DEVICE AND METHOD FOR NEUROSTIMULATION

Abstract

An efficient nanoscale semiconducting optoelectronic system, containing a P/N acceptor donor organic hetrojunction which is optimized for neuronal stimulationan organic electrolytic photocapacitor. The devices comprise a thin (80 nm) trilayer of metal and p-n semiconducting organic nanocrystals. When illuminated in physiological solution, these metal-semiconductor devices charge up, transducing light pulses into localized displacement currents that are strong enough to electrically stimulate neurons with safe light intensities.

Claims

1. A device for neurostimulating a tissue in a physiological medium, the device comprising a multilayer comprising at least one metal or a conductive material layer and p-n/n-p structure layered thereon, the p-n/n-p structure comprising semiconducting organic nanocrystals.

2. The device according to claim 1, wherein the p-n/n-p structure is in continuous contact with the at least one metal or the conductive material layer, wherein the at least one metal or the conductive material layer is configured to be in electrical contact with a physiological medium in which the device operates, the p-n/n-p structure comprising a combination of two or more organic semiconductor pigments, at least one being selected amongst electron donor materials (a p-type material) and at least one other selected amongst electron accepting materials (an n-type material).

3. The device according to claim 2, the device comprising a metal layer or a conductive material layer, a layer of at least one light-absorbing (electron donor) material that is in continuous contact with the metal or the conductive material layer and a layer of at least one electron acceptor material that is in continuous contact with the layer of the at least one light-absorbing material.

4. The device according to claim 2, the device comprising a metal layer or a conductive material layer, a layer of at least one electron acceptor material that is in continuous contact with the metal or the conductive material layer and a layer of at least one light-absorbing (electron donor) material that is in continuous contact with the layer of the at least one electron acceptor material.

5. The device according to claim 2, the device comprising a substrate, at least one metal layer or a conductive material layer formed onto one or more regions of the substrate, and a p-n/n-p structure formed on at least one of the metal layer or metal regions.

6. The device according to claim 2, the device comprising a metal layer or a conductive material layer, a layer consisting of at least one p-type organic pigment material or a layer consisting of at least one n-type organic pigment material that is stacked onto at least a region of said metal layer.

7. The device according to claim 2, being a photocapacitor.

8. A photocapacitor device for responding to a light of a selected wavelength, said device comprising a first region comprising at least one metal or conductive material; and a second region composed of a p-n/n-p structure, wherein the first region is formed on a substrate in the form of a layer or a film onto which the second region, being in the form of a film structure or a multi-layered structure, is formed.

9. The device according to claim 1, wherein the p-n/n-p structure is a p-n structure or an n-p structure.

10. (canceled)

11. The device according to claim 2, wherein the p-type material is selected amongst organic semiconductor pigments, optionally polymeric.

12. The device according to claim 11, wherein the p-type material is selected from metal-containing or metal-free phthalocyanine (H.sub.2Pc), 2,3-naphthalocyanine, benz[b]anthracene, 5,5-Bis(2,2-dicyanovinyl)-2,2:5,2: 5,2:5,2-quinquethiophene, bis(ethylenedithio) tetrathiafulvalene, 2-[(7-{4-[N,N-bis(4-methylphenyl)amino]phenyl}-2,1,3-benzothiadiazol-4-yl)methylene]propanedinitrile, 6,13-bis((triethylsilyl)ethynyl) pentacene, Coronene, dibenzotetrathiafulvalene, 5,5-Di(4-biphenylyl)-2,2-bithiophene, 3,3-Didodecyl-2,2:5,2:5,2-quaterthiophene, 5,5-Dihexyl-2,2-bithiophene, 3,3-dihexyl-2,2:5,2:5,2-quaterthiophene, 5,5-Dihexyl-2,2:5,2:5,2:5, 2:5, 2-sexithiophene, dinaphtho[2,3-b:2,3-f]thieno[3,2-b]thiophene, 2-[7-(4-diphenylaminophenyl)-2,1,3-benzothiadiazol-4-yl]methylenepropanedinitrile, 2,6-diphenylbenzo[1,2-b:4,5-b]dithiophene, 2,7-diphenyl[1]benzothieno[3,2-b][1]benzothiophene, 6,13-diphenylpentacene, 2-{[7-(5-N,N-ditolylaminothiophen-2-yl)-2,1,3-benzothiadiazol-4-yl]methylene}malononitrile, 2,6-ditolylbenzo[1,2-b:4,5-b]dithiophene, Merocyanine dye, 13,6-N-sulfinylacetamidopentacene, tris[4-(5-dicyanomethylidenemethyl-2-thienyl)phenyl]amine, Rubrene, -sexithiophene, tetrathiafulvalene, epindolidione, quinacridone, indanthrene, flavanthrone and violanthrone.

13. The device according to claim 12, wherein the p-type material is a metal containing or metal free phthalocyanine (H.sub.2Pc).

14. (canceled)

15. The device according to claim 2, wherein the n-type material is selected amongst organic semiconductor pigments, optionally polymeric.

16. The device according to claim 15, wherein the n-type material is selected from N,N-dimethyl perylenetetracarboxylic diimide (PTCDI), N,N-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenedicarboximide, 1,3,6,8(2H,7H)-tetraone, 2,7-dicyclohexylbenzo[lmn] [3,8]phenanthroline, 1,3,8,10(2H,9H)-tetraone, 2,9-bis(2-phenylethyl)anthra[2,1,9-def:6,5,10-def]diisoquinoline, fullerene, 5,10,15,20-Tetrakis(pentafluorophenyl)-21H,23H-porphine, 7,7,8,8-tetracyanoquinodimethane, 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane, 1,2,3,4,5,6,7,8-octafluoro-9,10-bis[2-(2,4,6-trimethylphenyl)ethynyl]anthracene, 1,2,3,4,5,6,7,8-octafluoro-9,10-bis[4-(trifluoromethyl)phenyl]anthracene, 1,4,5,8-naphthalenetetracarboxylic dianhydride, Indeno[1,2-b]fluorene-6,12-dione, 2,9-dipropylanthra[2,1,9-def:6,5,10-def]diisoquinoline-1,3,8,10(2H,9H)tetrone, N,N-dipentyl-3,4,9,10-perylene dicarboximide, 1,3-dimethyl-2-phenyl-2,3-dihydro-1H-benzoimidazole, 4-(2,3-dihydro-1,3-dimethyl-1H-benzimidazol-2-yl)-N,N-dimethylbenzenamine, N,N-dimethyl-3,4,9,10-perylenedicarboximide, 4-(1,3-dimethyl-2,3-dihydro-1H-benzoimidazol-2-yl)-N,N-diphenylaniline, 2,9-dihexylanthra[2,1,9-def:6,5,10-def]diisoquinoline-1,3,8,10 (2H,9H)tetrone, 2,7-dihexylbenzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetrone, 2,9-diheptylanthra[2,1,9-def:6,5,10-def]diisoquinoline-1,3,8,10(2H,9H) tetrone, 1,7-dibromo-3,4,9,10-tetracarboxylic acid dianhydride, 6,12-bis(2,4,6-trimethylphenyl) indeno[1,2-b]fluorine, 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine, 2,2-bis[4-(trifluoromethyl)phenyl]-5,5-bithiazole, 5,5-bis(tridecafluorohexyl)-2,2:5,2:5,2-quaterthiophene, N,N-bis(3-pentyl)perylene-3,4,9,10-bis(dicarboximide), 6,12-bis(2,3,4,5,6-pentafluorophenyl)indeno[1,2-b] fluorine, 2,9-bis[(4-methoxyphenyl)methyl]anthra[2,1,9-def:6,5,10-def]diisoquinoline 1,3,8,10(2H,9H)tetrone, bisbenzimidazo[2,1-a:2,1-a]anthra[2,1,9-def:6,5,10-def]diisoquinoline-10,21-dione, indigo, 6,6-dibromoindigo (tyrian purple), isoindigo, indanthrone, diindeno[1,2,3-cd:1,d,3-jk]pyrene, diindeno[1,2,3-de,1,2,3-kl]anthracene and dinaphth[1,2-a: 1,2-h]anthracene.

17. The device according to claim 16, wherein the n-type material is N,N-dimethyl perylenetetracarboxylic diimide (PTCDI).

18.-30. (canceled)

31. The device according to claim 1, the device being photoresponsive at a wavelength between 400 and 2,000 nm.

32. (canceled)

33. The device according to claim 1 being wire free.

34.-36. (canceled)

37. A method for stimulating an excitable tissue or cell, the method comprising placing or positioning into, onto or in the vicinity of a target excitable tissue or cell at least one photoresponse device according to claim 1; and focusing light with a wavelength between 400-2,000 nanometers onto the device, to thereby cause a photoresponse effect (electrical pulse), and stimulation of the excitable tissue or cells.

38. A method of generating an electrical pulse at the vicinity of a biological tissue or cell, the method comprising positioning into, onto or in the vicinity of a target excitable tissue or cell at least one photoresponse device according to claim 1; and focusing light with a wavelength between 400-2,000 nanometers onto the device, to thereby generate an electrical pulse.

39. A device according to claim 1 configured as a retinal implant.

40.-41. (canceled)

42. A photoresponse device comprising a Cr/Au bilayer and a p-n structure being in continuous contact with the Cr/Au layer, the p-n structure comprising phthalocyanine (H.sub.2Pc) and N,N-dimethyl perylenetetracarboxylic diimide (PTCDI).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0063] FIGS. 1A-E depict exemplary organic photocapacitor devices according to the invention: FIG. 1A is a schematic representation of a photocapacitor device consisting of sequentially deposited Cr/Au and H.sub.2Pc (p-type) and PTCDI (n-type) layers. FIG. 1B shows the molecular structures of the pigment semiconductors. Metal-free phthalocyanine (H.sub.2Pc) functions as the primary light-absorbing layer and p-type electron donor, while N,N-dimethyl perylenetetracarboxylic diimide (PTCDI) acts as the n-type electron-acceptor, which attains a negatively-charged surface upon illumination. FIG. 1C is anenergy band illustration of a metal-p-n photocapacitor device during the start of the illumination pulse when the capacitor charges. FIG. 1D is a two-dimensional slice of an electrostatic simulation of electrical potential distribution in electrolytic solution above a metal-p-n photocapacitor, when the p-n junction is charged to 250 mV. The positive potential is closely localized on the exposed metal film, while a negative potential plume extends from the top of the p-n heterojunction layer. Scale bar=200 m. FIG. 1E presents the mechanism of capacitive coupling of an illuminated photocapacitor with an adjacent cell.

[0064] FIGS. 2A-G depict photocapacitive charging of Cr/Au/H.sub.2Pc/PTCDI film type I. FIG. 2A presents a two photocapacitor measurement configurations for Type I samples (11 cm.sup.2 p-n area on a 1.51.5 cm.sup.2 gold coated glass slide): Grounded metal samples for voltage (V) and current (I) measurements, and floating samples for voltage transient (Vt) measurements. Numbers denote which figure panels show measurements in the given configuration. FIG. 2B shows the optical absorbance overlaid with spectral responsivity of Type I photocapacitors. FIG. 2C provides photoelectric characterization. c1 and c2 are photovoltage (V) and photocurrent (I), respectively, measured between the bath electrode and the grounded p-n-metal device. c3 is the photovoltage transient (Vt) measured 10 m above the p-n film, using a glass capillary electrode versus bath reference electrode. Vertical grey lines indicate onset and termination of the light pulses. FIG. 2D provides cathodic peak value of Vt (cpVt) is a function of peak anodic current divided by the spot size radius r.sub.(spot). FIG. 2E shows cpVt as a function of illumination intensity for two different electrolytes: phosphate-buffered saline (PBS) and artificial cerebrospinal fluid (aCSF). FIG. 2F presents a lateral profile of cpVt measured 10 m above the surface for two different light spots that are significantly smaller than the p-n region. Measurements start from the center of the light spot and are measured laterally at 25 m increments. Cathodic charging is strongest in the center of the spot, with Vt rapidly decaying outside of the directly illuminated region. FIG. 2G, Stress test results on grounded samples to evaluate the effects of different sterilization procedures. Measurement was done after sequential: oxygen plasma, triple treatment with absolute ethanol, storing overnight in buffer, UV sterilization, and second triple treatment with absolute ethanol.

[0065] FIGS. 3A-D depicts photostimulation of neuronal cultures. FIG. 3A, Cortical primary neurons cultured on PDL-coated petri-dishes, control sample (n=4). Scale bar=100 m. FIG. 3B, Cortical primary neurons cultured on type I devices (n=3). FIG. 3C, Calcium imaging traces (dF/F) of neurons cultured on PDL coated petridish. FIG. 3D, Ca imaging traces of neurons cultured on type I devices. Vertical red lines in (c) and (d) indicate a light stimulation of 100 consequative pulses (600 nm, 480 mW/cm.sup.2, pulse duration 5 ms, interpulse interval 10 ms).

[0066] FIGS. 4A-F depicts the evolution from films to pixels. FIG. 4A, Type II samples comprise p-n circular islands of varying size deposited on an infinitely large gold layer. Type III are devices where the size of both p-n islands and the underlying metal is varied. Scale bar below the pigment image=500 m. FIG. 4B, The effect of p-n island size. Cathodic peak values of voltage transients (cpVt) from type II samples as a function of p-n island size for three different illumination intensities. Vt is measured 10 m above the centre of the p-n islands. Light spot size is larger than the maximal island size. FIG. 4C. Lateral cpVt profile measured 10 m above a type III sample, showing the maximum value of Vt in the centre of the p-n island, with voltage changing sign above the metal film. Measurements are from the centre of the p-n island and moving aside at 25 m increments. FIG. 4D, The effect of gold size on Vt measured 10 m above the p-n film in type III samples with constant p-n island size and variable metal size. FIG. 4E and FIG. 4F. p-n circular islands of varying size deposited in between the electrodes of multielectrode arrays (MEA) with either infinitely large (e) or =480 m of circular (f) gold layer. Scale bars=1 mm.

[0067] FIGS. 5A-F depicts direct responses of retinal ganglion cells (RGC) in a light-insensitive retina. FIG. 5A, A piece of light insensitive embryonic chick retina was laid on the MEA shown in FIG. 4E. Light pulses illuminated only a pigment bilayer of 100 m marked by a red arrow using 40 objective. Electrical stimulation injected to electrode G4, marked by a blue arrow. Direct responses of the retinal ganglion cells (RGC) were detected only in red-circled electrodes for light stimulation and blue-circled electrodes for electrical stimulation. Corresponding regression lines for these electrodes are shown and their linear equation, with a slope of 38. FIG. 5B, An image of a retina placed on the MEA, with optical focus on the photoreceptor nuclear layer. The p-n island (marked with square in FIG. 5A) is clearly visible beneath the retina. FIG. 5C, The same image as in FIG. 5B focused on the nerve fibre layer. The orientation of the fibres is clearly seen in the image, found to be 38 by the FFT directionality histogram. FIG. 5D, Current pulse stimulation of the retina. Relative location of the injected electrode, G4, is marked by a blue arrow and circle. The latency of the response is increased when recorded from more distant electrodes to the stimulating electrode. This measurement serves as an internal control. FIG. 5E, Photostimulation of the retina. Relative location of the illuminated pigment is marked by a red arrow and circle. Electrodes H.sub.4 and G5, which are close to the source, record the electrical response of the pigment bilayer as well. These responses are seen in the overlaid red traces, recorded from pulses that did not evoke retinal responses. FIG. 5F. Direct responses to 5 ms light pulse of different intensities showing the intensity-response dependence. Vertical grey lines indicate onset and termination of the light pulses.

[0068] FIGS. 6A-B show scanning electron micrographs of Cr/Au/H.sub.2Pc/PTCDI (1/9/30/30 nm) device layers, showing the rough nanocrystalline morphology of the evaporated organic p-n layers at two different magnifications.

[0069] FIGS. 7A-D show the ability using electrochemical impedance spectroscopy (EIS) to calculate resistances and capacitances of the photocapacitor/electrolyte system. The upper panels show Bode plots (left FIG. 7A and right FIG. 7B) for p-n devices measured in dark (black squares) and illuminated (open circles), with lines showing fits to the equivalent circuit diagrams plotted below (top FIG. 7C and bottom FIG. 7D).

[0070] FIGS. 8A-B show MEA current injection voltage transients. FIG. 8A, Transient voltages, Vt, measured with a glass capillary electrode placed above a 30 m diameter MEA electrode (left inset) at 4 different distances. Biphasic current pulse of 5 uA, 300 is (0.2 mC/cm.sup.2, right inset) were injected into the MEA electrode. FIG. 8B, Cathodic peak of the transient voltages as a function of the injected current measured at four different distances above the MEA electrode. The equivalent charge density is shown above.

[0071] FIGS. 9A-B show that metal-containing phthalocyanines do not lead to stable operation. Measurement of photovoltages of the ZnPc/PTCDI materials pair illustrates the effects of delamination (FIG. 9A C18 free and FIG. 9B with C18).

[0072] FIGS. 10A-D show photothermal response of Cr/Au-H.sub.2Pc/PTCDI filn FIG. 10A, Calibration procedure by measuring electrode resistance as a function of bath temperature. Electrode resistance was calculated by measuring the electrode voltage drop during 10 nA current injection. FIG. 10B1-B4 and FIG. 10C1-C4 are the same traces, respectively, at different time and voltage scales. Sample was illuminated by a 660 nm LED, either continuously for 1.5 s (B1,2., C1,2) or 100 pulses of 5 ms, 10 ms interval (B3,4., C3,4). Intensity was either 1700 mW/cm.sup.2 (B1-3., C1-3) or 480 mw/cm.sup.2 (B4., C4: similar to the cell culture activation protocol, see FIG. 3). Electrode was either unbiased (B1., C1) or biased by 10 nA (B2-4., C2-4). The latter resulted in a voltage drop proportional to the electrode resistance at a bath temperature of 36.8 C., which was set to zero before illumination. Light pulses resulted in transient voltage changes typical to the photoelectrical response. Biased measurements also show a DC component that is proportional to the change in electrode resistance due to photothermal heat dissipation. Changes in temperature were calculated according to the calibration procedure in FIG. 10A: V1=0.6 mv, V2=0.5 mv, V3=0.2 mv. These corresponds to T1=0.83 C., T2=0.69 C., and T3=0.28 C. FIG. 10D, Characteristic photothermal response for 1.5 s constant illumination of 1700 mW/cm.sup.2 (as in B2, C2).

DETAILED DESCRIPTION OF EMBODIMENTS

[0073] Materials

[0074] Phthalocyanine H.sub.2Pc (Alfa Aesar), ZnPc (BASF) and CuPc (BASF) were each purified by three-fold temperature gradient sublimation in a vacuum of <110.sup.3 torr. PTCDI, N,N-Dimethyl-3,4,9,10-perylenetetracarboxylic Diimide (BASF), was likewise purified thrice by sublimation.

[0075] Device Fabrication

[0076] Photocapacitor devices were fabricated using physical vapour deposition processes either on clean microscope slide glass or on commercial multielectrode arrays (Multichannel Systems GmbH), with both metal and organic regions defined by stainless steel shadow masks. Both glass and MEA substrates, after solvent cleaning, were treated with UV-generated ozone and a layer of chromium (2 nm) followed by gold (18 nm) was evaporated at a base pressure of <110.sup.6 mbar at a rate of 0.2 /s and 3-5 /s, respectively. It is known that following these fabrication procedures gives primarily Cr.sub.2O.sub.3 rather than metallic Cr. Following evaporation, the samples were exposed to UV-generated ozone for 15 minutes and then placed into a chamber held at 75 C. containing vapour of n-octyltriethoxysilane (OTS) for 2 hr. Following OTS treatment the substrates were rinsed with acetone and water and placed in boiling acetone for 15 minutes to remove multilayers and excess silanisation physioadsorbed on the Cr/Au or TiN electrodes (the latter in the case of MEA). The OTS layer was found to improve the adhesion of the organic semiconductor layer and prevent delamination, and produced reliably higher photovoltage than bare Cr/Au. Following rinsing with isopropanol and water and drying under a nitrogen stream, the samples were placed with appropriate shadow masking in an organic materials evaporator. The pigment layers were evaporated at a rate of 0.5 /s for the p-type layer and 5-6 /s for the n-type at a base pressure of <110.sup.6 mbar, to give a total thickness of 60 nm consisting of 30 nm of p- and n-type.

[0077] Photo-Response Characterization

[0078] The illumination unit consisted of a light-emitting diode (LED) with a peak wavelength of 660 nm (Thorlabs) mounted on an Olympus upright microscope (BX51WI) using a 4 or water immersion objectives of 10, 20, and 40, resulting in illumination intensities within the range of 0.6-1725 mW/cm.sup.2. The measurement unit consisted of a current amplifier (model 1212; DL Instruments) or voltage amplifier (model ELC-03XS, npi electronic GmbH). A photogenerated voltage was measured between the underlying metal electrode and a reference electrode (either Au or Ag/AgCl) in phosphate buffered saline (PBS) or modified Tyrode's solution (5 mM KCl, 25 mM NaHCO.sub.3, 10 mM glucose, 1.2 mM MgSO.sub.4, 1.2 mM HEPES, 0.5 mM glutamine, 2.5 mM CaCl.sub.2). Voltage transients were recorded using a micropipette electrode filled with 3M KCl, mounted on a computer motorized micromanipulator (model PatchStar, Scientifica) vs. Ag/AgCl reference electrode in the electrolyte.

[0079] A Xenon-Discharge Lamp and Czerny-Turner Monochromator were used as light source to acquire the photocurrent spectra. The photocurrents were amplified using a Lock-in amplifier and chopper operated at 29 Hz. The current rms values were acquired as a function of wavelength and normalized for the light intensity as measured with a pyroelectric detector. Impedance spectra were acquired in 0.1 M KCl with a Metro-Ohm PGSTAT 204 at OCP conditions.

[0080] Electrostatic Modeling

[0081] Electric potential distribution of the device immersed in electrolyte was modeled using the Robin Hood Solver software package for complex 3D electrostatic problems using the Robin Hood calculation method. Charged photocapacitor devices were modeled as two concentric metal plateslarger bottom gold electrode fixed at 0V potential, and the smaller top electrode which represented an equipotential surface at the top of the p-n junction, and which could be set at arbitrary potentials depending on the modeled electrode. The electrodes in the model were separated by a thin dielectric layer with relative permittivity of 3, characteristic to the organic semiconductors used here. The dielectric layer in the model represented the p-n junction region of the device. The space surrounding the device was modeled as a dielectric with relative permittivity of 80.1, representing a water-based electrolyte. All the dimensions in the model were true to the experimentally measured devices.

[0082] Neural Culture

[0083] All mice were treated in accordance with the principles and procedures of the Israel National Institute of Health and the United States National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. Protocols were approved by the Institutional Animal Care and Use Committee of the Tel Aviv University. Dissociated cortical cultures were prepared as follows: the entire cortices of SV129-mice, post-natal 0-1, were removed. Cortical tissue was digested with 0.065% trypsin (Biological Industries) in PBS for 15 min, followed by mechanical dissociation by trituration. Cells were re-suspended in a modified essential medium (MEM) without phenol red and glutamine, 5% horse serum, 50 mM glutamine, 0.02 mM glucose, 0.5% Pen-Strep, 2% B-27, and 0.75% glutamax (Gibco) and plated on either poly-D-lysine (PDL, Sigma) covered petri dish (control) or on type I samples (experiment) with a cell density of 3000 cells/mm.sup.2 (70010.sup.3 cells per dish). Cultures were maintained at 37 C. with 5% CO.sub.2. Growth medium was partially replaced every 3-4 days. At 4 DIV, cultures were infected with AAV-CAG-GCaMP6s viral vector (prepared by the Tel Aviv University vector core facility).

[0084] Optical Recording Via Calcium Imaging

[0085] Calcium imaging recordings were performed on 14 DIV in buffered mice artificial cerebrospinal solution (mice aCSF: 10 mM HEPES, 4 mM KCl, 1.5 mM CaCl.sub.2, 0.75 mM MgCl2, 139 mM NaCl, 10 mM D-glucose, adjusted with sucrose to an osmolarity of 325 mOsm, and with NaOH to a pH of 7.4). Images were acquired with an EMCCD camera (Andor Ixon-885) mounted on an Olympus upright microscope (BX51WI) using a 20 water immersion objective (Olympus, LUMPLFL NA 0.4).

[0086] Fluorescent excitation was provided via a 120 W mercury lamp (EXFO x-cite 120PC) coupled to a GFP filter cube (Chroma T495LP). Images were acquired at 59 fps in 22 binning mode using Andor software data-acquisition card (SOLIS) installed on a personal computer, spooled to a high capacity hard drive and stored as uncompressed multi-page tiff file libraries.

[0087] Electrical Recordings from Retinas

[0088] Coupling between the tissue and the electrodes was improved by placing a small piece of polyester membrane filter (5 m pores; Sterlitech) and a ring weight on the retina. The filter was removed before light stimulation to minimize scattering. Retinas were kept at physiological conditions, at a temperature of 34 C., and perfused (2-5 mL/min) with oxygenated (95% O.sub.2, 5% CO.sub.2) chick aCSF solution (5 mM KCl, 25 mM NaHCO.sub.3, 9 mM glucose, 1.2 mM MgSO.sub.4, 1.2 mM HEPES, 0.5 mM glutamine, 2.5 mM CaCl.sub.2). Neuronal signals were amplified (gain 1100 MEA1060-UP; MultiChannel Systems), digitized using a 64-channel analogue to digital converter (MC_Card; MultiChannel Systems), and recorded (MC_Rack; MultiChannel Systems). Direct retinal responses were recorded with 30-m diameter TiN electrode MEAs, using electrical stimuli generated by an external stimulator. In vitro epiretinal stimulation was carried out using a biphasic pulse of 300 s and found a critical threshold for eliciting retinal responses of 0.4-1.4 mC/cm.sup.2, similar to what has been reported in the literature.

[0089] Organic Electrolytic Photocapacitors

[0090] The organic thin film electrolytic capacitor we introduce here is a photodiode which produces electrical double layers upon illumination in water (FIG. 1A). It is believed that the electrical potential difference induced in the surrounding electrolyte could affect the membrane potential of cells in the vicinity, even stimulating action potentials in excitable cells providing the voltage perturbation is large enough. The photocapacitors consist of a p-n heterojunction bilayer on top of a metallic back-contact. A surrounding physiological electrolyte is in contact with both the bottom metal and the top of the p-n junction (FIGS. 1A-C). Devices are fabricated by sequential physical vapour deposition through stencil masks, allowing control over geometries and compatibility with various substrates. In contrast to many semiconductor materials that are sensitive to water, hydrogen-bonded pigments are exceedingly stable in aqueous environments: they can be readily biofunctionalized using simple water-based chemistry and have recently been shown to be stable photoelectrocatalysts in a pH range from 1 to 12. The materials combination which emerged as most promising and was used throughout this study comprises a Cr/Au layer (2 nm/18 nm) followed by a 30 nm layer of metal-free phthalocyanine (H.sub.2Pc) and 30 nm layer of N,N-dimethyl Perylene-3,4:9,10-tetracarboxylic Bisimide, PTCDI for short (FIGS. 1A-B). In an aqueous electrolyte, the device band diagram (shown at the beginning of the light pulse in FIG. 1C) is that of a p-n donor-acceptor photodiode with the metal and the electrolyte forming the bottom and upper electrodes. Photogenerated excitons separate into free carriers at the donor-acceptor (p-n) interface. The electrons accumulate in the n-type semiconducting layer and give rise to an oppositely charged double layer at the semiconductor-liquid interface. Photogenerated holes are injected into the metal, and form an electrical double-layer with the surrounding electrolyte. The maximum possible photovoltage (U.sub.photo) is given by the difference between the quasi-Fermi level at equilibrium and the conduction band edge of the n-type material. To understand the electrical potential in the surrounding aqueous environment, it is convenient to use electrostatic models. The charge and potential distribution were calculated for different charging voltages. The resultant distribution of electrical potential around a concentric photocapacitor device is plotted in FIG. 1D. Perturbation of the potential has a magnitude of several tens of mV at tens of m above the surface of the p-n layer. Therefore, the photoinduced voltage that a cell in close contact with the photocapacitor will feel can in principle be large enough to directly induce action potential generation, via the capacitive coupling mechanism as shown in FIG. 1E. The choice of p-n, as opposed to n-p, gives a negative surface potential on the top of the organic layer, thereby leading to depolarization, as opposed to hyperpolarization, of the attached cell membrane. A further critical aspect of successful device design is the surface morphology of the p-n layer. Nanoscale structure allows for higher photocharge densities to be achieved. Scanning electron microscopy revealed that 60 nm-thick p-n layers have a rough truncated nanopillar-like morphology (FIG. 6A-B) with relatively high surface area.

[0091] For photocapacitor characterization, we first fabricated 1.51.5 cm metallized (Cr/Au) glass slides with 1 cm.sup.2 square p-n layer (denoted as type I samples). This arrangement was used to establish baseline parameters for photovoltage/photocurrent, spectral response, and stability. The gold electrode was wired to be grounded or floating (FIG. 2A). The spectral responsivity for photocathodic current was measured for grounded samples, showing strong photocapacitive current generation in the red region of the visible spectrum, 700-600 nm, correlating closely with optical absorbance of the p-n stack (FIG. 2B). FIG. 2C shows photovoltage (V, trace 1) and photocurrent (I, trace 2) values of the photocapacitors measured between the Cr/Au layer versus reference electrode (Ag/AgCl) immersed in the solution, using pulsed illumination (5 ms, 660 nm). These results provide benchmark values for the photovoltages that the bilayer device can generatearound 280 mV (FIG. 2C, trace 1). Corresponding displacement current values, I, are 400 A/cm.sup.2 for light intensities of 60 mW/cm.sup.2 (FIG. 2C, trace 2). The photocurrent profile has a capacitive transient shape, and by integrating charge of cathodic (charging) and anodic (discharging) phases, we obtain an equal value of charge, evidencing that the current is non-Faradaic in nature. We obtained more details on Type I devices using electrochemical impedance spectroscopy (EIS). In the dark, the p-n junction response is described by a geometric capacitance of 130 nF/cm.sup.2 that corresponds well to the layer thickness of the depleted n-type semiconductor (d=Eco/C.sub.g=33 nm with E=3). Under illumination (620 nm, 0.81 mW/cm.sup.2) the impedance drops as carriers are accumulated in the semiconducting layers. The EIS data allows us, on the basis of an equivalent circuit model (FIG. 7A-D), to extract the capacitance between the p-n layer and water (C.sub.dl=3.8 F/cm.sup.2) and the internal resistance of the illuminated p-n junction (R.sub.int=1.2 k/cm.sup.2). The resistance in the dark, meanwhile, is very high (G/cm.sup.2) since both p and n materials are intrinsic semiconductors. Photofaradaic processes that follow a purely resistive path through the junction have only a very small contribution in the impedance spectra and show charge-transfer resistances in darkness or under illumination of R.sub.CT>1.1 M/cm.sup.2, evidencing that the photocurrent is indeed capacitive in nature.

[0092] The photovoltage build-up created in solution was next studied above the photocapacitor, this parameter being defined as the transient voltage, V.sub.t. This is measured with a glass micropipette electrode in solution mounted on a micromanipulator. All measurements were taken with the micropipette tip 10 m above the pigment surface, versus a large Ag/AgCl bath reference electrode, to give a realistic impression of what voltage perturbations cells adhered to the devices will encounter (FIG. 2C, trace 3). These V.sub.t measurements are taken without the Cr/Au metal film being electrically grounded, the metal is instead in direct contact with electrolyte, allowing us to characterize the operation of the photocapacitors in a wireless, free-standing mode. This scenario reflects the working conditions of standalone implantable device. The measured electrical potential is in the order of a few millivolts, up to 25 mV (to be contrasted with 280 mV under the same illumination conditions when measuring the grounded sampleFIG. 2C, trace 1). Vt profile and intensity is positively correlated with current profile (FIG. 2C, trace 2). It was found that cathodic peak values of Vt (cpVt) are a function of peak anodic current divided by the spot size radius, r.sub.(spot), consistent with classic electrostatics for potential above a disk of charge (FIG. 2D). Thus, while a displacement current can be readily associated with a known injected charge value, V.sub.t can also be associated with a corresponding charge value. To empirically link between V.sub.t and electrophysiology-relevant charge injection values we recorded V.sub.t as a function of distance (0-30 m) from a standard TiN MEA electrode, during stimulation with known current values (using values above the critical threshold needed to achieve action potential stimulation in explanted retinas, 0.1 mC/cm.sup.2), (FIG. 8A-B). The same experiment was repeated, this time recording the photogenerated potentials at 10 m above the pigment as a function of light intensity, using 10 ms pulses. Photovoltage values were recorded in both phosphate-buffered saline (PBS) and artificial cerebrospinal fluid (aCSF), which mimics the electrolytic environment in the eye. The peak values (cathodic phase) are plotted in FIG. 2E, which shows that photovoltages suitable for direct retinal stimulation can be generated already with illumination values around 100 mW/cm.sup.2. We found that measuring type I samples while illuminating a limited area (through 20 and 40 objective), the photocathodic voltage is highest in the middle of the illumination spot and decays rapidly at the edges of the spotlight (FIG. 2E). The lack of lateral leakage current in the semiconductor layer is due to its intrinsic nature, we know from impedance analysis that the resistance of the layer in the dark is in the gigaohm range.

[0093] For proper operation in electrophysiological applications, devices must be stable in aqueous environments and compatible with sterilization procedures. We measured samples over several days in PBS solution without noting decrease of recorded photovoltage. Accelerated stress test involving sequential treatment with oxygen plasma, ethanol, incubation in cell culture medium, followed by UV sterilization treatment and repeated ethanol rinsing were performed to validate device stability (FIG. 2G). In this study, we fabricated also devices from the well-known metal-containing phthalocyanine derivatives with copper and zinc, CuPc and ZnPc. These performed initially at a similar level as H.sub.2Pc devices, however these devices were not stable with respect to delamination and failed during these stress-test experiments (FIGS. 9A-B). The Cr/Au/H.sub.2Pc/PTCDI device configuration routinely passed the entire stress test sequence without significant loss in photovoltage or visible delamination.

[0094] Photostimulation of Cultured Primary Neurons

[0095] Having established details on the relationships between device structure and photovoltage behaviour, we proceeded to demonstrate stimulation of primary neuronal cultures (FIG. 3). We compared dissociated mice cortical neurons cultured on type I sample (Cr/Au/H.sub.2Pc/PTCDI, n=3) with neurons cultured on standard petri-dishes coated poly-d-lysine (PDLa standard cell adhesion layer, n=3). After 4 days in vitro (DIV) we infected the cultures with a viral vector for expressing the calcium indicator GCaMP6 and imaged neural activity at DIV 14. All cultures on both types of substrates developed into viable neural network, exhibiting spontaneous activity as indicated by the fluorescent calcium imaging (FIGS. 3A-B). Using a pulsed light stimulation, composed of 100 pulses of 600 nm, 480 mW/cm.sup.2, 5 ms pulse duration, 10 ms interpulse interval, we were able to detect a clear response only in a neuronal network that was cultured on type I device samples (FIG. 3D). It is important to note that the kinetics of the calcium indicators are relatively slow and do not show reliable single action-potential-associated calcium signals. Therefore, only a burst of activity that result from a train of pulses can accumulate into a detectable signal. In order to evidence the photocapacitive mechanism behind the observed action potential generation, we evaluated the contribution of photothermal heating (FIG. 10A-D). We utilized a calibrated pipette conductometric technique.sup.36 to measure local heating at the p-n device surface. Using the same illumination protocol, with the pulse train of 5 ms pulses, we registered temperature increases of 0.28 C. over the timescale of 1.5 s. The magnitude of these temperature changes indicates that a photothermal effect cannot be responsible for the action potential generation observed in these neuronal cultures. These Ca imaging studies show the potential of the organic photocapacitors to stimulate action potentials and the stability of the devices in physiological environment, and furnish preliminary evidence that the materials are not detrimental to cell viability.

[0096] From Film to Pixels

[0097] While larger uniform films are a simple platform for stimulating neurons, patterned pixels offer several possible advantages including integration with recording electrode arrays and stimulation localization. Decrease in the lateral dimensions of the device is also required for effective retinal implants or other applications requiring electrical stimulation. To design devices for effective stimulation using isolated islands, samples with p-n areas of different sizes, ranging from 200 to 1000 m in diameter, on top of a large, (type II), or finite (type III) gold surface area were fabricated and their V.sub.t was measured as described before (FIG. 4A). We evaluated the dependence of cathodic photovoltage as a function of the sizes of both the p-n junction area and the underlying gold layer. First, we varied the size of p-n junction islands on a gold film which had hundredfold greater area than the p-n regions, which we refer to as the infinite gold condition (FIG. 4A, device type II). We found that photocathodic voltage scales linearly with p-n junction diameter, and that p-n junction diameter of less than 150 m is unlikely to yield effective stimulation (FIG. 4B). It is apparent that the gold in contact with surrounding electrolyte is necessary for accommodating the positive charges photogenerated by the p-n junction. By laterally scanning the micropipette electrode from the centre of the p-n junction onto the gold layer it is clear that the sign of the recorded potential shifts from negative on top of the p-n junction to positive over the metal, and remains positive to around 250 m away from the edge of the p-n layer (FIG. 4C). The measured potential in solution closely follows the electrostatic model, plotted together with experimental data, for potential in the vicinity of disks of charge. To quantify the effect of exposed gold on performance, the p-n junction diameter was held constant 200 m and we varied the underlying gold size (FIG. 4A, device type III). An increased area of exposed gold is a critical parameter to obtain higher photocathodic values (FIG. 4D). Using these findings, we modified commercial MEAs with p-n pixels on large gold traces (FIG. 4E) and on 470 m diameter gold disks (FIG. 4F), creating platforms for localized photostimulation and simultaneous neural recording.

[0098] Photostimulation of a Blind Retina Model

[0099] The embryonic chick retina is a well-established model for the development of the visual system and the retina in particular. At embryonic day 14 (E14), retinal cells are in an early maturation stage, but the retina is not yet sensitive to light. Opsins mRNA only begins to appear in a small region by then, while photoreceptor electrical activity in response to light is not detected before E17. Thus, at this stage of development, the chick retina serves as a light insensitive retinal model.

[0100] Retinas (E14) were placed on type II or type III device-modified MEAs (FIG. 5A). Outer nuclear layer (FIG. 5B) and the nerve fibre layer (FIG. 5C) are readily apparent in visual inspection with a light microscope. The intrinsic light-insensitivity is always verified prior to further experiment, though E14 seldom show any light sensitivity. To provide an internal control, we used a single MEA electrode in the mode of typical electrical stimulation, delivering 0.8 A over 300 s, generating a direct action-potential response in the retina (FIG. 5D). We find that the exact same direct responses are generated synchronously in ganglion cells and fibres at the vicinity of the illuminated photocapacitor device pixels by delivering a 2 ms light pulse through the objective (FIG. 5E). In the chick retina, direct responses are easily recognized as they propagate in both directions along the nerve fibres (retrograde and anterograde). The latency of a direct response becomes larger when detected on electrodes that are further away from the stimulating electrode. Indeed, measured propagation direction (red and blue regression lines in FIG. 5A) and speed of 0.330.045 m/s (calculated from the latency of the response between two adjacent electrodes, 500 m apart), correspond well with fibre layer alignment (FIG. 5C) and known action potential propagation speed in the chick retina. The photocapacitive pixels elicit the same direct response as current-injected MEA electrodes (n=4 retinas), verifying that the devices are photocapacitively evoking direct retinal responses. Since these spikes are synchronized, they are summed into a large electrical signal that is superimposed on the stimulating signal. The amplitude of the recorded response is a function of the amount of recruited somas and nerve fibres that is directly correlated with the stimulus light intensity (FIG. 5F). Both type II and type III samples were found to evoke direct responses in retinas. Successful stimulations were made with all pixels of 100 m diameter and above for pulse duration as short as 1 ms. The minimal intensities for detecting a response were 430 mW/cm.sup.2 and 130 mW/cm.sup.2 for 100 and 200 m diameter pixels, respectively. These results unambiguously show deterministic and rapid action potential generation in light-insensitive retinas.

Discussion

[0101] The devices studied are free-standing (electrically-floating structures) and are fabricated via scalable fabrication steps where the substrate remains at room temperature, which allows integration with arbitrary substrate materials. While silicon based photodiodes have so far played a dominant role in the realm of artificial photoelectrical stimulation of neurons, silicon devices have several shortcomings compared with organic pigment layers. First, pigment films have a higher absorbance coefficient, allowing them to efficiently absorb light. At 660 nm, used in this work, the absorbance coefficient of vacuum-evaporated H.sub.2Pc is 310.sup.5 cm.sup.1, while silicon is 2.5810.sup.3 cm.sup.1. This difference allows making thinner photoactive films much less invasive, as the devices can have thicknesses that are much smaller than single cells. Our devices are 500 times thinner than the thinnest state-of-the-art silicon diodes for retinal implants. Moreover, in our design, one has a nanostructured semiconductor surface in direct contact with the electrolytic medium/biological sample, there is no voltage drop on a passivation layer or on conducting interconnects in between. Silicon photocapacitive devices charge metal electrodeshere we have the semiconductor surface itself serving as the primary charge-carrying electrode. Secondary metal electrodes must be employed in the case of silicon since it is not stable in physiological aqueous media. It must be carefully encapsulated, and interconnects passivated using SiO.sub.2/Si.sub.3N.sub.4 layers, for example. The organic p-n layers can make direct contact with the physiological environment due to their durability. Organic crystalline pigments like phthalocyanine and perylene bisimides are famously indestructible in terms of chemical and photochemical stability. Further, the nontoxicity of both phthalocyanines and perylene pigments is well-documented. These materials are used in cosmetics, medical products, and tattoos. They are commercial colorants which belong to the lowest category of hazard and toxicity for consumer approved materials in the EU.

[0102] Achieving temporal control over neural stimulation requires activation with short latency of the response. In the case of retinal stimulation, such short latency responses are attributed to directly activating the retinal ganglion cells (RGCs), when the electrodes are placed on top of the inner limiting membrane (ILM) at the epiretinal side, or to the inner nuclear layer (INL), when the electrodes are positioned subretinally, next to the degenerated photoreceptors. Direct activation of RGCs means that each stimulation pulse produces short latency synchronized action potentials (AP) in several somas and axons of RGCs that are located at the vicinity of stimulating electrode. On the contrary, stimulation of inner retinal neurons results in the generation of bursts of unsynchronized spikes in the RGCs with much longer latency, due to synapse transmission. Therefore, a major challenge in neuronal activation, in particular with photosensitive nanostructures, is to understand and to control the mechanism by which the activation is achieved, aiming for a sufficient charge injection for obtaining direct electrical activation similar to that of the best-optimized silicon-based electronics. Moreover, such electrical stimulation should be capacitive, which is considered safe and can be used for extended duration, unlike faradaic stimulation and thermal activation that are not considered optimal and should be avoided.

[0103] The retinal setup involves laying the RGC on top of the organic pigment, as in the case of epiretinal stimulation, while illuminating from the direction of the photoreceptors. This light trajectory is opposite to what is normal physiologically, but it does not contradict with the focus of this work, showing that the photoelectric transduction of our device is sufficient to stimulate neural tissue in a direct electrical manner at safe light intensities. In terms of neuronal stimulation benchmark parameters, our ultrathin organic device reaches parity with the state-of-the-art silicon diode-based technologies. This presents the ability to evoke action potentials in retinas using the same light intensity range as triple-tandem silicon retinal stimulation diodes. The range of pulsed light intensities and durations we have used has been deemed two orders of magnitude below the safe limit for ocular stimulation. Moreover, 660 nm is within the biological tissue transparency window, which can enable a host of other applications in peripheral nerve stimulation.

[0104] To conclude, we demonstrated a new and advantageous concept to photostimulate neurons. Primary neurons were cultured on our photocapacitor devices for three weeks, demonstrating viability of both the devices and the cells. The latter could readily be photostimulated using short impulses of light. We next integrated photocapacitors onto commercial MEAs, enabling simultaneous photoexcitation and recording. Using this platform, we demonstrated effective direct photostimulation of light-insensitive embryonic chicken retinas. The MEA allows us to make an in situ control of conventional electrical stimulation, thereby we verify that the photocapacitor arrays and the electrical stimulation have the exact same retinal response. We experimentally discount the presence of photothermal heating effects. The culmination of this work are stand-alone photocapacitors with organic pixels of 100 m in diameter to locally and reproducibly evoke action potentials. Future research of this device concept should involve optimizing materials to afford higher responsivity and photovoltage, allowing smaller pixels and lower light intensities. Different nano- and microstructuring of the organic material must be explored to yield optimal coupling with cells. The technology is a new platform that can interact with living cells via a true capacitive coupling mechanism, thus enabling safe and versatile next-generation implant technologies, and already at the level demonstrated here is suitable for various in vivo applications in peripheral or central nervous system stimulation, for example in the context of traumatic injury. Success in these efforts requires deployment of the devices on implantable and/or bioresorbable substrates, and evaluation of their stability and performance in vivo.