KR100554407B1 - Method for the preparation of iridium nanofilm with high charge injection density, iridium namofilm prepared therefrom and electrode for stimulating neural cells using the iridium namofilm - Google Patents

Abstract

The present invention provides a method for producing an iridium (Ir) nano thin film having a high charge injection density, an iridium nano thin film prepared by such a method, and an electrode for nerve cell stimulation having such an iridium nano thin film.

Iridium, iridium oxide, nano thin film, bioelectrode.

Description

Method for the preparation of iridium nanofilm with high charge injection density, iridium namofilm prepared therefrom and electrode for stimulating neural cells using the iridium namofilm}             

FIG. 1A is a positive polarization curve in sulfuric acid, and FIG. 1B is a positive polarization curve in saline solution.

FIG. 2A is a cyclic voltage curve of a 30 nm thick Ir film with a stable iridium-iridium oxide interface, and FIG. 2B is a cyclic voltage curve of a 60 nm thick Ir film.

3A is a SEM micrograph of a 300-cycle activated Ir film, and FIG. 3B is a SEM micrograph of a 500-cycle activated Ir film.

FIG. 4A is a FE-SEM micrograph as it is deposited of a 60 nm thick Ir film, FIG. 4B is a FE-SEM micrograph after 800-cycle, and FIG. 4C is a FE-SEM micrograph after 1200-cycle.

5 shows high resolution TEM micrographs of activated Ir films.

FIG. 6 shows the cyclic voltammogram of Ir film activated in isotonic saline solution.

7A is a SEM micrograph after incubation of PC12 cells for 5 days without NGF treatment, and FIG. 7B is a SEM micrograph after incubation of PC12 cells with NGF treatment for 5 days.

8 is an optical micrograph of Swiss 3T3 fibroblasts in Ir film deposited CP Ti.

The present invention relates to a method for producing an iridium nano thin film having a high charge-injection density, an iridium nano thin film produced by such a method, and an electrode for nerve cell stimulation having such an iridium nano thin film.

For spinal cord injury patients, electrical stimulation is a promising technique for restoring function. Although complex, implantable neuromuscular stimulators have been developed for many clinical applications, including breathing, bowel and bladder, gait and manipulation. The implantable stimulator delivers current to the nerve by placing the electrode at or near the nerve. The delivered current depolarizes the nerves that can be electrically excited, generating an action potential in the nerves, which excite the peripheral reactors and muscles beyond the neuromuscular junction. Such systems generally have means for generating electrical pulses, leads for delivering pulses to muscles, and electrodes for delivering pulses to nerves.

Materials to be considered for use as electrically active parts of neural stimulation electrodes must meet a set of conditions. Stimulation should be carried out without causing irreversible inductive current reactions such as water electrolysis, metal melting, gas evolution or oxidation of organics. In addition, miniaturized electrode shapes are needed to control many different muscles in practical applications, and materials that provide higher current and charge carrying capacity are needed. Up to 32 muscles are activated for motor rehabilitation of biplegic and paraplegic patients, and up to 8 muscles are activated to restore hand deflection in quadriplegic patients.

In recent years, iridium has attracted attention as a candidate for stimulating neural tissue. Iridium exhibits high resistance to dissolution in corrosive media and good biocompatibility. It is necessary to successfully produce thinner films of stable Ir films on various substrates or substrates and to have higher charge densities.

The present inventors studied the application of an iridium nano thin film formed by Ir electron beam deposition to an electrode for nerve cell stimulation after removing an oxide from a substrate by applying an Ar ion beam bombardment. Stably coating an ultrathin layer of Ir nanothin film and repeatedly sweeping the potential in an acid solution over a specific potential range to form an oxide film in which a portion of the iridium nanothin film is oxidized and having such an oxidized Ir layer. Electrode using Ir nano thin film has a significant increase in charge input compared to bare oxidized electrode, thus exhibiting a suitable charge input ability for use in neurostimulation despite being nano thin film, and cytotoxicity including nerve cells The present invention was found to be absent.

In one aspect, the present invention provides a method for removing an oxide from a substrate by subjecting the substrate for neural electrodes to ion beam bombardment; Depositing iridium at a deposition rate of 0.1-0.5 dB / s using an iridium electron beam evaporator; Subjecting the iridium deposited substrate to ion beam bombardment; And cycle activating the iridium deposit in an acid solution with a potential from 0.0 V (SHE) to 1.45 V (SHE) in a cycle, thereby providing a method for producing an iridium nano thin film having an iridium oxide layer for use in neural electrodes. do.

In the process of the present invention, iridium deposition first removes the substrate from the substrate surface with an ion beam, preferably a 120V, 2A argon ion beam for 30 minutes, and then vapors of Ir atoms generated through an electron beam evaporator under vacuum. The flux is carried out by depositing at a rate of 0.1 to 0.5 kW / s, preferably 0.3 kW. The deposited Ir nano thin film is a stepwise continuous process of depositing Ir electron beam evaporates after Ar ion beam bombardment, and Ir electron beam deposits are performed alone without Ar ion beam bombardment. Subsequently, the Ir electron beam deposited Ir nano thin film is further exposed to 120 V, 2 A Ar ion beam bombardment for 20 minutes to further stabilize the Ir nano thin film.

The anode polarization curves of Ir thin films deposited according to the method of the present invention have higher open-circuit corrosion potential and lower anodic current density than uncoated samples in both sulfuric acid and saline solution. ) Was substantially the same as bulk Ir. This shows good adhesion without defects on the substrate.

In the process of the present invention, iridium is oxidized to iridium oxide for activation of the deposited Ir nanofilm, and the acid solution used therein is preferably sulfuric acid solution, more preferably 0.1M sulfuric acid solution.

According to the method described above, a stable iridium nano thin film of a thickness of iridium deposits, for example, 1 to 100 nm in thickness, can be produced, and the potential in the acid solution to increase the charge input density of the Ir thin film of this ultra thin layer is increased. The gap was activated in cycles to form an iridium oxide layer. The number of activation cycles depends on the desired charge density, the thickness of the deposited Ir nanofilm, etc. For example, a 30 nm thick Ir nanofilm reduces the charge input of Ir when activated at 800-cycles, and 60 nm. The Ir nano thin film of thickness decreased the charge input value of Ir when activated at 1600-cycle.

In order to produce an Ir nano thin film having a desired charge density, the thickness of the nano thin film and the number of activation cycles for forming an oxide thereof must be controlled mutually. As one specific example, the ion beam impact may be subjected to the above steps. And then activate the 30-nm-thick Ir thin film with less than 800 cycles, preferably 100-700-cycle, or 60-nm-thick Ir thin film with less than 1600 cycles, preferably 100-1500 By cyclic activation, an iridium nano thin film having an iridium oxide layer for use in neural electrodes can be produced.

In another aspect, the present invention provides an ion beam bombardment to a substrate for neural electrodes to remove oxides from the substrate; Depositing iridium at a deposition rate of 0.1-0.5 dB / s using an iridium electron beam evaporator; Subjecting the iridium deposited substrate to ion beam bombardment; And cycle activating the iridium deposit in an acid solution with a potential from 0.0 V (SHE) to 1.45 V (SHE) in a cycle, wherein the iridium deposit is non-toxic to neurons and prevents proliferation and differentiation of attached neurons. An inducible, iridium nano thin film having an iridium oxide layer is provided.

The substrate on which the iridium thin film is coated may be selected from various materials such as silicon wafer, silicon rubber, platinum, gold, copper, titanium, nickel, and the like, and these materials may take various forms such as plates and wires. If the substrate is ductile, the contact area can be widened and therefore can act at lower application potentials. As the material having ductility, silicone rubber can be preferably used.

The prepared iridium nano thin film has different charge densities represented in physiological saline solution depending on the thickness of Ir deposited and the degree of oxide formation thereof, and may have, for example, a charge density required for intracortical stimulation of 10 mC / cm 2.

The iridium nano thin film of the present invention can not only attach to living cells such as neurons but also proliferate and differentiate. Attachment and proliferation of PC12 cells to Ir-coated CP Ti up to 9 days was similar to that for smoothly treated CP Ti, and the network of neurons and axons developed well. 7 and 8 demonstrate the growth of axons and fibroblasts under the treatment of nerve growth factor (NGF) and did not show toxicity to neurons and fibroblasts. Attachment of such cells is useful for the delivery of stimulated charge densities.

In still another aspect, the present invention provides an electrode for nerve cell stimulation having an iridium thin film.

By using an electrode having an iridium thin film that does not exhibit such cytotoxicity, nerve cells acting through an electrochemical mechanism can be stimulated to improve the functions of breathing, bowel, bladder, and walking of spinal cord injury patients.

The present invention will be illustrated by illustrating the following specific implementation. However, these examples are for illustrative purposes only and are not intended to limit the invention.

Iridium Thin Film Formation and Generation of Iridium Oxide

Before depositing iridium, the surface of the substrate was impacted with an Ar ion beam for 30 minutes using a Mark II ^™ end-hall type ion gun (Commonwealth Scientific, Alexandria, VA, USA) fixed at 120V and 2A for 30 minutes. While the preformed oxide was removed. Iridium was then deposited onto commercially graded pure (CP) Ti and Ni-Ti smoothly treated metal substrates or Si wafers. The chamber was vacuumed using a cryopump (OB-10, Helix Technology, Mansfiend, MA, USA) at a base pressure of 2 x 10 ^-7 Torr, and the vapor flux of Ir atoms was transferred to an electron beam evaporator (Telemark, Fremont, CA, USA) and deposited onto the substrate at a deposition rate of 0.3 kW / s. The substrate was placed on a water-cooled substrate holder rotated at 8 rpm during deposition to increase the uniformity of the coating layer. The iridium deposited substrate was then bombarded with an Ar ion beam for 20 minutes at 120V, 2A to obtain a stable iridium film.

Anodic polarization curves and cyclic voltage curves (voltammogram) were measured using EG & G Princeton Applied Research Model 273 potentiostat. In order to evaluate the charge input capacity in 0.1MH ₂ SO ₄ , a linear scan was performed with a triangular waveform of 0 to 1.45V for a standard hydrogen electrode (SHE) at a rate of 100mV / s, and the resulting current density was linearly scaled. Plot. Samples were placed in a specially made Teflon ^™ sample holder. To measure the current density in saline, the Ir film was first activated with a cycle number selected from 0.1MH ₂ SO ₄ . Each electrode was then carefully cleaned and transferred to a saline solution, followed by a potential sweep of −0.4 to 0.8 V (SHE) at a scan rate of 100 mV / s.

FIG. 1A shows the anode polarization curves of Ni-Ti samples Ir deposited with bulk Ir, Ni-Ti, and Ti-6Al-4V in N ₂ -degenerated 1N H ₂ SO ₄ , FIG. 1B Shows the polarization polarization curves of Ni-Ti samples Ir deposited with bulk Ir, Ni-Ti, and Ti-6Al-4V in an isotonic saline solution. Each anode polarization curve was measured by increasing the potential from 50 mV below the open-circuit corrosion potential measured after soaking for 30 minutes at a scan rate of 0.33 mV / s. Ir deposited on Ni-Ti produced a more inert open-circuit corrosion potential and produced a lower anode current density relative to the Ni-Ti electrode. The anode polarization aspect of the Ir film was substantially the same as that of bulk Ir, indicating a dense, stable, and free of defects.

2A shows the cyclic voltage curve of a 30 nm-thick Ir thin film deposited on a Si wafer in 0.1MH ₂ SO ₄ . The curve shifted outwards as the number of cycles increased. This behavior was identical to bulk Ir.

Continuous potential cycling of the Ir film produced thicker oxides, with the 30 nm thick Ir film having the highest dosing capacity at 800-cycle, but this ability gradually decreased. During the positive portion of the cycle, the valence of the iridium oxide transitioned from Ir ⁺³ to Ir ⁺⁴ with the release of protons and electrons, and a reverse reaction occurred during the negative portion. In the oxidation process, electrons were removed from the oxide through the metal-oxide interface by applying a suitable anode potential to the iridium film. Charge repulsion causes an equal amount of protons to exit through the oxide-electrolyte interface, thereby maintaining the valence of the charge.

Since the deposited Ir film is a good conductor and forms a stable metal-oxide interface, electrons are easily removed from the oxide for oxidation. The volume of oxide increases with successive potential cycling by oxidation of iridium, and the deposited Ir layer becomes thinner. If the retained Ir layer loses its good conductivity, electron transfer is slow at the iridium-iridium oxide interface. The high charge input capacity of iridium oxide is due to the valence transition of iridium in the oxide, and the charge input value decreases when the conduction of retained iridium becomes a limiting factor for electron transfer.

This similar behavior was observed for Ir films with a thickness of 60 nm and only a decrease in charge density occurred at high cycles of 1600 as shown in FIG. 2B.

Surface Observation of Iridium Thin Films

The morphology of the thin films was observed by SEM (JSM-5310, JEOL, Tokyo, Japan) and high resolution TEM (JEM-4010, JEOL, Tokyo, Japan). X-ray photoelectron spectroscopy (XPS; PHI-5700, PHI, Minnesota, USA) was used to characterize the deposited Ir surface and the activated Ir surface.

3 shows a scanning electron micrograph of an Ir film activated at (a) 300 cycles and (b) 500 cycles in 0.1MH ₂ SO ₄ . The surface topography of the Ir film as deposited is smooth and uncharacteristic, but the Ir film activated at 300 cycles is somewhat different, making the surface less uniform and rough as the activation cycle increases.

The results of sweeping 60 nm thick Ir films with different number of cycles and observing their cross sections by FE-SEM are shown in FIG. 4. Figure 4a is deposited as is, Figure 4b after 800 cycles, Figure 4c is a FE-SEM micrograph of the film after 1200 cycles. It is evident that as the number of cycles increases, the thickness of the iridium oxide becomes thicker. The thickness of the iridium oxide was measured at about 100 nm and about 150 nm at 800 cycles and 1200 cycles, respectively, and the average growth rate of the iridium oxide was calculated to be 1.25 mA / cycle.

The interface of Ir metal and Ir oxide was analyzed by high resolution transmission electron microscopy (HRTEM), and the HRTEM micrograph is shown in FIG. 5. Oxide developed through the IR thin film. Growth of the oxide film in aqueous solution requires diffusion of metal cations to the oxide / solution interface or diffusion of hydroxide ions to the metal / oxide interface. Iridium does not diffuse easily through oxides, so hydroxide ions must diffuse through oxides during their growth. Unprocessed Ir can easily come into contact with the electrolyte due to the nano-scale porous form and the oxide can continue to increase as the cycle increases.

Charge input capability of activated iridium thin film

In terms of performance in saline, Ir films were first activated with selected cycle numbers of 0.0 V (SHE) to 1.45 V (SHE) in 0.1 MH ₂ SO ₄ . Each electrode was then carefully washed with distilled water and transferred to a saline solution. If the potential applied to the stimulating nerve electrode is too high, oxygen is generated, and if it is too low, hydrogen is generated. This reaction should be ruled out because of possible physical damage from the gas produced and local pH changes around the electrode. As a result, the upper and lower limits of the potential for charge injection of the Ir film in the isotonic saline solution were chosen to be 0.8 V (SHE) and -0.4 V (SHE). 6 shows the charge density of 'activated' Ir in saline solution as a function of the number of activation cycles in 0.1MH ₂ SO ₄ . The cycle voltage curve was recorded in the second cycle at a scan rate of 100 mV / s, but the oxide did not change up to 10,000 cycles because it did not grow under these conditions, and showed excellent stability. Ir films that are more active at 0.1MH ₂ SO ₄ can inject higher charge densities under the same stimulus of the applied cyclic potential, which is very important because charge injection can be controlled and optimized for the stimulation of different nerve fibers. Do. By activating the Ir film up to 1000 cycles in 0.1MH ₂ SO ₄ , a charge density of 10 mC / cm ² required for intracortical stimulation was obtained.

Use of iridium thin film to nerve electrode for stimulation

An essential feature of the material considered as an implant is that it should be non-toxic, so the cytotoxicity of Ir films deposited on CP Ti was evaluated.

Swiss 3T3 fibroblasts were evaluated and compared to CP Ti, which is FDA approved. Cytotoxicity testing using Swiss 3T3 fibroblasts was first performed by MTT [1-4 {4,5-dimethylthiazol-2yl} -3,5-diphenyl formazan, Sigma] method. Embryonic cortical neurons were cultured in iridium and iridium oxide without applying electrical signals. Cells were prepared from the Sprague-Dawley cortex by mechanical separation of 18 day old embryos from Ca ⁺² / Mg ⁺² free Hank balanced salt solution (HBSS), and the tips of each cell lightly finished in flames. Separation was carried out by grinding 10 times in 1 ml of HBSS using a 9 inch siliconized Pasteur pipette with. After stabilizing undispersed tissue for 3 minutes, the supernatant was transferred to a 15 ml tube and centrifuged at 200 × g for 1 minute. Pellets were neurobasal medium (Cat.No. 21103-049, Gibco) supplemented with 0.5 ml L-glutamine, 25 uM glutamate and B27 supplement (Cat. No. 17504, Gibco-BRL Life Technology, Paisley, UK). Carefully resuspended in BRL Life Technology, Paisley, UK). 2 × 10 ⁵ cells / ml of cells were then placed in iridium or iridium oxide along the control of the Si wafer in a 24-well plate. Incubated at 37 ° C. in a 5% CO ₂ incubator for 4 days. After incubation, the samples were coated with an ultra-thin layer of gold / Pt with ion sputters (E1010, HITACHI, Tokyo, Japan), and their forms were observed by scanning electron microscopy (S-800, HITACHI, Tokyo, Japan).

Since the cytotoxicity of Ir films is 106% compared to CP Ti, Ir films formed by electron beam deposition are considered non-toxic, and cells cultured in Ir films are shown in FIG. 8.

PC12 cells were obtained from the American Type Culture Collection, Rockville, MD, USA, and RPMI supplemented with 10% horse serum, 5% fetal calf serum, 100 U / ml penicillin, and 100 μg / mg streptomycin Incubated at 37 ° C. in 1640 medium under a humid atmosphere of 95% air and 5% CO ₂ . The cells were kept in log phase division for at least three generations. Cells were then aspirated off and then seeded on Ir-coated commercial grade pure (CP) Ti plates (10 mm ² ) at a density of 2 × 10 ⁵ cells / cm ² . The culture medium was replaced every other day in the log phase and daily in the stop phase. The number of cells was measured at appropriate time intervals in culture to assay cell adhesion and proliferation. Cell-attachment assay was performed using a hemocytometer and proliferative activity was measured by PreMix WST-1 cell proliferation assay system (Takara Bio Inc., Shiga, Japan).

To differentiate PC12 cells on Ir-coated CP Ti plates, cells were treated with NGF (R & D Systems Inc., Minneapolis, MN, USA) at a final concentration of 50 mg / ml, 95% air and 5% CO for 5 days. Incubated in a humid atmosphere of ₂ . The medium was changed daily. After 5 days, the Ir-coated Ti plate was coated with ultrathin layer of gold / Pt in an ion spotter (E1010, HITACHI, Tokyo, Japan), and the morphology of PC12 cells was determined by scanning electron microscopy (S-800, HITACHI, Tokyo, Japan). Japan).

Cell cultures using PC12 cells were performed on Ir-coated CP Ti phase without electrical stimulation and on smoothly treated CP Ti for comparison. Attachment and proliferation of PC12 cells to Ir-coated CP Ti up to 9 days was similar to that for smoothly treated CP Ti, indicating that neurons can adhere to the Ir thin film and survive.

Since the rate of proliferation is regulated by applying a constant potential and the gene expression for nerve growth factor (NGF) is electrically activated by selective potential, the rate of proliferation will be increased on the Ir film when charge input is induced. After incubation for 5 days, the form without NGF treatment of cultured PC12 cells on Ir-coated CP Ti is shown in FIG. 7A and the form with NGF treatment is shown in FIG. 7B. The network of neurons and axons is clearly visible, and growth cones can be clearly observed at the ends of the axons. The number of long libes in the growth cones is clearly evidence of the pathfinding of the axons. The morphology of PC12 cells was not affected by NGF treatment for at least 5 days of culture. Neural attachment on the Ir film is useful for the delivery of stimulated charge densities.

According to the present invention, an Ir nano thin film that is stably deposited using an Ir beam evaporator is repeatedly swept in an acid over a specific potential range to form an oxide film to form an oxide film. It provides a non-toxic iridium nano thin coating electrode.

Claims (7)

Applying an argon (Ar) ion beam bombardment to the substrate for neural electrodes to remove oxides from the substrate; Depositing iridium at a deposition rate of 0.1 to 0.5 kW / s using an iridium (Ir) electron beam evaporator; Subjecting the iridium deposited substrate to ion beam bombardment; And activating the iridium deposit in a sulfuric acid solution with a potential from 0.0 V (SHE) to 1.45 V (SHE) in cycles, thereby producing an iridium nano thin film having an iridium oxide layer for use in neural electrodes. The method of claim 1, wherein the deposition rate of the iridium electron beam deposit is 0.3 kW / s. The method of claim 1 wherein the iridium deposit is 30 nm thick and the deposit is activated from 100 to 700-cycle in sulfuric acid solution. The method of claim 1, wherein the iridium deposit is 60 nm thick and the deposit is activated 100 to 1500-cycle in sulfuric acid solution. Applying an ion beam bombardment to the substrate for neural electrodes to remove oxides from the substrate; Depositing iridium at a deposition rate of 0.1-0.5 dB / s using an iridium electron beam evaporator; Subjecting the iridium deposited substrate to ion beam bombardment; And activating the iridium deposit in a sulfuric acid solution with a potential from 0.0V (SHE) to 1.45V (SHE) in cycles, inducing proliferation and differentiation of neurons that are toxic to neurons and attached to them. An iridium nano thin film which can have an iridium oxide layer. 6. The iridium nano thin film of claim 5, wherein the substrate is selected from the group consisting of silicon wafers, silicon rubber, platinum, gold, copper, and titanium. A nerve cell stimulating electrode having an iridium nano thin film of claim 5.

Images