Conductive films

Abstract

Plasma enhanced chemical vapour deposited films are described. Methods of producing said films and their uses are also described. The films comprise one or more first regions and one or more second regions, said first regions being more electrically conductive than said second regions. Biomolecules or cells can be covalently attached or immobilised onto the films and the films can be used for the individual or simultaneous electrical and optical analysis of said biomolecules or cells.

Description

Conductive filmsField of the disclosure

[0001] The present disclosure relates to plasma enhanced chemical vapour deposited films, said films comprising at least carbon, oxygen and optionally nitrogen. The films comprise one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions. The present disclosure also relates to methods of producing said films, their biofunctionalisation, and their use, for example, in the manufacture of electrodes or microelectrodes.Background of the disclosure

[0002] Current electrophysiological studies are limited by the lack of suitable biofunctionalisable cell culture platforms for electrical or optical analysis. Trade-offs must be made between electrical read out and cellular observation, slowing the development of fundamental physiology, knowledge of neural and cardiac associated disease and patient specific drug development.

[0003] Microelectrode arrays (MEAs) for electrophysiological studies have evolved from gold to indium tin oxide and carbon-based electrodes, but the capability to easily biofunctionalise both electrically conductive surfaces that may be used as electrodes and non-conducting surfaces alike is still lacking.

[0004] Standard routes for electrode fabrication include masking, etching, chemical deposition or photolithographic techniques. These methods all include long processing times or extended delays between pattern modification and design fabrication.

[0005] Manufacturing constraints prevent ease of design iteration and the ability to customise electrode layouts for specific applications. Laser induced graphite is at the forefront of addressing these issues. Standard procedures utilise continuous CO2 lasers which are limited in electrode patterning precision due to large heating effects at the site of laser incidence. Due to the resolution and uniformity constraints of the current procedures, no laser patterning of microelectrode arrays for commercial use has been achieved, and hence these sensors are typically of millimetre scale.

[0006] Transparent platforms in particular are in high demand to allow concurrent electrical readout and cellular imaging, ultimately allowing multiplexing of electrical and optical analysis. In-vitro platform providers often claim to have transparent capabilities, however while the substrate is transparent the electrodes are opaque and block any analysis of the electrode-cell interface.

[0007] In terms of transparent micro electrode array technologies, there are a handful of transparent electrodes on the market, including graphite, titanium nitride (TiN), indium tin-oxide (ITO) and other organic polymer coatings such as PEDOT. ITO electrodes are extremely costly, well exceeding the prices of standard MEA platforms and have low levels of biocompatibility compared to organically based electrode materials.

[0008] Although becoming more utilised, graphene electrodes and organic polymers have limited processing available for patternable electrode design, needing wet chemical methods for deposition, and they lack a functional biomolecule binding mechanism. Currently, all commercially available transparent electrodes lack capability for facile reagent-free, on-contact, covalent surface functionalisation with bio signalling molecules.

[0009] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the disclosure

[0010] Disclosed herein are new plasma enhanced chemical vapour deposited films and processes for their preparation. The films comprise one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions. Cells, biomolecules, or small organic molecules may be covalently attached to or immobilised on the films.

[0011] In one aspect, the present disclosure provides a plasma enhanced chemical vapour deposited (PECVD) film, said film comprising at least carbon and oxygen;wherein said film comprises one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions.

[0012] In embodiments, the film further comprises nitrogen.

[0013] In another aspect, the present disclosure provides a plasma enhanced chemical vapour deposited (PECVD) film, said film comprising at least carbon, nitrogen and oxygen; wherein said film comprises one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions.

[0014] In embodiments of the films comprising nitrogen, the one or more first regions have a nitrogen content that is at least 10% lower than a nitrogen content in the one or more second regions.

[0015] In embodiments, the one or more first regions have an oxygen content that is at least 10% higher than an oxygen content in the one or more second regions.

[0016] In embodiments of the films comprising nitrogen, the nitrogemcarbon elemental ratio in the PECVD film is from about 0.01 :1 to about 1 :1 , or from about 0.1 :1 to about 2:3.

[0017] In embodiments, the one or more first regions have equivalent or higher radical concentration relative to the one or more second regions.

[0018] In other embodiments, the one or more first regions have equivalent or lower radical concentration relative to the one or more second regions.

[0019] In embodiments, the percentage elemental carbon as measured by XPS in the PECVD film is from about 50% to about 90% by atomic % composition. In other embodiments, the percentage elemental carbon as measured by XPS in the PECVD film is greater than about 70% by atomic % composition.

[0020] In embodiments, the percentage elemental nitrogen as measured by XPS in the PECVD film is from about 0% to about 50% by atomic % composition. In otherembodiments, the percentage elemental nitrogen as measured by XPS in the PECVD film is less than about 20% by atomic % composition.

[0021] In embodiments, the percentage elemental oxygen as measured by XPS in the PECVD film is from about 5% to about 40% by atomic % composition. In other embodiments, the percentage elemental oxygen as measured by XPS in the PECVD film is less than about 10% by atomic % composition.

[0022] In embodiments, the one or more first regions have an optical transmittance of greater than about 50%, or greater than about 70%, or greater than about 80%.

[0023] In embodiments, the PECVD film thickness is from about 10 to about 1000 nm. More preferably from about 50 to about 300 nm. In other embodiments, the PECVD film thickness is greater than 50 nm.

[0024] In embodiments, the PECVD film may be present on at least one surface of a substrate. The substrate may be any suitable substrate. For example, the substrate may be metal, glass (such as silicate glass, borosilicate glass or soda-lime glass), polymer (such as PDMS, PTFE, polycarbonate, polycaprolactone, polyethylene), quartz, chalcogenides, metal oxides (such as indium-tin oxide coated glass), or silicon (such as a silicon wafer).

[0025] In another aspect, the present disclosure provides a substrate coated with a PECVD film according to any one of the herein disclosed embodiments.

[0026] In some embodiments, the substrate comprises two or more different substrates.

[0027] In some embodiments, the substrate comprises one or more of metal, glass, polymer, quartz, chalcogenides, metal oxides, or silicon or diamond. Preferably, the substrate comprises one or more of diamond, indium-tin oxide, glass, gold, PDMS or quartz.

[0028] In another aspect, the present disclosure provides two or more substrates coated with a PECVD film according to any one of the herein disclosed embodiments, thereby providing an interface between said two or more substrates. In some embodiments the substrate is substantially transparent.

[0029] In some embodiments, the PECVD film may be present on at least one surface of two or more substrates, thereby spanning an interface between said two or more substrates. Advantageously, this provides an alternative arrangement of conduits between the PECVD film and potential external measurement devices.

[0030] In embodiments, the one or more first regions comprise one or more electrodes.

[0031] In embodiments, the one or more first regions comprise two or more electrodes, said electrodes having any suitable inter-electrode distance. For example, the interelectrode distance is greater than about 1 pm. For example from about 1 pm to about 50 mm, or from about 1 pm to about 10 mm, or from about 1 pm to about 1 mm, or from about 1 pm to about 500 pm, or from about 1 pm to about 300 pm, or from about 300 pm to about 50 mm, or from about 300 pm to about 10 mm, or from about 300 pm to about 1 mm, or from about 300 pm to about 500 pm, or from about 500 pm to about 50 mm, or from about 500 pm to about 10 mm, or from about 500 pm to about 1 mm, or from about 1 mm to about 50 mm, or from about 1 mm to about 10 mm, or from about 10 mm to about 50 mm. Other inter-electrode distances are contemplated.

[0032] In embodiments, the electrodes comprise an electrode pad and an electrode track connected to said electrode pad.

[0033] In embodiments, the electrode pad diameter is any suitable electrode pad diameter. For example, the electrode pad diameter may be greater than about 20 pm. For example from about 20 pm to about 100 mm, or from about 20 pm to about 10 mm, or from about 20 pm to about 1 mm, or from about 20 pm to about 500 pm, or from about 20 pm to about 250 pm, or from about 250 pm to about 100 mm, or from about 250 pm to about 10 mm, or from about 250 pm to about 1 mm, or from about 250 pm to about 500 pm, or from about 500 pm to about 100 mm, or from about 500 pm to about 10 mm, or from about 500 pm to about 1 mm, or from about 1 mm to about 100 mm, or from about 1 mm to about 10 mm, or from about 10 mm to about 100 mm. Other electrode pad diameters are contemplated.

[0034] In embodiments, the one or more electrodes in the one or more first regions may be micro electrodes.

[0035] In embodiments of the micro electrodes, the inter-electrode distance may be any suitable inter-electrode distance. For example from about 1 pm to about 500 pm, or from about 10 pm to about 300 pm.

[0036] In embodiments of the micro electrodes, the electrode pad diameter may be greater than about 20 pm. For example from about 20 pm to about 250 pm.

[0037] In embodiments, the PECVD film further comprises one or more biomolecules or organic small molecules covalently bonded to or immobilised on the one or more of the first and / or second regions of the PECVD film.

[0038] In embodiments, the one or more biomolecules comprise one or more organic small molecules, proteins, polysaccharides, nucleotides, oligonucleotides, antioxidants, growth factors, vitamins and lipids.

[0039] In embodiments, the PECVD film further comprises cells, wherein the cells are covalently bonded to or immobilised on one or more of the first and / or second regions of the PECVD film.

[0040] In another aspect, the present disclosure provides a process for producing a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising:(a) providing a precursor plasma enhanced chemical vapour deposited film, said precursor PECVD film comprising at least carbon and oxygen; and(b) patterning the one or more first regions into the precursor PECVD film by subjecting areas of said precursor PECVD film to a laser beam.

[0041] In embodiments of the process, the PECVD film further comprises nitrogen.

[0042] In another aspect, the present disclosure provides a process for producing a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising:a) activating one or more surfaces of a substrate by exposing said substrate to a plasma formed in the presence of one or more of helium, neon, oxygen, nitrogen, argon and xenon; b) depositing a precursor plasma enhanced chemical vapour deposited (PECVD) film on the one or more activated surfaces of the substrate by exposing said activated surface to a plasma formed in the presence of one or more organic gases, and one or more of nitrogen, helium, neon, argon and xenon; and c) patterning one or more first regions into the precursor PECVD film by subjecting areas of said precursor PECVD film to a laser beam.

[0043] In another aspect, the present disclosure provides a process for producing a substrate coated with a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising: a) activating one or more surfaces of a substrate by exposing said substrate to a plasma formed in the presence of one or more of helium, neon, oxygen, nitrogen, argon and xenon; b) depositing a precursor plasma enhanced chemical vapour deposited (PECVD) film on the one or more activated surfaces of the substrate by exposing said activated surface to a plasma formed in the presence of one or more organic gases, and one or more of nitrogen, helium, neon, argon and xenon; and c) patterning one or more first regions into the precursor PECVD film by subjecting areas of said precursor PECVD film to a laser beam.

[0044] In some embodiments, the substrate comprises two or more different substrates.

[0045] In embodiments, the total pressure in step a) is from about 25 mTorr (3.3 Pa) to about 500 mTorr (66.7 Pa).

[0046] In embodiments, the pressure in step a) is from about 25 mTorr (3.3 Pa) to about 150 mTorr (20.1 Pa).

[0047] In embodiments, the total pressure in step b) is from about 25 mTorr (3.3 Pa) to about 500 mTorr (66.7 Pa).

[0048] In embodiments, the total pressure in step b) is from about 50 mTorr (6.7 Pa) to about 350 mTorr (46.7 Pa), or from about 50 mTorr (6.7 Pa) to about 250 mTorr (33.3 Pa), or from about 50 mTorr (6.7 Pa) to about 150 mTorr (20.1 Pa).

[0049] In embodiments, the pressure in step b) is less than about 250 mTorr (33.3 Pa), or less than about 200 mTorr (26.7 Pa).

[0050] In embodiments, a plasma discharge in step a) is maintained from about 10 seconds to about 30 minutes, or from about 5 minutes to about 15 minutes.

[0051] In embodiments, a plasma discharge in step b) is maintained from about 10 seconds to about 30 minutes, or from about 30 seconds to about 25 minutes.

[0052] In embodiments, the laser is a He laser, a CO2 laser, a Nd:YVCU laser, a titaniurmsapphire laser or a ytterbium laser.

[0053] In embodiments, the plasma in step a) is formed in the presence of argon.

[0054] In embodiments, the plasma in step b) is formed in the presence of argon and optionally nitrogen.

[0055] In embodiments, the one or more organic gases comprise one or more of hydrocarbon and substituted hydrocarbon.

[0056] In embodiments, the one or more organic gases comprises one or more linear or branched alkane or cycloalkane, linear or branched alkene or cycloalkene, and linear or branched alkyne.

[0057] In embodiments, the one or more organic gases comprises acetylene or substituted acetylene.

[0058] In embodiments, the substituted hydrocarbon comprises one or more of hydroxyl substituted hydrocarbon, amino substituted hydrocarbon, and hydrocarbons substituted with sulphur-containing groups.

[0059] In embodiments, the volume ratio of the at least one organic gas fed to the plasma chamber to the sum of one or more of nitrogen, helium, neon, argon and xenon, is about 1 to 20 to about 1 to 2, or from about 1 to 15 to about 1 to 5.

[0060] In embodiments, the volume ratio of the at least one organic gas fed to the plasma chamber to the sum of argon and / or nitrogen is about 1 to 20 to about 1 to 2, or from about 1 to 15 to about 1 to 5.

[0061] In embodiments, the power supplied in step a) is from about 10 W to about 200 W, or from about 50 W to about 100 W.

[0062] In embodiments, the power supplied in step b) is from about 10 W to about 200 W, or from about 50 W to about 100 W.

[0063] In embodiments, the wavelength of the laser light is from about 200 nm to about 1500 nm, or from about 300 nm to about 1100 nm, or from about 450 nm to about 600 nm.

[0064] In embodiments, the laser is pulsed at pulse rates from about 100 fs to about 1 ns, or from about 200 fs to about 50 ps, or from about 150 fs to about 15 ps.

[0065] In embodiments, the laser operates at a frequency of about 1 kHz to about 5 MHz, or from about 200 kHz to about 2 MHz, or from about 200 kHz to about 1 MHz.

[0066] In another aspect the present disclosure provides a PECVD film according to any one of the herein disclosed PECVD film embodiments produced by the process of any one of the herein disclosed processes.

[0067] In another aspect, the present disclosure provides a method of covalently bonding or immobilising small organic molecules or biomolecules to a PECVD film, said film comprising at least carbon and oxygen, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said method comprising:(a) depositing small organic molecules or biomolecules on the PECVD film; and(b) covalently attaching or immobilising one or more small organic molecules or biomolecules to one or more of the first and second regions of the PECVD film.

[0068] In embodiments of the method of covalently bonding or immobilising small organic molecules or biomolecules, the PECVD film further comprises nitrogen.

[0069] In another aspect, the present disclosure provides a method of covalently bonding or immobilising cells to a PECVD film, said film comprising at least carbon and oxygen, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said method comprising:(a) depositing cells on the PECVD film; and(b) covalently attaching or immobilising the cells to one or more of the first and second regions of the PECVD film.

[0070] In embodiments of the method of covalently bonding or immobilising cells, the PECVD film further comprises nitrogen.

[0071] In another aspect, the present disclosure provides a method of covalently bonding or immobilising cells to the herein disclosed PECVD film, comprising: a) depositing cells in a cell culture medium on a PECVD film according to any one of the herein disclosed embodiments; b) covalently attaching biomolecules from the cell culture medium to one or more of the first and second regions of a PECVD film; and c) attaching the cells to the covalently attached biomolecules.

[0072] In another aspect, the present disclosure provides a PECVD film according to any one of the herein disclosed embodiments for use in a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule, and / or cell.

[0073] In another aspect, the present disclosure provides a substrate coated with a PECVD film according to any one of the herein disclosed embodiments for use in a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule, and / or cell.

[0074] In another aspect, the present disclosure provides the use of a PECVD film according to any one of the herein disclosed embodiments in the manufacture of a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule, and / or cell.

[0075] In another aspect, the present disclosure provides the use of a substrate coated with a PECVD film according to any one of the herein disclosed embodiments in the manufacture of a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule, and / or cell.

[0076] In another aspect, the present disclosure provides the use of a substrate coated with a PECVD film according to any one of the herein disclosed embodiments in the manufacture of a device for the simultaneous electrical and optical analysis of small organic molecules, biomolecules, cells, and / or populations of cells including organoids and explants.

[0077] In embodiments, the device is a sensing device.

[0078] In another aspect, the present disclosure provides a method of analysing small organic molecules, biomolecules or cells comprising: providing a PECVD film according to any one of the herein disclosed embodiments, said PECVD film comprising small organic molecules, biomolecules and / or cells covalently bonded or immobilised thereto; and one or more of optically imaging the small organic molecules, biomolecules, and / or cells; electrically stimulating the small organic molecules, biomolecules, and / or cells; recording an electrical output from the small organic molecules, biomolecules, and / or cells.

[0079] In another aspect, the present disclosure provides a method of analysing small organic molecules, biomolecules or cells comprising: providing a substrate coated with a PECVD film according to any one of the herein disclosed embodiments, said PECVD film comprising small organic molecules, biomolecules and / or cells covalently bonded or immobilised thereto; and one or more ofoptically imaging the small organic molecules, biomolecules, and / or cells; electrically stimulating the small organic molecules, biomolecules, and / or cells; recording an electrical output from the small organic molecules, biomolecules, and / or cells.

[0080] In another aspect, the present disclosure provides a microelectrode array (MEA) comprising a PECVD film according to any one of the herein disclosed embodiments.

[0081] In another aspect, the present disclosure provides a microelectrode array (MEA) comprising a substrate coated with a PECVD film according to any one of the herein disclosed embodiments.

[0082] In another aspect, the present disclosure provides a microelectrode array (MEA) comprising a substrate coated with a PECVD film, said film comprising at least carbon and oxygen; the film further comprises one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions; the one or more first regions comprising one or more electrodes or micro electrodes; and wherein the substrate comprises one or more substrates, said one or more substrates comprising one or more of metal, glass, polymer, quartz, chalcogenides, metal oxides, silicon or diamond.

[0083] In embodiments, advantages of the presently disclosed PECVD films, methods of fabrication and methods of covalently bonding or immobilising small organic molecules, biomolecules, or cells include one or more of the following:• The PECVD films can form attachments with small organic molecules, biomolecules, or cells without the need for chemical linkers or other reagents.• The PECVD films may be optically transparent and have low auto-fluorescence.• The first regions of the PECVD films have sufficiently high electrical conductivity for electrical applications.• The method of fabrication provides fast production micro- or macro-scale patterning of conductive regions in the PECVD films, with customisable feature design and manufacture.• The PECVD films provide concurrent high quality cell imaging and electrical stimulation / recording.

[0084] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0085] Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0086] Figure 1. X-ray Photoelectron Spectroscopy data showing the change in elemental concentrations (in atomic % composition) for a given fabrication fluence condition. Dotted lines annotated with atomic symbols indicate the elemental percentages present in pristine films before laser treatment. Peak conductivity occurs at -0.046 J / cm2.

[0087] Figure 2. Raman spectra evolution with incident laser fluence condition. All spectra are shifted arbitrarily in intensity to aid observation of peak evolution. The most prominent peaks at 1350 nnr1and 1580 nnr1correspond to D and G peaks respectively, which indicate the presence of sp2and sp3hybridisations within the film structure. The formation of the peak at -1100 nnr1is attributed to the presence of silicon, which becomes more evident with film thinning / densification as fluence of the treatment increases.

[0088] Figure 3. D / G peak intensity ratios observed via Raman spectroscopy of treated films with fluence variation.

[0089] Figure 4. Absorption of PECVD films with varied incident laser treatment fluence. All values are normalised to a comparison standard microscope glass baseline.A) Absorption spectra of selected laser fluence treatments on 4:10:3 SCCM C2H2:N2:Ar films illustrating electrode transparency under varied manufacturing laser fluence conditions. Initial untreated PECVD film shown in solid line; B) Absorption of electrodes with various manufacturing fluence conditions. The displayed wavelengths (488 nm, 543 nm, 592 nm and 635 nm) relate to common fluorescent microscopy applications.

[0090] Figure 5. Electron Paramagnetic Resonance spectra showing increased radical concentration with increased incident laser fluence.

[0091] Figure 6. Graph showing the determination of the ablation threshold by single shot ablation studies at various film thicknesses.

[0092] Figure 7. Relationship between thickness and ablation threshold within the film (N=1 line) and the fluence required for conductive enhancement (N = 992 line) with projected incubation trend ( F con d).

[0093] Figure 8. A) Conductivity of 4:10:3 C2H2:N2:Ar SCCM PECVD films laser treated at varied laser fluence. All transient scanning parameters are fixed; B) Conductivity of films with constant laser fluence and varied scan fluence is governed by hatch spacing and laser scan speed.

[0094] Figure 9. Micro electrode tracks imaged using an optical microscope. A) Phase contrast image showing electrode detail; B) Image showing complete microelectrode array (MEA) arrangement; C) A commercial MEA from Multi-Channel Systems shown for comparison, note the dark tracks arising from lack of optical transparency.

[0095] Figure 10. AFM analysis of various scan Fluence conditions; A) is an untreated, pristine PECVD film; B, C and D show scan parameters (raster pitch, scan speed) of (2pm, 50mm / s), (2pm, 5mm / s) and (5pm, 50mm / s) respectively.

[0096] Figure 11. BSA binding fluorescence studies for varied scan fluence conditions before and after detergent washing (SDS) to remove physisorbed protein. Fluorescence signal of all PECVD films with / without laser treatment is much brighter when compared to bare glass after detergent washing, indicating the strength of biomolecular binding and presence of covalent attachment mechanisms regardless of treatment condition. Reference samples are shown to the right of the parameter grid.

[0097] Figure 12. A) Fluorescence intensity of the parameter study compared to the baseline of pristine PECVD films. Quantification of the average fluorescence for each condition sampled at 3 regions upon each surface of the corresponding parameter condition treatment. Dotted horizontal lines from top to bottom represent a pristine PECVD film, glass surface and no BSA sample conditions. All scanning conditions have fluorescence above that observed for bare glass; B) Variance of the fluorescence measurement demonstrating the variation of the protein binding across an electrode patterned into a PECVD film and a pristine PECVD film.

[0098] Figure 13. Illustration of transparency in biological sample applications. Fluorescent images of fibroblast culture on PECVD films with laser patterned conductive tracks, illustrating no interference of the electrode tracks with imaging processes. A) Actin Red stained cytoplasm of Fibroblasts; B) NucBlue™ stained nuclei; C) Inverted image of electrode tracks; D) Superimposed image of A, B and C demonstrating the visibility of cells on top of electrode tracks.

[0099] Figure 14. A) graph showing the ablation threshold of PECVD films with different underlying substrate materials; B) ablation threshold characterisation of PECVD films with different underlying substrate materials.

[0100] Figure 15. Raman spectroscopy of PECVD films on a range of underlying substrates with characteristic peaks of graphite like structures (dotted), traces from top to bottom are PDMS, gold, glass, ITO and diamond.

[0101] Figure 16. Multi-material substrate PECVD laser induced transparent conductive (LITC). Left: PECVD LITC on glass substrate connected to underlying gold electrodes for robust readout; Right: Close-up of PECVD LITC interface. The underlying glass (5) and gold (3) regions have LITC (1 ,4) and pristine PECVD film (2) showing transformation of the thin film on different underlying substrates.

[0102] Figure 17. Electrochemical data showing performance of PECVD films. A) Illustrates comparison of PECVD LITC films to typical ITO films. B) Phase (+) and Impedance (x) values for the PECVD LITC film illustrating impedance for comparison to standard electrodes. C) Cyclic voltammetry data shows stability of the LITC electrode in a wet-chemical environment, supporting the use of the electrode for stimulation and measurement regimes.

[0103] Figure 18. Electro-chemical measurements showing the charge injection capability of the film when compared to ITO electrode materials. A) Input stimulation waveform and measured waveform from both PECVD LITC and ITO electrodes; B) Integrated signals showing the accumulation of charge delivered to the cells atop the application targeted electrodes.Detailed description of the embodiments

[0104] It will be understood that the disclosure described and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

[0105] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. It will be understood that the disclosure described and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

[0106] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0107] For the purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

[0109] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an organic gas” means one organic gas or more than one organic gas.

[0110] As used herein, the term “and / or”, e.g., “X and / or Y” will be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0111] As used herein, the term “about” refers to a quantity, value, dimension, size, or amount that varies by as much as 10%, 5%, 1% or 0.1 % to a reference quantity, value, dimension, size, or amount.

[0112] Throughout the present disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0113] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0114] It will be understood that the disclosure described and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text. All of these different combinations constitute various alternative aspects of the disclosure.

[0115] Disclosed herein are new plasma enhanced chemical vapour deposited films and processes for their preparation. The films comprise one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions. Cells, biomolecules, or small organic molecules may be covalently attached to or immobilised on the films.Plasma enhanced chemical vapour deposited films

[0116] The present disclosure relates to a plasma enhanced chemical vapour deposited (PECVD) film, said film comprising at least carbon and oxygen; wherein said film comprises one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions.

[0117] The film may further comprise nitrogen.

[0118] The present disclosure further relates to a plasma enhanced chemical vapour deposited (PECVD) film, said film comprising at least carbon, nitrogen and oxygen; wherein said film comprises one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions.

[0119] The one or more first regions of the PECVD film may have a nitrogen content that is at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70% lower than a nitrogen content in the one or more second regions.

[0120] The one or more first regions of the PECVD film may have an oxygen content that is at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70% higher than an oxygen content in the one or more second regions.

[0121] The PECVD film may have a nitrogemcarbon elemental ratio of about 0.01 :1 to about 1 :1 . For example the film may have a nitrogemcarbon elemental ratio of about about 0.1 :1 to about 1 :1 , or about 0.15:1 to about 1 :1 , or about 0.2:1 to about 1 :1 , or about 0.25:1 to about 1 :1 , or about 0.3:1 to about 1 :1 , or about 0.35:1 to about 1 :1 , or about 0.4:1 to about 1 :1 , or about 0.45:1 to about 1 :1 , or about 0.5:1 to about 1 :1 , or about 0.55:1 to about 1 :1 , or about 0.6:1 to about 1 :1 , or about 0.65:1 to about 1 :1 .

[0122] The PECVD film may comprise unpaired electrons. These unpaired electrons may be on or near the surface of the first and second regions. The unpaired electrons may be at a depth of 40 nm or less from the surface of the first and second regions, or within about 30, 20 or 10 nm of the surface, or may be from about 10 to about 40 nm from the surface, or from about 10 and 30, 20 and 40, or 20 and 30 nm from the surface, or about 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nm from the surface. They may be at a variety of depths from about 0 to about 40 nm. In some instances they may be at depths of greater than 40 nm. They may be throughout the volume of the first and second regions. This may render the regions capable of reacting with another material, such as a biomaterial, so as to covalently bond said material to the first or second regions.

[0123] The one or more first regions of the PECVD film may have equivalent or higher radical concentration relative to the one or more second regions.

[0124] The one or more first regions of the PECVD film may have equivalent or lower radical concentration relative to the one or more second regions.

[0125] Radical concentration and in turn the functionalisation or bio-functionalisation of the film surface occurs when there are free radicals present throughout the film structure. Without being bound by theory, radical concentrations can be increased, or “reawakened” in older films via annealing. As annealing / restructuring occurs during laser treatment the radical concentration may increase up to a certain laser power level. Once the laser power reaches a point at which graphitisation occurs, these radicals may become incorporated within the graphite structure. Therefore radical density may decrease as laser power increases. There is therefore a trade-off between conductivity and radical density which can be tuned appropriately for the desired application.

[0126] The percentage elemental carbon as measured by XPS in the PECVD film may be from about 50% to about 90% by atomic % composition, or from about 60% to about 90% by atomic % composition, or from about 70% to about 90% by atomic % composition, or from about 50% to about 80% by atomic % composition, or from about 60% to about 80% by atomic % composition, or from about 70% to about 80% by atomic % composition.

[0127] The percentage elemental nitrogen as measured by XPS in the PECVD film may be from about 0% to about 50% by atomic % composition, or from about 0% to about 40% by atomic % composition, or from about 0% to about 30% by atomic % composition, or from about 0% to about 25% by atomic % composition, or from about 0% to about 10%, or from about 10% to about 50% by atomic % composition, or from about 10% to about 40% by atomic % composition, or from about 10% to about 30% by atomic % composition, or from about 10% to about 25% by atomic % composition.

[0128] The percentage oxygen as measured by XPS in the PECVD film may be from about 5% to about 40% by atomic % composition, or from about 5% to about 30% by atomic % composition, or from about 5% to about 25% by atomic % composition, or from about 5% to about 15% by atomic % composition, or from about 10% to about 40% by atomic % composition, or from about 10% to about 30% by atomic % composition, or from about 10% to about 25% by atomic % composition, or from about 10% to about 15% by atomic % composition.

[0129] The nitrogen content of the PECVD film prior to graphitisation through laser treatment is dependent on the conditions used during the plasma deposition process. Generally lower carbon content, higher nitrogen content films are thinner in comparison to higher carbon content films formed using similar deposition times, meaning longer deposition times may be required to have sufficient carbon content in the bulk film for graphitisation. As conductivity is dependent on the removal of nitrogen from the surface to allow carbon bond restructuring, a balance may be struck with conductivity to deliver a nitrogen content for a desired application. Outside of the graphitised electrodes, the surrounding areas of the deposited film may be tailored to provide nitrogen levels, and in turn zeta potentials, relevant to the application. Nitrogen present in the PECVD film may not be required for certain applications and so the PECVD film may have relatively low levels of nitrogen present.

[0130] In addition to a film’s original composition, external factors such as the atmosphere of laser treatment can contribute to differing elemental percentages present in the film. Oxygen levels may fluctuate dependent on the atmosphere the laser treatment is performed in, i.e. in vacuum or another inert gas. Therefore, the percentage of carbon, oxygen, and optionally nitrogen as measured by XPS in the film may be dependent on the atmosphere within which the laser treatment is performed.

[0131] Without being bound by theory, laser treatment in air would produce a PECVD film higher in oxygen content due to the incorporation of atmospheric oxygen into the film during treatment. Conversely, laser treatment in argon would not lead to incorporation of atmospheric oxygen and thus the films would have a lower oxygen content.

[0132] Films may be formed in the presence of nitrogen and fragments of this gas may be incorporated into the film during deposition or laser treatment. For example, the use of nitrogen may result in the presence of amine, imine or nitrile groups, or a mixture thereof, in the first or second regions of the film. Thus, the first and second regions of the film disclosed herein may comprise various percentages of nitrogen. Films formed without the presence of nitrogen would comprise low levels of nitrogen. Low nitrogen films may lead to improved effectiveness of the laser treatment, but may reduce the advantages of having nitrogen present for some of the presently disclosed applications. Therefore, the nitrogen content in the PECVD film may be tuned to application specific requirements.

[0133] The one or more first regions may have an optical transmittance of greater than about 50%, or greater than about 70%, or greater than about 80%.

[0134] The PECVD film may have a thickness from about 10 to about 1000 nm, or from about 50 to about 300 nm. In other embodiments, the PECVD film thickness is greater than 50 nm.Substrates

[0135] Formation of the PECVD film may be performed on at least one surface of a substrate, said substrate being selected from any suitable substrate. For example, metal, glass (such as silicate glass, borosilicate glass or soda-lime glass), polymer (such as PDMS, PTFE, polycarbonate, polycaprolactone, polyethylene), quartz, chalcogenides, metal oxides (such as indium-tin oxide coated glass), or silicon (such as a silicon wafer). Other suitable substrates are contemplated.

[0136] In alternative embodiments, the substrate may be selected from diamond, indium-tin oxide, glass, gold, PDMS or quartz.

[0137] In some embodiments the substrate is substantially transparent.

[0138] As used herein, “substantially transparent” refers to a material or region with an optical transmittance of greater than about 50%, or greater than about 70%, or greater than about 80%, or greater than 90%.

[0139] Any substrate, including metals, polymers or dielectrics, that are chosen for a specific application can have a precursor PECVD film formed thereon and the film graphitised. Each substrate material will have its own laser characterisation based on the absorption of the laser light, but ultimately the film deposition and conductive enhancement processes remain the same.

[0140] Metals may also act as a substrate, and the present disclosure provides a biofunctionalised film that may be used to modify commercially available micro electrode array systems due to the microscale patterning capability and conductive enhancement, replacing, for example, the un-functionalised epoxy-based SU8 photoresist commonly used for these insulator film applications.

[0141] The presently disclosed films may also have the advantage of providing a uniform surface between the more and less conductive regions, due to no difference in manufacturing material in each region, unlike current commercially available platforms. This uniformity across the platform’s surface may provide no variation in cell culture conditions which might otherwise impact the analysis of electrophysiological phenomena.

[0142] In some embodiments, the PECVD film may be deposited on at least one surface of two or more substrates, thereby providing an interface between said two or more substrates.Electrodes

[0143] The PECVD film may comprise one or more electrodes in the one or more first regions.

[0144] The one or more first regions may comprise two or more electrodes.

[0145] When a PECVD film comprises two or more electrodes in the one or more first regions, the inter-electrode distance may be any suitable inter-electrode distance. For example, the inter-electrode distance may be greater than about 1 pm. For example from about 1 pm to about 50 mm, or from about 1 pm to about 10 mm, or from about 1pm to about 1 mm, or from about 1 pm to about 500 pm, or from about 1 pm to about 300 pm, or from about 300 pm to about 50 mm, or from about 300 pm to about 10 mm, or from about 300 pm to about 1 mm, or from about 300 pm to about 500 pm, or from about 500 pm to about 50 mm, or from about 500 pm to about 10 mm, or from about 500 pm to about 1 mm, or from about 1 mm to about 50 mm, or from about 1 mm to about 10 mm, or from about 10 mm to about 50 mm. The person skilled in the art would appreciate there is no particular upper limit for the inter-electrode distance, and this parameter will depend on the application in question and laser parameters used to fabricate the PECVD film.

[0146] The electrodes may comprise an electrode pad and an electrode track connected to said electrode pad.

[0147] The electrode pad diameter may be any suitable electrode pad diameter. For example, the electrode pad diameter may be greater than about 20 pm. For example from about 20 pm to about 100 mm, or from about 20 pm to about 10 mm, or from about 20 pm to about 1 mm, or from about 20 pm to about 500 pm, or from about 20 pm to about 250 pm, or from about 250 pm to about 100 mm, or from about 250 pm to about 10 mm, or from about 250 pm to about 1 mm, or from about 250 pm to about 500 pm, or from about 500 pm to about 100 mm, or from about 500 pm to about 10 mm, or from about 500 pm to about 1 mm, or from about 1 mm to about 100 mm, or from about 1 mm to about 10 mm, or from about 10 mm to about 100 mm. The person skilled in the art would appreciate there is no particular upper limit for the electrode size, and this parameter will depend on the application in question and laser parameters used to fabricate the PECVD film.

[0148] The one or more electrodes in the one or more first regions may be micro electrodes.

[0149] For micro electrodes, the inter-electrode distance may be any suitable interelectrode distance. For example from about 1 pm to about 500 pm, or from about 10 pm to about 300 pm.

[0150] For micro electrodes, the electrode pad diameter may be greater than about 20 pm. For example from about 20 pm to about 250 pm.

[0151] It will be appreciated by the person skilled in the art that depending on the type, duration, power, trace speed and raster pitch of the laser treatment that the dimensions of the electrodes patterned into the one or more first regions are highly tuneable depending on the required application. The person skilled in the art would be able to take the teachings of the present disclosure and apply them to create a PECVD film with both macroscale and microscale electrode patterning. For example, microscale patterning may be used in applications such as micro electrode arrays, whereas macroscale patterning may be used for coating the surfaces of cell culture wells for cell stimulation and electrical response readout and / or the coating of a range of substrates for sensing applications.PECVD Films containing biomolecules / cells

[0152] The PECVD films as described in the present disclosure may further comprise one or more biomolecules or organic small molecules covalently bonded to or immobilised on one or more of the first and / or second regions of the PECVD film.

[0153] Without wishing to be bound by theory, it is envisaged that radicals within the PECVD film facilitate the covalent bonding or immobilisation of biomolecules or organic small molecules to provide biomolecules or organic small molecules covalently bonded or immobilised on regions of the PECVD film.

[0154] The one or more biomolecules may comprise one or more proteins, polysaccharides, nucleotides, oligonucleotides, antioxidants, growth factors, vitamins and lipids. Other biomolecules are contemplated.

[0155] The PECVD films as described in the present disclosure may further comprise cells, wherein the cells are covalently bonded or immobilised on one or more of the first and / or second regions of the PECVD film.Process for preparing PECVD films or substrates coated with a PECVD film

[0156] The present disclosure further relates to a process for producing a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising:(a) providing a precursor plasma enhanced chemical vapour deposited film, said precursor PECVD film comprising at least carbon and oxygen; and(b) patterning the one or more first regions into the precursor PECVD film by subjecting areas of said precursor PECVD film to a laser beam.

[0157] In a process for producing a PECVD film, the PECVD film may further comprise nitrogen.

[0158] The present disclosure further relates to a process for producing a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising: a) activating one or more surfaces of a substrate by exposing said substrate to a plasma formed in the presence of one or more of helium, neon, argon and xenon; b) depositing a precursor plasma enhanced chemical vapour deposited (PECVD) film on the one or more activated surfaces of the substrate by exposing said activated surface to a plasma formed in the presence of one or more organic gases, and one or more of nitrogen, helium, neon, argon and xenon; and c) patterning one or more first regions into the precursor PECVD film by subjecting areas of said precursor PECVD film to a laser beam.

[0159] The present disclosure further relates to a process for producing a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising: a) activating one or more surfaces of a substrate by exposing said substrate to a plasma formed in the presence of one or more of helium, neon, oxygen, nitrogen, argon and xenon; b) depositing a precursor plasma enhanced chemical vapour deposited (PECVD) film on the one or more activated surfaces of the substrate by exposing saidactivated surface to a plasma formed in the presence of one or more organic gases, and one or more of nitrogen, helium, neon, argon and xenon; and c) patterning one or more first regions into the precursor PECVD film by subjecting areas of said precursor PECVD film to a laser beam.

[0160] In another aspect, the present disclosure provides a process for producing a substrate coated with a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising: a) activating one or more surfaces of a substrate by exposing said substrate to a plasma formed in the presence of one or more of helium, neon, argon and xenon; b) depositing a precursor plasma enhanced chemical vapour deposited (PECVD) film on the one or more activated surfaces of the substrate by exposing said activated surface to a plasma formed in the presence of one or more organic gases, and one or more of nitrogen, helium, neon, argon and xenon; and c) patterning one or more first regions into the precursor PECVD film by subjecting areas of said precursor PECVD film to a laser beam.

[0161] In another aspect, the present disclosure provides a process for producing a substrate coated with a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising: a) activating one or more surfaces of a substrate by exposing said substrate to a plasma formed in the presence of one or more of helium, neon, oxygen, nitrogen, argon and xenon; b) depositing a precursor plasma enhanced chemical vapour deposited (PECVD) film on the one or more activated surfaces of the substrate by exposing said activated surface to a plasma formed in the presence of one or more organic gases, and one or more of nitrogen, helium, neon, argon and xenon; andc) patterning one or more first regions into the precursor PECVD film by subjecting areas of said precursor PECVD film to a laser beam.

[0162] Processes for generating suitable plasmas are described in PCT / AU2022 / 051135 and PCT / AU2023 / 050317, the entirety of which are incorporated herein by reference.

[0163] In some embodiments, the substrate comprises two or more different substrates.

[0164] The total pressure in step a) may be from about 25 mTorr (3.3 Pa) to about 500 mTorr (66.7 Pa).

[0165] The total pressure in step b) may be from about 25 mTorr (3.3 Pa) to about 500 mTorr (66.7 Pa).

[0166] The plasma discharge in step a) may be maintained from about 10 seconds to about 30 minutes, or from about 5 minutes to about 15 minutes.

[0167] The plasma discharge in step b) may be maintained from about 10 seconds to about 30 minutes, or from about 30 seconds to about 25 minutes.

[0168] The plasma in step a) may be formed in the presence of argon.

[0169] The plasma in step b) may be formed in the presence of argon and optionally nitrogen.

[0170] The capacitively coupled radio frequency power used to generate the plasma may have a power of about 5 to about 500 W, or about 5 to 100, 5 to 200, 5 to 300, or 5 to 400 W, e.g., about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450 or 500 W.

[0171] The pulsed bias voltage may have a frequency of about 1 Hz to about 50 kHz, or about 1 Hz to 20 kHz, 1 Hz to 10 kHz, 1 Hz to 5 kHz, 5 Hz to 50 kHz, 10 Hz to 50 kHz, 20 Hz to 50 kHz, 10 Hz to 20 kHz or 20 Hz to 30 kHz.

[0172] The bias voltage may be from about -1000V to about 1000 V, or -500 to 500, -200 to 200, -100 to 100, -50 to 50, -1000 to 0, -500 to 0, -200 to 0, -100 to 0, -50 to 0, 0 to 50, 0 to 100, 0 to 200, 0 to 500 or 0 to 1000V, e.g., about -1000, -900, -800,-700, -600, -500, -400, -300, -200, -100, -50, 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 V. In embodiments, negative bias voltages are preferred, for example, from about -100 V to about -1000 V, or from about -200 V to about -1000 V.

[0173] The pulse duration may be from about 1 to about 150 microseconds, or about 1 to 100, 1 to 50, 1 to 20, 1 to 10, 10 to 150, 20 to 150, 50 to 150, 100 to 150, 10 to 100, 10 to 50 or 50 to 100 microseconds, e.g., about 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 microseconds.

[0174] The ratio between off time and on time of the pulses may be from about 1 (i.e., 1 :1 ) to about 20, or about 10 to 15, 15 to 20 or 13 to 17, e.g., about 10, 13, 15, 17 or 20. In some instances it may be greater than 10, optionally up to 100, or up to 90, 80, 70, 60, 50, 40 or 30.

[0175] The flow rates of gases into the plasma chamber may be varied to achieve a desired pressure and residence time within the chamber. Flow rates are dependent on the scale of operation and the size of the plasma chamber.

[0176] The deposition time may be from less than 1 minute to about 30 minutes, or from 1 to 20, 1 to 10, 1 to 50, 5 to 30, 10 to 30, 20 to 30, 5 to 20, 5 to 10 or 10 to 20 minutes, e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 minutes or may be longer than 30 minutes.

[0177] It will be appreciated that any of the flow rate, pressure and power may be varied according the specifically desired properties of the film to be produced. Thus, any of the numerical values or ranges exemplified herein for each of the pressure and power may be used together, in any combination. For example, in one embodiment of the deposition step, a pressure of about 20 Pa and a power of about 50 W to about 100 W may be used. All other possible combinations are envisaged herein.Organic gases

[0178] In forming the plasma, the gas comprises at least one gas which is organic, i.e., contains carbon and is not carbon dioxide. It may comprise a carbon-carbon double bond and / or a carbon-carbon triple bond. It may be an alkene or an alkyne. It may be a mixture of such gases. The organic gas may be polymerisable under the conditions of the process. The gas may comprise more than one organic gas. In the present context,a gas is taken to be a substance which is in the gaseous state at the temperature and pressure prevailing at the time in the plasma chamber.

[0179] In forming the plasma in step b), the one or more organic gases may comprise one or more of hydrocarbon and substituted hydrocarbon.

[0180] The one or more organic gases may comprise one or more linear or branched alkane or cycloalkane, linear or branched alkene or cycloalkene, and linear or branched alkyne.

[0181] The one or more organic gases may comprise acetylene or substituted acetylene.

[0182] The substituted hydrocarbon may comprise one or more of hydroxyl substituted hydrocarbon, amino substituted hydrocarbon, and hydrocarbons substituted with sulphur-containing groups.

[0183] The volume ratio of the at least one organic gas fed to the plasma chamber to the sum of one or more of nitrogen, helium, neon, argon and xenon is about 1 to 20 to about 1 to 2, or from about 1 to 15 to about 1 to 5.

[0184] In embodiments, the volume ratio of the at least one organic gas fed to the plasma chamber to the sum of argon and / or nitrogen is about 1 to 20 to about 1 to 2, or from about 1 to 15 to about 1 to 5.Laser patterning

[0185] Subjecting regions of the precursor PECVD film to laser treatment leads to an increase in carbon and oxygen content in these regions, and a decrease in nitrogen content (if present), when compared to untreated regions of the PECVD film. Without being bound by theory, the increase in carbonisation of a region of the PECVD film that has been subjected to laser treatment may result from chemical restructuring of the PECVD film at the treated region. The restructured carbon atoms rebond to produce a more ordered graphite structure, which leads to improved electrical conductivity.

[0186] In the process for preparing the PECVD film of the present disclosure, any suitable laser may be used for laser patterning. The type of laser used may in part be selected based on the substrate selected for deposition of the PECVD film, or thethickness of the PECVD film to be deposited on the substrate. For example, in the process for preparing the PECVD film of the present disclosure, the laser may be a He laser, a CO2 laser, a Nd:YVO4 laser, a titaniurmsapphire laser, or a ytterbium laser. Other suitable laser types known in the art are contemplated.

[0187] The wavelength of the laser light may be from about 200 nm to about 1500 nm, or from about 300 nm to about 1100 nm, or from about 450 nm to about 600 nm.

[0188] The laser may be pulsed from about 100 fs to about 1 ns, or from about 200 fs to about 50 ps, or from about 150 fs to about 15 ps.

[0189] Laser interactions are largely dependent on the pulse length of the laser. Longer pulse length (>15 ps) interactions are highly thermal, with single photon absorption limiting interactions of the laser to materials with high absorbance in the specific wavelength used. As the treatment process of these longer pulse length lasers is thermal, they can invoke graphitisation at high rates with fast processing times. This process allows efficient fabrication of large area electrode surfaces. Such films may be used in biosensing applications, where the electrode features are typically in the mm resolution scales and do not require concurrent optical observation.

[0190] Ultrafast lasers (<15ps) are no longer governed by single photon absorption. Multiphoton absorption can occur, allowing photo-ionisation, i.e. non-thermal restructuring of the film. This decreases the thermal damage present and allows better patterning resolution on the surface - delivering micro-scale resolution. Multi-photon absorption also allows thin, transparent films to be targeted, allowing the development of nanoscale thickness transparent electrodes. Hence these treatments are better suited for microscale applications, such as micro electrode arrays used in electrophysiological studies, with the added benefit of optically observing the cells throughout the electrical stimulation and readout processes.

[0191] The laser may operate at a frequency of about 1 kHz to about 5 MHz, or from about 200 kHz to about 2 MHz, or from about 200 kHz to about 1 MHz.

[0192] The laser may have a scan speed from about 5 mm / s to about 250 mm / s, or from about 5 mm / s to 200 mm / s, or from about 5 mm / s to 150 mm / s, or from about 5 mm / s to 100 mm / s, or from about 5 mm / s to 50 mm / s.

[0193] The present disclosure also provides a PECVD film as described by any one of the herein disclosed embodiments may be produced by a process according to any one of the presently disclosed processes.

[0194] The present disclosure also provides a substrate coated with a PECVD film according to any one of the herein disclosed embodiments.Method of attaching biomolecules and / or cells to the substrate

[0195] The present disclosure provides a method of covalently bonding or immobilising small organic molecules or biomolecules to a PECVD film, said film comprising at least carbon and oxygen, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said method comprising:(a) depositing small organic molecules or biomolecules on the PECVD film and(b) covalently attaching or immobilising one or more of the small organic molecules or biomolecules to one or more of the first and second regions of the PECVD film.

[0196] In embodiments of the method of covalently bonding or immobilising small organic molecules or biomolecules to a PECVD film, the film further comprises nitrogen.

[0197] In another aspect, the present disclosure provides a method of covalently bonding or immobilising cells to a PECVD film, said film comprising at least carbon and oxygen, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said method comprising:(a) depositing cells on the PECVD film; and(b) covalently attaching or immobilising the cells to one or more of the first and second regions of the PECVD film.

[0198] In embodiments of the method of covalently bonding or immobilising cells to a PECVD film, the film further comprises nitrogen.

[0199] The present disclosure also provides a method of covalently bonding or immobilising cells to a PECVD film comprising: a) depositing cells in a cell culture medium on a PECVD film according to any one of the herein disclosed embodiments; b) covalently attaching biomolecules from the cell culture medium to one or more of the first and / or second regions of the PECVD film; and c) attaching the cells to the covalently attached biomolecules.

[0200] The present disclosure provides a PECVD film according to any one of the herein disclosed embodiments for use in a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule and / or cell.

[0201] The present disclosure provides a substrate coated with a PECVD film according to any one of the herein disclosed embodiments for use in a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule, and / or cell.

[0202] The present disclosure provides a substrate coated with a PECVD film according to any one of the herein disclosed embodiments for use in a device for the simultaneous electrical and optical analysis of small organic molecules, biomolecules, cells and / or populations of cells including organoids and explants.

[0203] The present disclosure provides the use of a PECVD film according to any one of the herein disclosed embodiments in the manufacture of a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule and / or cell.

[0204] The present disclosure provides the use of a substrate coated with a PECVD film according to any one of the herein disclosed embodiments in the manufacture of a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule, and / or cell.

[0205] The present disclosure provides the use of a substrate coated with a PECVD film according to any one of the herein disclosed embodiments in the manufacture of adevice for the simultaneous electrical and optical analysis of small organic molecules, biomolecules, cells and / or populations of cells including organoids and explants.

[0206] The device may be a sensing device.

[0207] The present disclosure provides a method of analysing small organic molecules, biomolecules or cells comprising: providing a PECVD film according to any one of the herein disclosed embodiments, said PECVD film comprising small organic molecules, biomolecules, and / or cells covalently bonded or immobilised thereto; and one or more of optically imaging the small organic molecules, biomolecules, and / or cells; electrically stimulating the biomolecules, and / or cells; recording an electrical output from the small organic molecules, biomolecules, and / or cells.

[0208] The person skilled in the art would be able to determine suitable electrical analyses that could be conducted using a PECVD film of the present disclosure. For example, electrical conductivity, electrical resistivity, intra and extracellular signal recording and analysis, stimulation, impedance spectroscopy, and cyclic voltammetry for biosensing applications. Other suitable electrical analyses are contemplated.

[0209] The person skilled in the art would be able to determine suitable optical analyses that could be conducted using a PECVD film of the present disclosure. For example, atomic force microscopy (AFM), fluorescence, colorimetry, chemiluminescence and surface-enhanced Raman spectroscopy (SERS). Other suitable optical analyses are contemplated.

[0210] The present disclosure provides a method of analysing small organic molecules, biomolecules or cells comprising: providing a substrate coated with a PECVD film according to any one of the herein disclosed embodiments, said PECVD film comprising small organic molecules, biomolecules and / or cells covalently bonded or immobilised thereto; and one or more of optically imaging the small organic molecules, biomolecules, and / or cells;electrically stimulating the small organic molecules, biomolecules, and / or cells; recording an electrical output from the small organic molecules, biomolecules, and / or cells.

[0211] The present disclosure provides a method of analysing small organic molecules, biomolecules, cells and / or populations of cells including organoids and explants, comprising: providing a substrate coated with a PECVD film according to any one of the herein disclosed embodiments, said PECVD film comprising small organic molecules, biomolecules and / or cells covalently bonded or immobilised thereto; and one or more of optically imaging the small organic molecules, biomolecules, and / or cells; electrically stimulating the small organic molecules, biomolecules, and / or cells; recording an electrical output from the small organic molecules, biomolecules, and / or cells.

[0212] In some embodiments, according to any one of the herein disclosed aspects, the cell or cells may form part of a cell population. For example, the cell population may include organoids and / or explants. Other cell populations are contemplated.

[0213] In another aspect, the present disclosure may provide a microelectrode array (MEA) comprising a PECVD film according to any one of the herein disclosed embodiments.

[0214] In another aspect, the present disclosure provides a microelectrode array (MEA) comprising a substrate coated with a PECVD film, said film comprising at least carbon and oxygen; the film further comprises one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions; the one or more first regions comprising one or more electrodes or micro electrodes;wherein the substrate comprises one or more substrates, said one or more substrates comprising one or more of metal, glass, polymer, quartz, chalcogenides, metal oxides, silicon or diamond.Examples

[0215] Optically transparent and electrically conducting films can be created on a substrate using plasma-enhanced chemical vapour deposition (PECVD) followed by a laser treatment. The film formed in a PECVD process with energetic ion bombardment has an amorphous structure with embedded radicals containing unpaired electrons in an environment that helps stabilise the unpaired electrons. The embedded radicals are chemically reactive and enable reagent-free covalent attachment of organic small molecules or biomolecules on contact. A subsequent laser treatment restructures the amorphous carbon by partial removal of hydrogen, oxygen, or optionally nitrogen, atoms leading to a more electrically conductive structure, increasing the electrical conductivity of the PECVD film without eliminating the radical content and reagent-free covalent binding capability.Example 1 - MethodsSubstrate preparation

[0216] The PECVD films described in the present disclosure can be created on a wide variety of substrates (glass, silicon wafer, metal sheet) chosen according to the desired applications.

[0217] Substrates were prepared for coating by a PECVD film with the following washing protocol: 3x Ethanol (80%) washes, 1x MilliQ wash with 5 minutes of sonication. All characterisation was performed on standard borosilicate glass microscope slides (Livingstone Pathology Grade).Plasma Film Deposition

[0218] The PECVD process was performed in a purpose-built chamber. Vacuum (base pressure of 5 x10-5Torr) was supplied by a dual pump system (Edwards Backing Pump and Next400 Turbo-Molecular Pump) and monitored within the chamber by a PFEIFFER PKR251 pressure gauge. The plasma was created using a capacitively coupled radio frequency generator (Eni OEM-6). Prior to film deposition the substratesurface was cleaned and activated using Argon plasma, generated with a RF power (13.56 MHz Eni Power System OEM-6 RF Generator) of 75 W for 10 minutes at 75 mTorr. Voltage pulses of 20 ps duration at -500 V at a frequency of 3000 Hz are applied to the sample holder for the surface activation, via a RUP3 pulse generator (GBS Elektronik GmbH, Dresden, Germany).

[0219] Once surface cleaning and activation was complete, the chamber was evacuated back to base pressure for deposition. Gaseous precursors of argon, acetylene and optionally nitrogen were introduced to the chamber in various ratios (as desired). The chamber pressure was kept at 110 mTorr while deposition occurred by adjusting the opening of a connection valve between the pumps and the plasma chamber. The mass flow and pressure of input gas precursors were monitored using Flowvision software coupled to Allicat Scientific mass flow controllers. Capacitively coupled RF power of 50 W was used to create the plasma in the chamber. High voltage pulses of -500 V with a frequency of 3000 Hz and pulse width of 20 ps were applied to the substrate using a RUP3 pulser via pulse generator (DATAPULSE 1 , 100A Pulse Generator, Division of Systron-Donner Corporation) to accelerate ions to the substrate surface. Deposition was then performed for 20, 15, 10, 5, 2, and 1 minute intervals to produce PECVD films with average thicknesses of 216.17, 97.67, 74.46, 38.18, 15.72 and 11 .70 nm respectively.Laser Patterning of Electrodes

[0220] Ablation thresholds of the PECVD films were determined using a 532nm (green), 10 ps pulsed laser (COHERENT Super Rapid-He) with Gaussian beam profile operating at a frequency of 200 kHz. Single shots were fired onto the surface in 3 x 10 dimensional arrays allowing observation of the ablated crater diameter via optical microscopy (OLYMPUS). Diameters were analysed using Python software with Hough Circles package. The ablation threshold provides an upper limit to the range of fluences suitable for film restructuring using laser processing. To determine this value, single pulse shots of varied fluence were delivered to the surface, and the relationship between crater diameter and the pulse energy is compared according to the equation:where R describes the diameter of ablated craters, t O is the laser spot radius, F is the fluence of laser treatment (j / cm2) and Energy is the energy of the laser treatment (J). Here the subscript “th” describes the value of the parameter at ablation threshold. By considering the extrapolation of the trend to the point at which crater diameter is zero, the threshold fluence can then be determined (Fth), often described as the ablation threshold.

[0221] Laser treatment on plasma deposited films was conducted using a 532 nm (green), 10 ps pulsed laser (COHERENT Super Rapid-He) with Gaussian beam profile operating at a frequency of 1 MHz. The laser line overlap was 90%, achieved by setting scan speed to 50 mm / s with raster pattern devised to have a 2 pm spacing, equidistant contour hatch pattern. Laser fluence values between the limits of 0-0.5 J / cm2were used in an attempt to promote chemical restructuring and enhance the electrical conductivity of the amorphous carbon films.

[0222] Laser patterning was achieved using any suitable AutoCAD pattern as required by the application. Hatch spacing was required to be 2 pm with minimum feature size of 16 pm width in accordance with the laser spot size. Here, MEAs mimicking the layout of commercially available products were designed to illustrate a commercially applicable patterning process. Laser treated PECVD films were analysed using Optical microscopy (OLYMPUS) to observe the patterning achieved by the method.Surface / Elemental Characterisation

[0223] Elemental compositions of laser treated films were recorded using a K- Alpha+TM X-ray photoelectron spectrometer (Thermo-Fisher Scientific, UK). A survey scan was performed as well as higher resolution scans of the C1 s, N1 s, 01 s and Si2p peaks allowing quantification and deconvolution of various bonding states of these elements. All spectra measured were analysed using Avantage Software (ThermoFisher Scientific), with the carbon peak deconvolution assuming C-C, C-O, C=O and O- C=O peaks are separated by 1 .5 eV and each peak has identical full width half maximum and ratio of Lorentzian / Gaussian line shape. Atomic % compositions quoted represent the atomic % composition assuming all non-hydrogen elements form 100% of the atomic composition.

[0224] Raman spectra were obtained using a Renshaw inVia Raman Microscope with a 532 nm laser. Spectra were obtained with 5 seconds exposure of 1% laser power for 15 accumulations. Low laser power was used to ensure film damage was minimised, as the measurement wavelength was identical to that used for film restructuring aimed at creating the conducting tracks.

[0225] Scanning Electron Microscopy was performed with a Phenom XL G2 Desktop SEM at 5kV operating in Back Scattered Electron (BSE) imaging mode.

[0226] Atomic Force Microscopy was performed using a Bruker Innova. Scans of areas 5 pm x 5 pm were conducted for parameter combinations of 2 pm 50 mm / s (B), 2 pm 5 mm / s (C) and 5 pm 5 mm / s (D), as well as a pristine film surface (A).Electrical Characterisation

[0227] DC resistance measurements were performed using a digital multi meter (Keithley DMM6500) attached to a microprobe station (Micromanipulator Co. Inc, USA 820944). Measurement probes (Au coated Tungsten 15 pm tip width) were placed 2.5 mm apart upon the surface at each laser treatment location and the conductivity value calculated from the measured resistance, accounting for the film thickness, measurement probe diameter and the probe spacing.Free Radical Density Measurement

[0228] Radicals present within the PECVD films before and after the laser treatment were analysed using electron paramagnetic resonance (SpinScanX, Adani, Belarus). Films were prepared on quartz slides (40x50x0.9 mm) and completely laser treated at fluences between 0.038-0.055 J / cm2a minimum of one week after deposition.Measurements were conducted at room temperature with a microwave frequency of 9.40 MHz and a central magnetic field of 336 mT with a sweep width of 30 mT and a microwave power of 0.946 mW. Measured signal peaks were integrated using Python MatPlotLib and Numpy software to determine radical content in the laser treated films.Protein Attachment

[0229] Fluorescently labelled BSA (excitation wavelength 555nm) was attached upon samples with pristine PECVD films and arrays of parameter scans investigating optimal laser trace speed and hatch spacing combinations. Each sample was left with 150 uL of0.1 mg / ml BSA for 24 hours to allow the protein to attach to the surface. To demonstrate the covalent protein binding ability of the surface after laser treatment, samples were compared before and after SDS washing (1 hr at room temperature) and the quantity of fluorescence analysed (OLYPMUS).Fibroblast Culture and Imaging

[0230] Fibroblasts (3T3-L1 ) between passage 15-20 were sub-cultured upon the plasma film electrode surface, plated at - 4 x 104cells / cm2. The fibroblasts were allowed to attach for 2 hours before the sample was completely immersed in media (whole media DMEM 4.5 g / L glucose + 10% foetal bovine serum). After overnight incubation at 37 °C, 5% CO2 the media was removed and samples rinsed once with PBS before cells were fixed for 10 minutes in a 10% neutral buffered formalin. After being rinsed 2x with PBS the fibroblasts were stained with nucBlue™ (Thermofisher) and ActinRed™ (Thermofisher) and mounted on a microscope with coverslip. Microscopic images were taken on a Nikon Ti-E widefield fluorescent microscope using a 10x objective and DS-Qi2 monochrome camera.Example 2 - Changes of chemical composition of the PECVD film as a function of laser treatment fluence

[0231] Laser treatment was performed in ambient atmosphere for Example 2. Without being bound by theory, the inventors believe that if the treatment was performed in vacuum, or other inert atmosphere, oxidation during this process would be minimised.

[0232] Films were observed to have a large reduction in nitrogen content (-50%) when present, before reaching a conductive state as analysed by X-ray Photo-electron Spectroscopy (Figure 1). There was also an increase in oxygen percentage, attributed to oxidising of the surface in the atmospheric laser treatment conditions. These results are aligned with that observed in laser induced graphitization (LIG) processes, where oxygenation of the surface is present in atmospheric treatment conditions.

[0233] Reduction of the nitrogen content occurs following the breaking of lower energy single bonds within the surface structure. The freed atoms form volatile species such as N2 which leave the film, allowing the remaining carbon atoms to rebond and produce a more ordered graphite structure. As conductivity increased, nitrogen proportions decreased around 30%, indicating removal of this element from the film and in turnrestructuring. Oxygen content slightly increased due to restructuring processes providing an opportunity for oxidation. Carbon proportions increased due to removal of nitrogen and oxygen from the film via formation of volatile species which escape the sample structure and diffuse into surrounding atmosphere. Carbon then decreased with thinning of the film and removal due to excess sublimation in overexposed regions. The increase in the silicon signal occurred with thinning of the film following removal of the constituents, restructuring and densification.Example 3 - Changes of carbonized structure after laser treatment (Raman spectroscopy)

[0234] The structure of the film in terms of sp2and sp3bond arrangement was illustrated by Raman spectroscopy and the data shown in Figure 2. The Raman measurement was performed with low laser power due to the overlap of the 532 nm laser used for spectroscopic analysis with the identical wavelength used in the laser induced graphitisation process of the PECVD film. The deposited carbon-based films comprise variously arranged sp2 and sp3 carbon bond formations.

[0235] Peaks at 1350 nm’1and 1580 nm’1are the D and G peaks attributed to the type of carbon bonding. Increasing D / G ratios implies structural re-organisation forming more ordered sp2crystalline graphite-like regions within the film (Figure 3). The peak arising at 1100nm’1was attributed to silicon, and the growth of this peak with ongoing laser treatment suggests the thinning of the treated films, i.e. compression / densification or ablation of the carbon structure, bringing the glass substrate into the depth range of analysis.Example 4 - Optical Characterisation

[0236] Absorption of common microscopy excitation wavelengths by the laser patterned PECVD films, as can be seen in Figure 4, remains low when compared to the standard microscope slide, allowing -90% of all incident light through the conductive track when directly compared. A peak in absorbance can be seen about the regions of treatment conditions that give higher conductivity. This was a result of the higher proportions of graphitised structure within the film, creating a more reflective surface.

[0237] As shown in Figure 4a, absorption increased (transparency decreased) at wavelengths lower than 450nm as conductivity is enhanced, attributed to the reflectivityincrease with the graphitic property enhancement. Once above peak conductivity, absorption decreased - likely due to film thinning. All treated films had high levels of transparency -90% (low absorption) about the wavelengths >500nm.

[0238] As shown in Figure 4b, as enhanced conductivity peaked, the absorption of the fabricated electrode also peaked, following enhancement of graphite like structure with higher reflectivity. Following the optimal fluence, absorption decayed due to film thinning. All conditions are <0.2 Absorption, that is >80% transmission for all common excitation wavelengths used in optical imaging analysis.Example 5 - Radical Measurement

[0239] Radical concentration within the laser treated samples was found to increase after laser annealing, shown by the high intensity peak in the EPR spectra (Figure 5). Higher peak integrals suggest large absorption due to free radicals within the film and hence the biomolecular binding capability of the treated electrode sites is maintained, if not increased relative to the pristine films.Example 6 - Characterisation of Treatment Regimes

[0240] Single shot ablation thresholds for the plasma activated films were determined as 0.18 J / cm2to 0.4 J / cm2, depending on film thickness (controlled via deposition time). Ablation threshold studies are shown in Figure 6 and Figure 7 as a function of film thickness, with similar trends found for conductivity enhancement with thickness variation due to the linear shift downwards in required fluence following consecutive pulse incubation effects.

[0241] Figure 7 shows that the multi-shot ablation fluence of N=992 shots demonstrated the conditions used within treatment overlap. The fitted Fth (N=Treatment) showed the ultimate upper limit under such treatment conditions for each film thickness, with minimum film thicknesses allowing conductive enhancement to be ~70nm as observed with the plotted Fcond. The multi-pulse incubation threshold illustrated the dominating incubational effects within thin films when compared to incubation throughout thick film treatment, providing evidence for substrate contribution to the films ablation threshold once the film attenuates too little laser energy at lower thickness values.Example 7 - Electrical Characterisation

[0242] DC conductivity values within the ranges of 0 to 12 S / cm were established for incident laser fluences of 0.036 to 0.056 on a film thickness of ~200 nm. Assuming incubational effects within the film are similar for both ablation and structural reformation, the relationship between the laser fluence (Fcond) required for conductive enhancement and the film thickness can be described by the power law:Fcond= 0-736 x Thickness-'24— 0.156

[0243] Films can be conductively enhanced knowing the average thickness used in the application dependent film properties (Figure 8). Conductivity occurs once oxygen and optionally nitrogen from within the film are sufficiently removed, increasing with continued treatment with the removal inferred from Raman and XPS analysis. Conductivity then drops once the films become significantly thinner, as shown by increased substrate silicon signal in both Raman and XPS analysis. This was also the case for overall scan fluence, governed by the pulse and line overlap density.Example 8 - Laser Patterned Electrode Fabrication

[0244] Micro electrode arrays were fabricated as shown in Figure 9 (A and B). These demonstrate the patterning ability of the technology with no restructuring of the insulating surface and no cracking or delamination of the PECVD film. Here the electrode pad diameter was 50 pm and the inter-electrode distance (IED) was 200 pm.Example 9 - Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) Analysis of Electrode Sites

[0245] SEM analysis demonstrated the overall surface uniformity in terms of conductivity and topology. There were two main trends arising from the analysis of the Fscanstudy, which allowed preferred parameters for pattern treatment to be determined according to the required application.

[0246] At lower trace speeds (5-10 mm / s) induced graphite has a roughened structure, implying a destructive and fast evaporation of N and O from the surface, occurring after some threshold is reached, as reported in the literature (M. Abdulhafez et al., ACS Applied Nano Materials, vol.4, no.3, pp. 2973-2986 (2021 )). The 25 mm / s seriesappears to be on the cusp of this region, with graphitic clusters or surface contaminants often providing the apparent seeding conditions necessary to prompt graphite formation as reported in the literature (B. Kulyk et al., Advanced Materials Technologies, vol. 7, no. 7, (2022)).

[0247] At trace speeds of 50-100 mm / s there was no evidence of destructive evaporation or cracking, and the laser irradiated pattern was visibly smooth with no identifiable surface features.

[0248] Analysis of the film of various scan fluence conditions is shown in Figure 10. This highlights that there was a change in surface structure and roughness following laser treatment. Roughness provides enhanced surface properties for various biological applications.

[0249] This analysis demonstrates the ability to tune surface topology of PECVD films using the laser, allowing tailoring of the surface roughness specific to cellular study requirements while maintaining uniform graphitisation and covalent binding of the electrode region.Example 10 - Covalent Attachment of Protein Demonstration

[0250] Fluorescent imaging of surface adsorbed BSA prior and post detergent (SDS) washing shows the maintaining of covalent binding capabilities in laser treated PECVD films (Figure 11). A suitable monolayer of the BSA protein, representative of cell signalling proteins that are needed for optimising cell culture, can be observed after the SDS wash. The parameter sweep of laser trace speed and hatching separation resulted in visible delamination of the film when heating becomes non-uniform, and hence appropriate parameters for hatching spacing and trace speed were found to be within the 25 and 50 mm / s categories with hatch spacing between 2 and 10 pm for a complete pattern coverage.

[0251] Here the protein binding can be further observed to vary largely on films that are undergoing different regimes of laser interaction. As varied laser parameters result in conditioning, roughening or deformation of the film, protein can vary in attachment, with increased surface area leading to higher proportions of binding sites. Considering the topography demonstrated with SEM analysis, BSA appears to become well absorbed to areas with surface roughness, demonstrating this as an avenue for further biomoleculeattachment on the already covalently binding PECVD electrodes. Surfaces produced uniform fluorescence when the laser treatment created a more uniform film graphitisation.

[0252] The fluorescence intensity of the surfaces (Figure 12a) and their variance (Figure 12b) demonstrate the variation of protein binding across an electrode patterned into a PECVD film when compared to a pristine PECVD film. Increased surface area due to roughening or cracking in the film will trap protein or provide increased binding area on the surface and cause higher fluorescence than the uniformly bound conditions. It is hence important to observe the spread of fluorescence data at each condition (indicated by the associated error bars) to optimise uniform protein binding upon an electrode region in the PECVD film. Horizontal dotted lines indicate reference sample signal intensity, with all treatment conditions falling well above the glass reference sample, indicating that covalent attachment was present.

[0253] Figure 12b shows that variation of parameters such as raster pitch and trace speed can be altered and still provide conditions for increased protein binding when compared to a pristine PECVD film.Example 11 - Imaging of Fibroblasts on Electrodes

[0254] Fibroblast culture upon a sample micro-electrode array pattern is shown in Figure 13 and illustrates the use of the film as a culture platform. Here there were no visible electrode tracks in the fluorescent cell images, either in the range 540 / 565 nm of a ActinRed™ stain (Figure 13a) or 360 / 460 nm of a nucBlue™ stain (Figure 13b), demonstrating the successful application of plasma deposited laser annealed electrodes as micro electrode array tracks. The image demonstrates the ability to visualise the fibroblasts upon the surface of the electrodes with little interference from the underlying electrodes.

[0255] The observation of all cell components regardless of placement upon the electrodes (shown in Figure 13c and Figure 13d) demonstrated the transparency under typical imaging environments, as all cell components were able to be seen upon the electrodes with no apparent attenuation of the fluorescent signal.Example 12 - formation of PECVD films on different substrates

[0256] To diversify applications, alternative substrates were investigated to explore how various underlying substrates affect the responses of deposited PECVD films to laser treatment by analysing their ablation thresholds under picosecond laser irradiation (Figure 14). The analysis revealed that the film's ablation response depends on the substrate's heat capacity, allowing adjustment of the laser parameters to optimise laser induced conductivity according to the substrate material.

[0257] This means the laser settings can be fine-tuned to create transparent conducting electrode tracks in the PECVD film irrespective of the underlying material. This approach is valuable for coating commercially available microelectrode platforms and for fabricating standalone in vitro systems that interface effectively with external measurement devices.

[0258] Data recorded included the associated damage thresholds of PECVD films on different materials showing the impact of substrate properties on the laser interaction. Raman Spectroscopy of the PECVD film on each material showed changes in graphite like bonding within the film (Figure 15). These results facilitate use of the technique on a range of underlying substrate materials, with adjustments to treatment protocols informed by the material properties.Example 13 - Multi-material substrate PECVD coatings

[0259] The ability to create laser induced transparent conductive regions in PECVD films deposited on a wide range of substrate materials enables more complex device architectures to be utilised. This includes creating transparent electrode tracks between multiple substrate materials, allowing robust interfacing to external measurement devices. This allows for laser modified PECVD films that can be easily interfaced with other instruments and implemented in a wider range of applications.

[0260] Figure 16 shows an exemplified embodiment of a multi-material substrate PECVD laser induced transparent conductive (LITC). The right hand diagram of Figure 16 shows a close up of the PECVD LITC interface between an underlying glass and gold substrate. Regions on the underlying glass (5) and gold (3) substrates have been treated to create LITC regions on each substrate material (1 & 4). A pristine PECVD film (2) show the thin film on the different underlying substrates.

[0261] Coating of different underlying substrate regions allowed a completely functionalised surface to be produced, that holds all properties of a single substrate device as previous described. The formation of conductive regions spanning between different substrate regions allowed electrodes to preserve properties of desired substrate function, for example transparency while on glass, and conduction to the underlying substrate when on a conducting substrate material such as gold.

[0262] Interfacing with conductive materials allowed electrical connection to external devices for measurement, stimulation and analysis. The coating could also be applied to commercial CMOS devices allowing functionalisation of the silicon regions and gold microelectrodes while facilitating conductivity at the cell-electrode interface.Example 14 - Comparison of electrochemical data between PECVD films and ITO films

[0263] Figure 17 shows a comparison between PECVD films of the present disclosure and typical ITO films. The electrochemical data and characterisation strongly support the electrodes capability for use in wet environments to provide an electrochemical readout.

[0264] PECVD films and ITO films were both tested for stem cell stimulation. The comparison of the PECVD films to the ITO films suggested good agreement of signal transfer and response when the transparent organic electrode materials are compared to an ITO alternative (Figure 18). This supports the use of the fabrication method to create transparent organic electrode alternatives for cell stimulation purposes.

Claims

CLAIMS1 . A plasma enhanced chemical vapour deposited (PECVD) film, said film comprising at least carbon and oxygen; wherein said film comprises one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions.

2. The PECVD film according to claim 1 , further comprising nitrogen.

3. The PECVD film according to claim 2, wherein the one or more first regions have a nitrogen content that is at least 10% lower than a nitrogen content in the one or more second regions.

4. The PECVD film according to any one of claims 1 to 3, wherein the one or more first regions have an oxygen content that is at least 10% higher than an oxygen content in the one or more second regions.

5. The PECVD film according to any one of claims 2 to 4, wherein the nitrogemcarbon elemental ratio in the PECVD film is from about 0.01 :1 to about 1 :1 , or from about 0.1 :1 to about 2:3.

6. The PECVD film according to any one of claims 1 to 5, wherein the one or more first regions have equivalent or higher radical concentration relative to the one or more second regions, or the one or more first regions have equivalent or lower radical concentration relative to the one or more second regions.

7. The PECVD film according to any one of claims 1 to 6, wherein the percentage elemental carbon as measured by XPS in the PECVD film is from about 50% to about 90% by atomic % composition.

8. The PECVD film according to any one of claims 1 to 7, wherein the percentage elemental nitrogen as measured by XPS in the PECVD film is from about 0% to about 50% by atomic % composition.

9. The PECVD film according to any one of claims 1 to 8, wherein the percentage elemental oxygen as measured by XPS in the PECVD film is from about 5% to about 40% by atomic % composition.

10. The PECVD film according to any one of claims 1 to 9, wherein the one or more first regions have an optical transmittance of greater than about 50%, or greater than about 70%, or greater than about 80%.11 . The PECVD film according to any one of claims 1 to 10, wherein the PECVD film thickness is from about 10 to about 1000 nm.

12. The PECVD film according to any one of claims 1 to 11 , wherein the one or more first regions comprise one or more electrodes.

13. The PECVD film according to claim 12, wherein the one or more first regions comprise two or more electrodes, said electrodes having an inter-electrode distance greater than about 1 pm, or from about 1 pm to about 50 mm.

14. The PECVD film according to claim 12 or claim 13, wherein the electrodes comprise an electrode pad and an electrode track connected to said electrode pad.

15. The PECVD film according to claim 14, wherein the electrode pad diameter is greater than about 20 pm, or from about 20 pm to about 100 mm.

16. The PECVD film according to any one of claims 1 to 15, further comprising one or more biomolecules or organic small molecules covalently bonded to or immobilised on one or more of the first and / or second regions of the PECVD film.

17. The PECVD film according to claim 16, wherein the one or more biomolecules comprise one or more organic small molecules, proteins, polysaccharides, nucleotides, oligonucleotides, antioxidants, growth factors, vitamins and lipids.

18. The PECVD film according to any one of claims 1 to 17, further comprising cells, wherein the cells are covalently bonded to or immobilised on one or more of the first and / or second regions of the PECVD film.

19. A substrate coated with a PECVD film according to any one of claims 1 to 18.

20. The substrate according to claim 19, wherein the substrate comprises two or more different substrates.21 . The substrate according to claim 19 or claim 20, wherein the substrate comprises one or more of metal, glass, polymer, quartz, chalcogenides, metal oxide, silicon or diamond.

22. A process for producing a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising: a) providing a precursor plasma enhanced chemical vapour deposited film, said precursor PECVD film comprising at least carbon and oxygen; and b) patterning the one or more first regions into the precursor PECVD film by subjecting areas of said PECVD film to a laser beam.

23. The process according to claim 22, wherein the precursor PECVD film further comprises nitrogen.

24. A process for producing a PECVD film, or a substrate coated with a PECVD film, said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said process comprising: a) activating one or more surfaces of a substrate by exposing said substrate to a plasma formed in the presence of one or more of helium, neon, argon and xenon; b) depositing a precursor plasma enhanced chemical vapour deposited (PECVD) film on the one or more activated surfaces of the substrate by exposing said activated surface to a plasma formed in the presence of one or more organic gases, and one or more of nitrogen, helium, neon, oxygen, nitrogen, argon and xenon; and c) patterning one or more first regions into the precursor PECVD film by subjecting areas of said PECVD film to a laser beam.

25. The process according to any one of claims 22 to 24, wherein the substrate comprises two or more different substrates.

26. The process according to any one of claims 22 to 25, wherein the laser is a He laser, a CO2 laser, a Nd:YVO4 laser, a titaniurmsapphire laser, or a ytterbium laser.

27. The process according to any one of claims 22 to 26, wherein a wavelength of the laser light is from about 200 nm to about 1500 nm, or from about 300 nm to about 1100 nm, or from about 450 nm to about 600 nm.

28. The process according to any one of claims 22 to 27, wherein the laser is pulsed from about 100 fs to about 1 ns, or from about 200 fs to about 50 ps, or from about 150 fs to about 15 ps.

29. The process according to any one of claims 22 to 28, wherein the laser operates at a frequency of about 1 kHz to about 5 MHz, or from about 200 kHz to about 2 MHz, or from about 200 kHz to about 1 MHz.

30. A PECVD film according to any one of claims 1 to 18, or a substrate coated with a PECVD film according to any one of claims 19 to 21 , produced by the process according to any one of claims 22 to 29.

31. A method of covalently bonding or immobilising small organic molecules or biomolecules to a PECVD film, said film comprising at least carbon, oxygen, and optionally nitrogen said film comprising one or more first regions and one or more second regions, said one or more first regions being more electrically conductive than said one or more second regions, said method comprising:(a) depositing small organic molecules or biomolecules on the PECVD film according to any one of claims 1 to 18; and(b) covalently attaching or immobilising one or more of the small organic molecules or biomolecules to one or more of the first and second regions of the PECVD film.

32. A method of covalently bonding or immobilising cells and / or populations of cells comprising:(a) depositing cells in a cell culture medium on a PECVD film according to any one of claims 1 to 18;(b) covalently attaching biomolecules from the cell culture medium to one or more of the first and second regions of the PECVD film; and(c) attaching the cells to the covalently attached biomolecules.

33. A PECVD film according to any one of claims 1 to 18, or a substrate coated with a PECVD film according to any one of claims 19 to 21 , for use in a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule, and / or cell.

34. The use of a PECVD film according to any one of claims 1 to 18, or a substrate coated with a PECVD film according to any one of claims 19 to 21 , in the manufacture of a device for the simultaneous electrical and optical analysis of a small organic molecule, biomolecule, and / or cell.

35. A method of analysing small organic molecules, biomolecules, cells, and / or populations of cells comprising: providing a PECVD film according to any one of claims 1 to 18, said PECVD film comprising small organic molecules, biomolecules, cells and / or populations of cells covalently bonded or immobilised thereto; and one or more of optically imaging the small organic molecules, biomolecules, cells, and / or populations of cells; electrically stimulating the small organic molecules, biomolecules, cells, and / or populations of cells; recording an electrical output from the small organic molecules, biomolecules, cells, and / or populations of cells.

36. An electrode array, or microelectrode array (MEA), comprising a PECVD film according to any one of claims 1 to 18, or a substrate coated with a PECVD film according to any one of claims 19 to 21 .

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