- are conducting well enough to be readily used as effective ductivity is not critically important and values in the order of conducting traces for our application. several kilo ohms are acceptable. To validate the CNT film quality and to quantify the 3.3 Extracellular neuronal recording and stimulation electrical conductivity values, CNT films were characterized using the flexible CNT MEA using Raman spectroscopy (RS) and direct electrical measure- ments respectively. RS was performed to characterize the Having established the electrical as well as the electrochemical nature and the quality of the MWCNT films (Fig. 2f). properties of the CNT films, we now turn to describe the electro- Raman spectrum of the CNT films show two distinct peaks physiological performances of the flexible electrodes. An elec- at 1,360 (D-band) and 1,580 cm−1(G-band)(Thomsen and trode array compatible with a standard multi-electrode array Reich 2007). We used the ratio between the D and the G band recording and stimulation setup was realized on a printed circuit (I /I ) as a crude characterization of the defect density and board (PCB) support (Fig. 3a). The array consists of 16 elec- D G each CNT film was measured at 20 different sites. The I /I trodes on a medical tape support each connected to an external D G for all films was higher than one, indicating fairly poor film pad. A top PDMS passivation layer, 150 μm thick and with quality associated with the highly entangled CNTs. However, 50 μm diameter holes, was used to define the effective size of we have extensively used similar films in the past to perform the electrodes (Fig. 3a, inset). The flexible array was then recording from dissociated neurons (Gabay et al. 2007; Shein mounted onto the PCB carrier to accommodate the link between et al. 2009) and from mouse retina (Shoval et al. 2009)with the electrodes and external amplifiers. A glass cylinder was excellent results. The obtained films are thus very well suited glued to the PCB support to serve as a well for the physiological for neuronal stimulation. To validate the durability of the CNT medium. films upon mechanical stress we have tested the electrical Embryonic chick retina (day 14) was used as a neuronal properties of the CNT films following repeated cycles (up to model. The retina was extracted and transferred to the medium chamber under physiological conditions. The retina was then 30 cycles) of folding and winding. No significant change in Biomed Microdevices (2014) 16:43–53 49 Fig. 3 Flexible CNT MEA for extracellular neuronal recording and stimulation. a The flexible CNT electrode array mounted on a PCB support linking the electrodes to external amplifiers (scale bar: 5 mm). The array consists of 16 electrodes and a top passivation layer with 50 μm-diameter holes which define the electrode effective size. An embryonic chick retina (day 14) was flattened on the electrode array. The edge of the retina is marked with a dashed line. Inset: enlargement of the electrodes area (area marked with a solid line; scale bar: 200 μm). b Acircuit model for extracellular recording and stimulation from a neural tissue using the flexible electrode array. The model demonstrates the electrochemical interface resistance and capacitance of the CNT electrode and the solution derived shunt capacitance as well as the point of stimulation flattened on the electrode array (Fig. 3a), with the retinal 4 Discussion and conclusions ganglion cell (RGC) layer facing down (as in an epi-retinal implant) and was anchored with a weight. Figure 3b illustrates We can now turn to look at how our new CNT electrodes rank a circuit model for extracellular recording and stimulation of compared with previously reported technologies. Table 1 neuronal tissue using the micro electrode array, depicting the summarizes specific DC capacitance, stimulation threshold electrochemical interface resistance and the capacitance of the and SNR values obtained with other CNT and flexible elec- CNT electrode as well as the solution derived shunt capaci- trode technologies. The table refers to studies that demonstrat- tance and the stimulation point. ed either recording or stimulation of neuronal activity. DC −2 At day 14 the embryonic retina is still at an early develop- capacitance values of 1–10 mFcm were measured from mental stage and clear spontaneous activity waves were most CNT electrodes on both rigid and flexible substrates. recorded demonstrating the overall functionality of the device The all CNT flexible MEA presented in this study is well −2 and the setup. We next tested the CNT electrodes suitability to within this range with 2 mFcm , exceeding both CNT elec- −2 evoke electrical activity in the retina tissue. Stimulation was trodes grown directly on flexible polyimide with 0.1 mFcm achieved at currents as low as 4 μA(Fig. 4a and b)and (Hsu et al. 2010;Chenetal. 2011b) aswellasPtelectrodes stimulation pulse width of 1 ms. With nearly perfectly capac- coated with SWCNT (drop coating) on a rigid Pyrex substrate −6 −2 itive electrodes, these values are well within the limits of safe with 4.5·10 mFcm (Gabriel et al. 2009). It should be noted stimulation. The observed electrical response is typical for that SNR and stimulation threshold values depend on the pre-synaptic cells activation. Validation of the synaptic pro- examined tissue as well as on the size and shape of the cesses was achieved with the use of the synaptic blockers 6- electrode. Therefore they cannot be used as a direct measure cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5- of MEA devices. The SNR in particular, provides only a phosphonovaleric acid (APV). 400 s after the introduction of validation for the acceptable performance of the electrodes. the synaptic blockers no retinal ganglion cell activation was A stimulation threshold of 4 nC measured by our flexible measured (Fig. 4c). CNT MEA is lower than that reported by other CNT MEA 50 Biomed Microdevices (2014) 16:43–53 rigid and flexible technologies, our device benefits from the advantages of a very clean and simple fabrication scheme and most importantly a seamless integration between the electrode and the circuit, ultimately supporting a reliable and scalable fabrication of state of the art flexible MEAs. To conclude this discussion, our novel flexible CNT electrodes, as other clean carbon basedelectrodes, aredistinguishedbyhavinga clear capacitive nature. Being produced by a simple, clean and robust process, these electrodes properties surpass previously de- scribed technologies. Notwithstanding these promising results, some improve- ments in the fabrication scheme are desirable. For exam- ple, the 150 μm insulation layer locates our electrodes at a significant distance from the tissue, limiting the spatial reso- lution of the device as reflected in the relative low amplitude of the recorded signals (Fig. 4a). Reducing the thickness of the insulation layer will also improve the adhesion of the tissue to the electrodes, further promoting the spatial resolution. To summarize, a new scheme based on CNTs was presented and demonstrated as an advantageous approach to form high performance neuronal electrode array devices. The electrodes gain their performances from the combination of several dif- ferent CNT properties. Foremost, CNTs films have extremely large surface area making them very effective electrochemical electrode with capacitive charge injection mechanism. CNTs are also inert and strong, making the electrodes stable in biological conditions. As CNT films are suitable to withhold bending, they are very well suited for flexible electronic appli- cations. In the realm of multi-electrode arrays, this feature is particularly important as flexible MEA devices are of great interest for implantable applications. Unlike other coatings Fig. 4 Electrical recording and stimulation of chick retina with that may tend to crack and disconnect from the flexible flexible CNT MEA. a Evoked activity using a biphasic cathodic first substrate during bending, CNT films are durable owing to pulse (arrowhead). The large signal at t=0 is an artifact of the stimulation. their remarkable mechanical properties and the unique struc- b Firing rate of evoked activity at different stimulation intensities (3–10 ture of the MWCNTs film. The entangled bundles of tubes, nC). c Firing rate of evoked activity after synaptic blockers CNQX and APV application (stimulation was applied every 10 s). After 400 s no forming a dense and continuous yet porous film, make these retinal ganglion cells activation is observed films particularly optimal for neuronal applications. Additio- nally, the adhesion between the CNTs and the polymeric substrate is strong, making the CNT film an integrated part technologies (both rigid and flexible) and is similar to values of the substrate. Since CNTs are chemically inert they are obtained with TiN commercial devices, further demonstrating also durable against corrosion, a very common challenge in the overall high quality of our devices. conventional metal technology in biological applications. The We have shown that our new flexible all CNT MEA perfor- circuit structure is seamless and all elements, connecting pads, mances are equivalent to rigid CNT technologies with the conducting traces and electrodes are made of CNT. This is an obvious major advantage of being flexible. Two flexible CNT enormous advantage for both in vivo and in vitro long term use technologies used for neuronal recording and stimulation were since it eliminates delamination of the coatings and the for- reported before. Lin and co-workers fabricated a vertically mation of cracks. These cracks result with leakage currents aligned CNT (VACNT) MEA embedded in Parylene-C film and failure of the device as often occurs with layering and (Lin et al. 2009) while Hsu and co-workers used low temper- connection of different materials. All these properties are ature CVD (i.e. 400 °C) to directly grow CNT MEA on added to the relatively simple and robust fabrication process polyimide (Hsu et al. 2010;Chenet al. 2011b). The specific discussed above. This fabrication process can be easily ex- capacitance of our CNT MEA is significantly higher than that tended to include elements such as photodiodes and allows for stacking of different functionality layers, make the all-carbon- of the directly grown CNTs on polyimide. Compared with both Biomed Microdevices (2014) 16:43–53 51 Table 1 Neuronal recording and stimulation multi-electrode technologies Reference Electrode description In vitro testing scheme Area (μm ) Specific DC Stimulation SNR −2 capacitance (mFcm ) threshold Rigid CNT Wang et al. (2006) Vertically aligned MWCNT (CVD) MEA on Embryonic rat hippocampal cells 2,500–10,000 1.6 10 nC NA MEAs a quartz substrate with PEGPL coating Gabay et al. (2007) MWCNT (CVD) MEA on a Si substrate Rat cortical cultures 5,024 10 NA 135 Commercial TiN MEA; not coated with CNT Rat cortical cultures 706 2.5 NA 4 Keefer et al. (2008) ITO MEAcoatedwithMWCNT-Au Mice frontal cortex cultures 314 3.24 195 mV Recording (electrochemical deposition) Gabrieletal. (2009) Pt MEA coated with SWCNT (drop coating) on Isolated rabbit retinas 1,256 0.000045 NA 21 aPyrex substrate Su et al. (2010) Cone-shaped Si MEA coated with MWCNT Crayfish giant neurons 10–2,000 2.5 NA 42.3 (CVD) after O plasma Fuchsberger et al. (2011) TiN MEA coated with MWCNT (micro-contact Rat postnatal hippocampal 5,024 2.5 NA Recording printing). cultures Flexible CNT Lin et al. (2009) Vertically aligned CNT electrodes embedded in Crayfish nerve cord 1,962 Not available NA 257 MEAs Parylene C film Hsu et al. (2010) CNT MEA (CVD) on polyimide after Crayfish giant neurons 3,600–40,000 0.1 NA 150 UV-ozone modification Chen et al. (2011b) CNT MEA (CVD) on polyimide after UV-ozone Crayfish caudal photoreceptor 7,850–125,600 0.21 NA 6.2 modification In vivo, EcoG of rat motor cortex NA 8.68 This paper CNTs on a medical tape Embryonic chick retina 1,962–125,664 2 4 nC 20 Other flexible Blau et al. (2011) PEDOT:PSS+5 % ethylene glycol (v/v) Embryonic rat and mice hearts 11,304 Not available NA 100 MEAs electrodes on PDMS Rat cortico-hippocampl cultures 5 Chen et al. 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