Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
  • C08J7/12—Chemical modification
  • C08J7/123—Treatment by wave energy or particle radiation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D1/00—Processes for applying liquids or other fluent materials
  • B05D1/62—Plasma-deposition of organic layers
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
  • C23C16/02—Pretreatment of the material to be coated
• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
  • C23C16/22—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the deposition of inorganic material, other than metallic material
  • C23C16/26—Deposition of carbon only
• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
  • C23C16/44—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating
  • C23C16/50—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating using electric discharges
  • C23C16/505—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating using electric discharges using radio frequency discharges
• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
  • C23C16/44—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating
  • C23C16/50—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating using electric discharges
  • C23C16/515—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating using electric discharges using pulsed discharges
• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C4/00—Coating by spraying the coating material in the molten state, e.g. by flame, plasma or electric discharge
  • C23C4/04—Coating by spraying the coating material in the molten state, e.g. by flame, plasma or electric discharge characterised by the coating material
• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C4/00—Coating by spraying the coating material in the molten state, e.g. by flame, plasma or electric discharge
  • C23C4/12—Coating by spraying the coating material in the molten state, e.g. by flame, plasma or electric discharge characterised by the method of spraying
  • C23C4/134—Plasma spraying
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D2201/00—Polymeric substrate or laminate
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2325/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Derivatives of such polymers
  • C08J2325/02—Homopolymers or copolymers of hydrocarbons
  • C08J2325/04—Homopolymers or copolymers of styrene
  • C08J2325/06—Polystyrene
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment

Definitions

• the present invention relates to methods for covalently binding biomolecules to plastic.
• the present invention relates to plasma-activated coatings of plastic wells or dishes to which biomolecules covalently bind.
• the invention is not limited to this particular field of use.
• Microplates provide a fast and convenient platform to study biological processes in high throughput assays, through immobilisation of biomolecules or cells on a solid support. Common applications include enzyme-linked immunosorbent assays (ELISA) and biomolecule interaction studies.
• ELISA enzyme-linked immunosorbent assays
• the standardised microplate format facilitates robust characterisation via fluorescence and colorimetric detection in automated optical microplate readers with small sample volumes.
• Commercially available microplates are typically made from plastic (such as polystyrene) that is modified by a tissue culture surface treatment which reduces hydrophobicity and enhances cell adhesion.
• Biomolecules of interest include proteins, such as antibodies and streptavidin, and smaller molecules, such as DNA and peptides.
• DNA probes are immobilised on microplates for use in accurate DNA detection and as scaffolds for DNA- directed assembly of multienzyme complexes.
• biomolecule immobilisation methods such as physical adsorption and chemical activation. [0005] Most commonly, physical adsorption is used to attach biomolecules such as DNA and streptavidin to microplate surfaces through hydrophobic or electrostatic interactions.
• biomolecules bind weakly to microplates with random orientation and are sensitive to changes in ionic concentration, pH and heat, resulting in 1004836263 reduced reproducibility. Proteins can also be denatured by physical adsorption, for example reducing monoclonal antibody activity to less than 10%.
• Stronger and more specific binding can be achieved through chemical activation of microplates, which increases stability, activity, and resistance to washing, and reduces competitive protein exchange.
• Silane chemistry for example aminopropyltriethoxylsilane (APTES), is often used for activation of surfaces for biomolecule binding. This surface activation approach has been used in DNA microarrays where the DNA is functionalised with thiol or amine groups prior to chemical coupling to the surface.
• APTES aminopropyltriethoxylsilane
• Plasma immersion ion implantation has previously been used to immobilise of biomolecules on plastic (Bilek & McKenzie, Biophysical Reviews 2010, 2, 55–65; Nosworthy et al., Acta Biomaterialia 2007, 3, 695–704; Kosobrodova et al., ACS Applied Materials & Interfaces 2018, 10, 227–237).
• the method is fast and reproducible, and the reactivity of the plastic surface is retained for long periods. Additionally, the coverage of reactivity is significantly improved compared to other plasma activation methods (such as PIII) making it suitable for use in Petri dishes and the wells of microplates.
• plasma activation methods such as PIII
• the present invention provides a method for coating a plastic surface, the method comprising: (a) treating the plastic surface with ions under a pulsed bias; and (b) depositing plasma on the plastic surface by plasma enhanced chemical vapour deposition (PECVD) of a gas mixture comprising acetylene and argon under a pulsed bias, wherein a mesh is positioned above the plastic surface such that the plasma passes through the mesh before being deposited on the plastic surface, and wherein the method produces a plasma-activated coating (PAC) on the plastic surface that covalently binds a biomolecule.
• PECVD plasma enhanced chemical vapour deposition
• PAC plasma-activated coating
• the ions are from noble gases.
• the ions are argon ions.
• the gas mixture further comprises nitrogen.
• the atmospheric percentage of nitrogen in the gas mixture is less than 35%.
• the atmospheric percentage of nitrogen in the gas mixture is about 21%.
• the treatment with ions is conducted for about 0.5 to about 30 minutes at RF power of about 5 to about 300 W and negative pulsed bias voltage of about 1 to about 1000 V.
• the treatment with ions is conducted for about 1 to about 15 minutes at RF power of about 50 to about 100 W and negative pulsed bias voltage of about 300 to about 700 V [0020] In one embodiment, the treatment with ions is conducted for about 2 minutes at RF power of about 75 W and negative pulsed bias voltage of about 500 V. [0021] In one embodiment, the plasma deposition is conducted for about 10 to about 30 minutes with a plasma discharge of about 5 to about 300W and a negative pulsed bias voltage of about 1 to about 1000 V. [0022] In one embodiment, the plasma deposition is conducted for about 10 to about 30 minutes with a plasma discharge of about 10 to about 100W and a negative pulsed bias voltage of about 300 to about 700 V.
• the plasma deposition is conducted for about 10 to about 30 minutes with a plasma discharge of about 50 W and a negative pulsed bias voltage of about 500 V.
• the gas mixture is maintained at about 110 mTorr.
• the negative pulsed bias is applied with a frequency of about 0.5 to about 10 kHz and pulse duration of about 1 to about 100 ⁇ s.
• the negative pulsed bias is applied with a frequency of about 1 to about 5 kHz and pulse duration of about 5 to about 30 ⁇ s.
• the negative pulsed bias is applied with a frequency of about 3 kHz and pulse duration of about 20 ⁇ s.
• the mesh is formed from a conductive metal.
• the mesh forms the lid of a Faraday cage enclosing the plastic surface.
• the mesh has 50 openings per linear inch.
• the conductive metal is stainless steel.
• the plastic surface has non-flat geometry. 1004836263 [0033] In one embodiment the plastic surface is a microplate well or a Petri dish. [0034] In one embodiment, the plastic is polystyrene.
• the biomolecule is a nucleic acid. [0036] In one embodiment, the biomolecule is a protein.
• the protein is streptavidin or laminin.
• the PAC has a surface nitrogen concentration of about 4 to about 14%.
• the PAC has a relative density of radicals of about 0.016 to about 0.021.
• the PAC has a mean absorption at 450nm of about 0.052 to about 0.056 a.u.
• the PAC has a thickness of about 20nm.
• the PAC hybridises DNA at a concentration of about 2x10 11 to about 5x10 11 molecules/cm 2 .
• the PAC immobilises streptavidin at a concentration of about 2x10 11 to about 9x10 11 molecules/cm 2 .
• the Faraday cage with a mesh lid comprises an open container, a mesh lid and a cap for securing the mesh lid to the open container, wherein the container, mesh lid and cap are all formed from a conductive metal.
• the present invention provides a plastic surface coated with a plasma-activated coating (PAC) when produced by the method of the invention.
• the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC has a surface nitrogen concentration of about 4 to about 14%.
• the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC has a relative density of radicals of about 0.016 to about 0.021. 1004836263 [0048] In another aspect, the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC has a mean absorption at 450nm of about 0.052 to about 0.056 a.u. [0049] In another aspect, the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC has a thickness of about 1nm to about 10nm.
• PAC plasma-activated coating
• the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC has a thickness of about 2nm.
• the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC hybridises DNA at a concentration of about 2x10 11 to about 5x10 11 molecules/cm 2 .
• the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC immobilises streptavidin at a concentration of about 2x10 11 to about 9x10 11 molecules/cm 2 .
• the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC binds laminin.
• PAC plasma-activated coating
• Figure 1 (a) Percentage surface elemental concentration of UT, TC, PIII and PAC treated PS microplates. (b) Relative electron paramagnetic resonance (EPR) integral intensity be- tween UT PS, PIII and PAC treated PS. Measured EPR signal was integrated and normalised by the depth of radical penetration (PIII) or deposition (PAC). (c) Effective depth of the PIII treatment on polystyrene and PAC thickness obtained from ellipsometry measurement of PAC treated silicon wafers.
• EPR Relative electron paramagnetic resonance
• Figure 2 (a) Schematic diagram showing oligonucleotide immobilisation methods and an example of data plotted on a graph. Fluorescent intensity was measured at steps 1, 3, 5, 7, 8, 9 and 11, and shown on the graph. (b) Distribution map of hybridised DNA on PAC treated microplate by the microplate reader. (c) Baseline subtracted Alexa647 fluorescence intensity of DNA immobilised (pH 4) and hybridised on TC, PIII and PAC treated microplates. [0056] Figure 3: (a) DNA immobilisation and hybridisation on PIII treated microplates at pH 3-8. (b) DNA immobilisation and hybridisation on PAC treated microplates at pH 3-8.
• Figure 6 Three different conditions, each with 3 mesh sizes, used for plasma treatment to reduce nanoparticle deposition in cell culture plate wells
• Figure 7 The Faraday cage plate holder from top view (A), bottom view (B) and a sketch with dimensions (C).
• Figure 8 Diagram showing plasma treatment chamber containing a plate to be treated with plasma.
• Figure 9 Nanoparticle count using flow cytometry. 1004836263
• Figure 10 SEM images taken on silicon wafers located inside 24 well plates, plasma treated without a mesh (A, B) and plasma treated with a Faraday cage with 50 mesh lid (C, D). Images were taken at 1000x magnification (A, C) and 5000x magnification (B, D).
• Figure 11 Thickness of PAC measured on silicon wafers located in various wells and plasma deposited using different treatment conditions.
• Figure 12 Comparison of PAC thickness deposited on 24-well plates without a mesh and with a Faraday cage with 50 mesh lid. Shading is used to illustrate the different ranges of thickness.
• Figure 13 Comparison across all three plates showing the percentage surface area coverage of beating colonies observed in each well.24-well plate with no plasma treatment (Untreated), plasma treated without a mesh (-Mesh) and plasma treated with a Faraday cage with 50 mesh lid (+Mesh). All the plates were incubated with Laminin 521 before cell seeding. Cells were cultured for up to 41 days.
• the term “subject” includes any human or non-human animal.
• the term “non-human animal” includes all vertebrates, for example mammals and non- mammals, such as non-human primates, horses, cows, dogs, etc.
• the words “comprise”, “comprising” and the like are to be construed in their inclusive, as opposed to their exclusive, sense, that is in the sense of “including, but not limited to”. 1004836263
• the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances.
• biomolecule means a molecule found in living organisms, such as amino acids, lipids, carbohydrates, proteins, polysaccharides and nucleic acids.
• culture plate means a plate that may contain, e.g., 6, 12, 24, 48, 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix. Each well may hold from nanolitres to millilitres of liquid, and can be circular or square.
• Petri dish and “culture dish” mean a shallow cylindrical dish that may be used to hold growth medium in which cells can be cultured.
• Non-flat geometry refers to structures that are not completely prone or confined on a single plane. Such structures are not substantially flat in shape and possess more than 2 dimensions (e.g., wall(s) and a base). Non-flat geometries include U-shaped, V- shaped and flat bottomed wells, ELISA plates, culture dishes and Petri dishes.
• pulse duration means the time over which the pulsed bias is applied in each pulse.
• Faraday cage means a container formed from a conductive material.
• microplates are made from polystyrene (PS). These microplates have wells with diameter approximately 6 mm and depth approximately 11 mm. There is a 2-3 mm gap between the holder and the bottom of the well when the microplate is placed on either a PIII or a PAC stainless steel holder. Aluminium foil was moulded to fill the gap in order to improve contact to the holder. Microplates were cut into quarters with a hot wire cutter before plasma treatment, so they could be mounted on the sample holder in our small prototype plasma reactor, and were taped back together for DNA and streptavidin immobilisation and fluorescence detection.
• PS polystyrene
• Example 2 – PIII treatment was performed using inductively coupled radio frequency (RF) power at 13.56 MHz (ENI radio frequency power generator) to generate plasma and a negative voltage bias applied to the stainless-steel sample holder to accelerate positive ions towards the sample.
• RF radio frequency
• the pressure inside the chamber was evacuated to less than 5x10 -5 Torr and high purity nitrogen gas was introduced and maintained at 2 mTorr during the treatment.
• the RF forwarded power was 100 W with a reverse power of 12 W when matched.
• Microplates, with or without aluminium foil, were taped on the stainless-steel holder with stainless-steel mesh placed 5 cm from the holder and electronically connected to it.
• Nitrogen ions were accelerated through the mesh towards the stainless-steel holder with 20 kV negative bias pulses of 20 ⁇ s duration at a frequency of 50 Hz.
• the microplates were treated for 400 or 800 seconds (PIII 400 or PIII 800).
• Example 3 – PAC treatment [0085] PAC treatment was performed in a separate plasma system using capacitively coupled RF power at 13.56 MHz (Eni OEM-6) and a negative pulsed bias generated by RUP6 1004836263 pulse generator (GBS Elektronik GmbH, Dresden, Germany). Microplates were positioned on a stainless-steel sample holder connected to RUP6 with aluminium foil placed under the microplates to eliminate air gaps between the sample holder and the plate and strengthen the accelerating electric field at the surface of the plate.
• the chamber was evacuated to less than 5 x 10 -5 Torr. Prior to the plasma coating, microplates were activated with argon ions to facilitate coating adhesion for 2 minutes at RF power of 75 W and an applied bias of 500 V. The pressure of argon inside the chamber was maintained around 70-80 mTorr.
• PAC deposition a reactive gas mixture of acetylene, nitrogen and argon was introduced into the chamber. The ratios between gases were controlled by a mass flow controller (Allicat Scientific) and the pressure inside the chamber during the coating deposition was maintained at 110 mTorr. Plasma deposition was conducted with a plasma discharge of 50 W and a negative bias voltage of 500 V for 10 or 30 minutes.
• Negative bias from RUP 6 was applied with a frequency of 3 kHz and pulse duration of 20 ⁇ s.
• Four different gas ratios were chosen for comparison: no nitrogen (0% nitrogen), low nitrogen (21% nitrogen), moderate nitrogen (71% nitrogen), and high nitrogen (93% nitrogen) (Table 1).
• Separate PAC treatments were performed on smooth silicon wafers with a native oxide layer to measure the coating thickness.
• Table 1 PAC treatments with four different gas flow rate recipes.
• Example 4 Chemical composition of PAC and PIII modified microplates
• Ellipsometric spectroscopy was used to calculate the coating thickness of PAC. Silicon wafers were used as substrates to measure coating thickness and coatings were deposited with the various PAC recipes shown in Table 1. Ellipsometric data were collected at three angles of incidence (65°, 70°, and 75°) using a J.A Woollam M2000 V spectroscopic ellipsometer. A model consisting of a silicon substrate, silicon oxide layer (2 nm), and a Cauchy layer to represent the PAC layer was used to fit the data to obtain the film thickness.
• Polystyrene film was used as a proxy for the microplate, which did not fit in the measurement instrument.
• the microwave absorption by unpaired electrons from the samples were measured using an electron paramagnetic resonance (EPR) spectrometer (Bruker EMX X-band). Measurements were done at room temperature with a microwave power of 2 mW and a frequency of 9.8 MHz. A magnetic field was scanned with a central value of 3523 G and a sweep width of 200 G. Ten scans were measured on each sample. Similar measurement was applied to 2,2-diphenyl-1-picrylhydrazyl (DPPH) powder containing EPR tube with known radical density to calculate the number of unpaired electrons on each sample.
• DPPH 2,2-diphenyl-1-picrylhydrazyl
• Polar components of PIII treated microplates (23.7 ⁇ 0.3 - 30.1 ⁇ 0.3 mJ/m 2 ) were found to be significantly higher than TC-treated microplates (4.8 ⁇ 0.1 mJ/m 2 , one-way ANOVA, p ⁇ 0.0001), and increased with treatment time (Figure 1e).
• the wettability 1004836263 and polar surface energy component values for the PIII treated microplates had a stronger correlation to the relative density of radicals as described above, than to the surface nitrogen and oxygen composition.
• the chemical composition of PAC microplates was determined by XPS, comparing four different gas recipes containing increasing amounts of nitrogen; no nitrogen, low nitrogen, moderate (mod) nitrogen and high nitrogen ( Figure 1a).
• PAC treated microplates had significantly higher amounts of surface nitrogen compared to PIII treated microplates (PIII 0.5-0.8%, PAC 4-14.6%), while PIII treated microplates had higher relative density of radicals (PIII 0.10-0.24, PAC 0.016-0.021) and were more hydrophilic (37.0°-45.5°) compared to all PAC (50.7°-65.1°) except the high nitrogen treatment (42.1°).
• the relative density of radicals appeared to have a greater correlation with surface hydrophilicity than surface nitrogen and oxygen compositions.
• PAC treated microplates had much smaller increase in absorbance (1.13-1.22 fold) compared to PIII treated microplates (3.36-4.5 fold), with no significant difference between PAC gas treatments (0.052- 0.056 a.u., one-way ANOVA, p>0.1).
• PIII resulted in a large (288%) increase in absorbance at 450 nm relative to untreated microplate, while PAC had a more modest (17.5%) relative increase.
• aromatic polymers have high autofluorescence at shorter wavelength due to low energy ⁇ to ⁇ * transitions.
• DNA solution was replaced with 200 ⁇ L of 1% BSA in 10 mM PBS at pH 7.4 for one hour at room temperature on a shaker to block the remaining active surface. BSA solution was removed and the wells were washed with 200 ⁇ L of 2% SDS three times with vigorous shaking. After rinsing with 200 ⁇ L of MilliQ water three times, DNA hybridisation was performed by adding a 21-nt complementary DNA strand with 3’ Alexa647 fluorophore modification (IDT DNA).
• IDTT DNA 21-nt complementary DNA strand with 3’ Alexa647 fluorophore modification
• Complementary DNA was added to a hybridisation buffer (consisting of 2 mM magnesium chloride (Sigma-Aldrich), 1 x Tris EDTA (Sigma-Aldrich), 1% BSA and 0.6% SDS) to a final concentration of 0.8 ⁇ M.
• a hybridisation buffer consisting of 2 mM magnesium chloride (Sigma-Aldrich), 1 x Tris EDTA (Sigma-Aldrich), 1% BSA and 0.6% SDS.
• washing buffer 1 (2 x saline-sodium citrate (SSC) + 0.6% SDS), washing buffer 2 (0.2 x SSC + 0.6% SDS), and washing buffer 3 (0.1 x SSC + 0.5% Tween 20).
• washing buffer 1 (2 x saline-sodium citrate (SSC) + 0.6% SDS
• washing buffer 2 0.2 x SSC + 0.6% SDS
• washing buffer 3 0.1 x SSC + 0.5% Tween 20.
• each well was filled with 40 ⁇ L of 10 mM PBS for fluorescence intensity measurement.
• the fluorescence intensity of the Alexa647 modification on the complementary DNA was measured with an 635-20/680-20 optic module on PHERAstar FSX.
• the top optic was used to scan a 10 x 10 matrix of well diameter 3-5 mm with 10 flashes at each scan point. Focal height was adjusted for each sample and the gain was set at 2000.
• wash steps were required to remove non-specifically bound DNA. Wash steps were included to remove non-covalently bound ssDNA (step 4, Figure 2a) and non-hybridised ssDNA from the microplates (step 7-11, Figure 2a).
• Washing was found to be effective at reducing the fluorescence signal of negative controls back to the baseline level (t-test, p ⁇ 0.0001), for both controls with no immobilising ssDNA (step 2) or addition of the wrong sequence of hybridising DNA (step 6, Figure 2c). For example, giving a 50% decrease from step 7 to 11, which is equivalent to approximately 8.4 x 10 10 DNA molecules removed from the surface.
• the density of hybridised DNA was found to be higher in the centre of the microplate wells compare to the edges ( Figure 2b). Well edges have less efficient fluid mixing and experience lower flux of implanting ions due to shadowing and the reduction of sheath area to wall area ratio, which would limit DNA density near the edge.
• DNA hybridisation was characterised for all recipes of PIII (PIII-400, PIII-800) and PAC (no, low, mod and high nitrogen). Greater fluorescence signal, indicating higher DNA hybridisation density, was observed for all plasma treated samples compared to the negative controls ( ⁇ 3.1 x 10 10 molecules/cm 2 ), and was also higher on PAC than PIII treated mi- croplates (PIII 1.3 x 10 11 molecules/cm 2 , PAC 3.4 x 10 11 molecules/cm 2 , one-way ANOVA, p ⁇ 0.0001) ( Figure 2c, Table 2).
• DNA hybridisation was approximately 2-5 fold denser on PAC compared to PIII (1.1-1.6 x 10 11 molecules/cm 2 , one-way ANOVA, p ⁇ 0.0001), and no nitrogen and low nitrogen PAC recipes had the greatest amount of hybridised DNA overall (4.7-4.8 x 10 11 molecules/cm 2 , one-way ANOVA, p ⁇ 0.0001). There was no significant difference between the four different PIII surfaces, indicating that the aluminium foil and the treatment time did not affect DNA immobilisation.
• the effect of pH on DNA immobilisation was tested by incubating immobilising ssDNA in pH 3 to 8 (step 2, Figure 2a). The highest hybridisation density was observed for pH 3 and 4 for almost all PIII and PAC treated microplates ( Figure 3a, b).
• DNA 1004836263 immobilisation capability of PIII treated microplate decreased one month after the PIII treatment, but there was no further reduction up to three months (Figure 3c). All PAC samples retained the same DNA immobilisation capability up to three months ( Figure 3d).
• DNA immobilisation and hybridisation on no and low nitrogen PAC samples were further optimised by using freshly prepared hybridisation solution and measuring fluorescence intensity from 3 mm diameter instead of 5 mm diameter. After further optimisation, the average hybridised density on no and low nitrogen increased to 5.2 x 10 12 molecules/cm 2 ( Figure 4). [00107] Interestingly, the measured relative density of radicals was found not to correlate with the amount of DNA hybridised to the surface.
• PIII microplates had 5-15 fold higher relative density of radicals than PAC, but a 2-5 fold lower DNA hybridisation capacity. This demonstrates that the low amount of radicals on PAC surfaces was sufficient for dense DNA immobilisation.
• Table 2 Hybridised DNA and immobilised streptavidin densities of TC, PIII and PAC microplates
• PAC surfaces had higher DNA density than PIII, which could be explained by the higher composition of nitrogen that could be protonated on PAC surfaces leading to more favourable electrostatic interactions. The highest DNA density was found on PAC surfaces with lower composition of surface nitrogen.
• Hydrophilicity of the surface is another important factor that could affect the intermolecular interaction with DNA. Both hydrophilic and hydrophobic surfaces can attract DNA.
• Hydrophobic surfaces can attract nearby DNA through short range vdW forces, and hydrophilic surfaces can attract longer-range DNA via dipole-dipole and hydrogen bonding. However, hydrophilic surfaces are more susceptible to form a dense hydration layer which competes with intermolecular attraction between the surface and DNA via hydrogen bonding. 1004836263 Thus, higher DNA density on the no nitrogen and low nitrogen PAC surfaces compared to other samples could be explained by their relative hydrophobicity compared to mod and high nitrogen PAC and all PIII conditions, which minimises formation of the hydration layer and maximises vdW forces.
• Example 7 Streptavidin immobilisation for PIII and PAC modified microplates
• Streptavidin immobilised on plasma treated surfaces was directly detected by the fluorescence of covalently modified Cy3 streptavidin.
• PAC surfaces it was also indirectly detected by binding with dual biotin-modified Alexa647-modified DNA strands. While the former quantifies protein immobilised, the latter will only detect active protein on the surface.
• each well was washed with 200 ⁇ L of 10% Triton in 10 mM PBS three times.
• the wells were rinsed with 200 ⁇ L of 10 mM PBS a further three times, then the wells were incubated with 2 ⁇ M 5’ biotin-modified ssDNA (IDT DNA, Table 3) in 10 mM PBS at pH 7.4 for one hour at room temperature on a shaker.
• IDT DNA IDT DNA, Table 3
• the hybridised surface was washed with 200 ⁇ L of washing buffer 4 (2 x SSC + 0.05% Tween 20) three times before rinsing with 10 mM PBS.
• washing buffer 4 (2 x SSC + 0.05% Tween 20)
• optic modules 540/580 and 635-20/680-20 was used to detect immobilised streptavidin-Cy3 and the hybridised complementary Alexa647-DNA, respectively.
• the top optic was used to scan 10 x 10 matrix of well diameter 3-5 mm with 10 flashes at each scan point. Focal height was adjusted for each sample and the gain was set at 1000.
• low nitrogen PAC had the highest amount of bound streptavidin (no nitrogen 2.2 x 10 11 molecules/cm 2 , low nitrogen 9.0 x 10 11 molecules/cm 2 , mod nitrogen 4.3 x 10 11 molecules/cm 2 , high nitrogen 4.0 x 10 11 molecules/cm 2 ), whereas for DNA both low and no nitrogen PAC were similarly high.
• This PAC trend was reproduced for biotin-DNA binding, indicating that the immobilised protein remains active (Figure 5b, no nitrogen x 10 11 , low nitrogen 12.2 x 10 11 , mod nitrogen 7.3 x 10 11 , high nitrogen 7.8 x 10 11 biotin-DNA molecules/cm 2 ).
• Plasma-generated nanoparticles were observed to accumulate in PAC coated 24 well plates. To prevent plasma-generated nanoparticles falling into the wells, the configuration of the sample holder was modified. Plasma treatments on 24 well plates were conducted using 3 conditions described below and shown in Figure 6. ⁇ Polymer mesh: square mesh with 3 sizes (3, 4 and 5 mm spacing) were modified from the lid of tissue culture plate using laser cutting and fit on the well plate during plasma treatment.
• ⁇ Electrically conductive mesh stainless steel mesh with 3 sizes, 4 mesh (i.e., 4 openings per linear inch), 10 mesh (i.e., 10 openings per linear inch) and 50 mesh (i.e., 50 openings per linear inch), was cut to cover the plate.
• the meshes are made of stainless-steel wire woven to create square gaps with the spacing size varying from 6 mm to 0.45 mm.
• a polymer fitting cut out from the lid of tissue culture plate was used to secure the mesh on top of the plate.
• the stainless steel mesh was not electrically connected to the sample holder.
• ⁇ Faraday cage with mesh lid The Faraday cage with mesh lid consists of 3 parts: an open container, a mesh lid and a cap for securing the mesh lid to the open container.
• the open container is made of a conductive metal (e.g., steel, brass, aluminium etc) and sized to contain the plate or dish to be coated.
• the cap is also made from a conductive metal, conforms to the shape of the open container and is used to electrically connect the stainless-steel mesh lid to the open container (i.e., mesh lid is sandwiched between the cap and the open container). This forms a Faraday cage around the plate or dish.
• Three sizes of mesh, 4 mesh, 10 mesh and 50 mesh were investigated for effect of plasma treatment using the same sample holder and other parameters. The configuration and sizes of a Faraday cage that is suitable for holing a plate are shown in Figure 7. [00115] The plasma treatment was performed using a custom-made three-chamber system (Figure 8).
• a culture plate (Corning Costar, 24 tissue culture plates) was positioned on the sample holder, either on a metal plate or in Faraday cage with mesh lid. The system was evacuated until a base pressure of 5x10 -5 Torr was reached.
• the plasma treatment consisted of two steps: surface activation with argon plasma and plasma deposition. Both steps utilized plasma generated by an RF power supply, with settings of 75 W for surface activation and 50 W for deposition.
• a pulsed negative bias of 500 V, at 3 kHz, and with duration of 20 ⁇ s was applied to the sample holder.
• the pressure of argon was 1004836263 regulated within the range of 70-80 mTorr.
• a gas mixture comprising acetylene, nitrogen, and argon was maintained at 110 mTorr, with flow rates of 1, 3, and 10 sccm, respectively. Surface activation was performed for durations of 2, 5, and 10 minutes, while the deposition step had a fixed duration of 10 minutes.
• the measurement of PAC thickness and the quantification of nanoparticles were performed using silicon wafers and glass coverslips placed inside the wells. The PAC thickness was determined using spectroscopic ellipsometry (J.A. Woollam M2000), while the number of nanoparticles was counted using an Olympus BMX-10 optical microscope.
• Conductive meshes are more efficient to prevent the particles compared to polymer mesh, especially when the mesh spacing size gets smaller.
• the Faraday cage with 50 mesh lid shows particle counts comparable to an untreated control plate (Table 1).
• Table 4 Number of particles counted on each image on coverslips located on different wells from each condition of plasma treatment. The results are the average of 3 images taken on each glass coverslip. 1004836263 [00118] Flow cytometer Cytek Aurora (USA) was used to count the number of particles in solution.
• Figure 9 compares the number of nanoparticles detected in 15 ⁇ l liquid using a flow cytometer, showing a significantly drop of particle count when plasma treatment was conducted with a Faraday cage with 50 mesh lid compared to conventional treatment without a mesh.
• the high density of nanoparticles in the liquid samples removed from no mesh treated plates indicates that these nanoparticles were loosely bound to the PAC and can easily be removed with low shear force such as the still method.
• Nanoparticles forming on the coating were visualized from SEM images ( Figure 10). They are present as single particles or clumps of particles on the coating obtained from no mesh plasma treatment. In contrast, when using a Faraday cage with 50 mesh lid, very few nanoparticles can be found on the clear and smooth coating.
• the coating thickness was determined by placing silicon wafers inside the wells and using spectroscopic ellipsometry to measure the PAC deposited on the surface of the silicon.
• Figure 11 compares the PAC thickness when silicon wafers were positioned at various locations across the plate.
• the coating thickness was generally thicker using stainless steel meshes compared to polymer meshes, especially as the mesh size decreased. It is hypothesised that the polymer mesh cannot conduct away the charges that land on it so a 1004836263 positive charge builds up over time that reduces the electric field created by the negative bias and therefore the force drawing ions from the plasma to create the coating is also reduced. This is less of an issue for stainless steel meshes as the charges are conducted away.
• Figure 12 provides a comprehensive comparison of the PAC thickness between treatment without a mesh and treatment with a Faraday cage with 50 mesh lid. Without using a mesh, the PAC thickness was measured to be 1.13 ⁇ 0.35 nm. However, when a mesh was used, the PAC thickness increased to 1.89 ⁇ 0.54 nm.
• Example 10 Comparison of cell culture in plasma treated wells [00124] The effect of plasma treatment on the differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CM) was examined on 24 well plates that were untreated (UT), plasma treated without mesh (-Mesh) and plasma treated with a Faraday cage with 50 mesh lid (+Mesh), and then coated with Laminin 521 (LN521).
• iPSCs induced pluripotent stem cells
• CM cardiomyocytes
• Laminin-521 (LN521) was diluted in PBS (containing Ca 2+ and Mg 2+ ) to a concentration of 10 ⁇ g/mL and 200 ⁇ L added to each well of UT, -Mesh and +Mesh treated wells. After overnight incubation at 4°C the wells were washed.
• iPSCs Human induced pluripotent stem cells (Gibco Episomal iPSC Line; A18945) that had been cultured on Matrigel in a 6-well plate (Corning Costar) in mTeSR Plus Basal Medium with Plus 5X Supplement added (STEMCELL, #100-0276) were passaged and plated into the wells at a density of approximately 3x10 5 cells/well.
• day 0 CM differentiation media A (STEMCELL, #05010) was added for two days.
• CMs beating cardiomyocytes
• FIG. 13 shows that +Mesh provides a stable surface on which iPSCs can grow and differentiate into CMs. +Mesh is superior to –Mesh, which is in turn superior to UT. It is hypothesised that this is due to the increased number of nanoparticles in the UT and -Mesh treated wells to which the laminin binds and are washed away during media exchange – exposing areas without laminin coating. In contrast +Mesh wells have a even PAC coating to which the laminin covalently binds and fewer nanoparticles. 1004836263

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