Methods for covalently attaching biomolecules to plastics

Abstract

The present invention relates to a method for covalently attaching biomolecules to plastics, and in particular to a plasma-activated coating on plastic wells or dishes to which biomolecules are covalently attached.

Description

[Technical Field]

[0001] This application claims priority to Australian Provisional Patent Application No. 2022902232 (filed 9 August 2022), the contents of which are incorporated herein in their entirety.

[0002] The present invention relates to a method for covalently attaching biomolecules to plastics. In particular, the invention relates to plasma activated coating of plastic wells or dishes to which biomolecules are covalently attached. However, it will be understood that the invention is not limited to this particular field of use. [Background technology]

[0003] Any discussion of prior art throughout this specification should not be taken as an admission that such prior art is widely known or forms part of the common general knowledge in the art.

[0004] Microplates provide a rapid and convenient platform for studying biological processes in high-throughput assays by immobilizing biomolecules or cells on a solid support. Common applications include enzyme-linked immunosorbent assays (ELISAs) and biomolecular interaction studies. Standardized microplate formats facilitate robust characterization using small sample volumes via fluorescent and colorimetric detection in automated optical microplate readers. Commercially available microplates are typically made of plastics (e.g., polystyrene) modified with tissue culture surface treatments that reduce hydrophobicity and enhance cell adhesion. However, due to the inert properties of polystyrene, additional surface functionalization is required to immobilize biomolecules for solid-phase assays. Biomolecules of interest include proteins such as antibodies and streptavidin, as well as smaller molecules such as DNA and peptides. For example, DNA probes are immobilized on microplates for use in accurate DNA detection and as a scaffold for the assembly of DNA-directed multienzyme complexes. However, current biomolecule immobilization methods, such as physical adsorption and chemical activation, have limitations.

[0005] Physical adsorption is most commonly used to attach biomolecules such as DNA and streptavidin to the surface of a microplate through hydrophobic or electrostatic interactions. However, physically adsorbed biomolecules bind weakly to the microplate in a random orientation and are highly sensitive to changes in ion concentration, pH, and heat, resulting in poor reproducibility. Proteins are also denatured by physical adsorption, reducing the activity of, for example, monoclonal antibodies to less than 10%.

[0006] Stronger and more specific binding is achieved through chemical activation of microplates, which increases stability, activity, and washing resistance, and reduces competitive protein exchange. Silane chemistry, such as aminopropyltriethoxysilane (APTES), is often used to activate surfaces for biomolecule attachment. This surface activation approach has been used with DNA microarrays, where DNA is functionalized with thiol or amine groups before chemically binding to the surface. However, chemical activation methods are multi-step processes, often require modified biomolecules, such as amine- or thiol-modified DNA oligonucleotides, and may contain cytotoxic reagents that must be thoroughly removed before downstream applications in cell culture.

[0007] Plasma immersion ion implantation (PIII) for immobilizing biomolecules on plastics PIII has been used previously for polymeric surface preparation (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). In PIII, energetic ions of carbon-free gases such as nitrogen, argon, or helium bombard the polymer surface, generating reservoirs of radicals below the polymer surface to which biomolecules can be covalently attached. This method is fast and reproducible, and the reactivity of these surfaces remains constant over time. However, it is not suitable for treating microplate wells because shadowing caused by the well walls can lead to uneven reactivity across the plastic surface.

[0008] Alternative plasma-based methods, such as argon plasma activation and nitrogen plasma activation, have been used on microplates (North et al., ACS Applied Materials & Interfaces 2010, 2, 2884-2891; Boulares-Pender et al., Journal of Applied Polymer Science 2009, 112, 2701-2709). However, these methods require a chemical activation step and specific functional groups on the biomolecules. Furthermore, the density of active groups incorporated by these approaches is limited by the type of gas mixture required to produce a mechanically robust surface coating. Another approach involves direct deposition of sprayed proteins, such as collagen, onto microplates using dielectric barrier discharge plasma (O'Sullivan et al., ACS Omega 2020, 5, 25069-25076). However, this method requires large amounts of protein and does not allow all biomolecules incorporated into the coating to access the surface.

[0009] Therefore, there is a need for a rapid and reproducible method for covalently binding biomolecules to microplates over long periods of time.

[0010] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. Summary of the Invention

[0011] Surprisingly, plasma-enhanced chemical vapor deposition (PECVD) using a carbon-containing gas mixture under pulsed bias has been found to produce plasma-activated coatings (PACs) on plastic surfaces, which enable the covalent immobilization of biomolecules. This method is rapid and highly reproducible, and the reactivity of the plastic surface is maintained over long periods of time. Furthermore, the reactivity range is significantly improved compared to other plasma activation methods (e.g., PIII), making it suitable for use on Petri dishes and microplate wells.

[0012] In one aspect, the present invention provides a method for coating a plastic surface, comprising: (a) treating a plastic surface with ions under a pulsed bias; (b) depositing plasma onto the plastic surface by plasma-enhanced chemical vapor deposition (PECVD) of a gas mixture comprising acetylene and argon under a pulsed bias; A method is provided in which a mesh is placed on a plastic surface, whereby plasma passes through the mesh before being deposited on the plastic surface, thereby producing a plasma activated coating (PAC) on the plastic surface, which covalently bonds to biomolecules.

[0013] In one embodiment, the ions are from a noble gas.

[0014] In one embodiment, the ions are argon ions.

[0015] In one embodiment, the gas mixture further comprises nitrogen.

[0016] In one embodiment, the atmospheric percentage of nitrogen in the gas mixture is less than 35%.

[0017] In one embodiment, the atmospheric percentage of nitrogen in the gas mixture is about 21%.

[0018] In one embodiment, the treatment with ions is carried out at an RF power of about 5 to about 300 W and a negative pulse bias voltage of about 1 to about 1000 V for about 0.5 to about 30 minutes.

[0019] In one embodiment, the treatment with ions is carried out at an RF power of about 50 to about 100 W and a negative pulse bias voltage of about 300 to about 700 V for about 1 to about 15 minutes.

[0020] In one embodiment, the ion treatment is performed at an RF power of about 75 W and a negative pulsed bias voltage of about 500 V for about 2 minutes.

[0021] In one embodiment, the plasma deposition is carried out with a plasma discharge of about 5 to about 300 W and a negative pulsed bias voltage of about 1 to about 1000 V for about 10 to about 30 minutes.

[0022] In one embodiment, the plasma deposition is carried out with a plasma discharge of about 10 to about 100 W and a negative pulsed bias voltage of about 300 to about 700 V for about 10 to about 30 minutes.

[0023] In one embodiment, the plasma deposition is carried out with a plasma discharge of about 50 W and a negative pulsed bias voltage of about 500 V for about 10 to about 30 minutes.

[0024] In one embodiment, the gas mixture is maintained at about 110 mTorr.

[0025] In one embodiment, the negative pulse bias is applied at a frequency of about 0.5 to about 10 kHz and a pulse duration of about 1 to about 100 μs.

[0026] In one embodiment, the negative pulsed bias is applied at a frequency of about 1 to about 5 kHz and a pulse duration of about 5 to about 30 μs.

[0027] In one embodiment, the negative pulse bias is applied at a frequency of about 3 kHz and a pulse duration of about 20 μs.

[0028] In one embodiment, the mesh is formed from a conductive metal.

[0029] In one embodiment, the mesh forms the lid of a Faraday cage that surrounds the plastic surface.

[0030] In one embodiment, the mesh has 50 openings per linear inch.

[0031] In one embodiment, the conductive metal is stainless steel.

[0032] In one embodiment, the plastic surface has a non-flat topography.

[0033] In one embodiment, the plastic surface is a microplate well or a petri dish.

[0034] In one embodiment, the plastic is polystyrene.

[0035] In one embodiment, the biomolecule is a nucleic acid.

[0036] In one embodiment, the biomolecule is a protein.

[0037] In one embodiment, the protein is streptavidin or laminin.

[0038] In one embodiment, the PAC has a surface nitrogen concentration of about 4 to about 14%.

[0039] In one embodiment, the PAC has a relative radical density of about 0.016 to about 0.021.

[0040] In one embodiment, the PAC has an average absorbance of about 0.052 to about 0.056 au at 450 nm.

[0041] In one embodiment, the PAC has a thickness of about 20 nm.

[0042] In one embodiment, the PAC is about 2×10 11 ~Approx. 5×10 11 molecules / cm 2 The DNA is hybridized at a concentration of 1:1.

[0043] In one embodiment, the PAC is about 2×10 11 ~Approx. 9×10 11 molecules / cm 2 Streptavidin is immobilized at a concentration of 1000 mg / ml.

[0044] In one embodiment, a Faraday cage with a mesh lid includes a lidless container, a mesh lid, and a cap for securing the mesh lid to the lidless container, wherein the container, mesh lid, and cap are all formed from a conductive metal.

[0045] In another aspect, the present invention provides a plastic surface coated with a plasma activated coating (PAC) when produced by the method of the present invention.

[0046] In another aspect, 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%.

[0047] In another aspect, the present invention provides a plastic surface coated with a plasma activated coating (PAC), wherein the PAC has a relative radical density of about 0.016 to about 0.021.

[0048] In another aspect, the present invention provides a plastic surface coated with a plasma activated coating (PAC), wherein the PAC has an average absorbance at 450 nm of about 0.052 to about 0.056 au.

[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 1 nm to about 10 nm.

[0050] 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 2 nm. provide.

[0051] In another aspect, the present invention provides a plastic surface coated with a plasma activated coating (PAC), wherein the PAC has a surface roughness of about 2×10 11 ~Approx. 5×10 11 molecules / cm 2 A plastic surface is provided on which DNA is hybridized at a concentration of 1000 uM.

[0052] In another aspect, the present invention provides a plastic surface coated with a plasma activated coating (PAC), wherein the PAC has a surface roughness of about 2×10 11 ~Approx. 9×10 11 molecules / cm 2 A plastic surface is provided on which streptavidin is immobilized at a concentration of

[0053] In another aspect, the present invention provides a plastic surface coated with a plasma activated coating (PAC), wherein the PAC is bound to laminin. [Brief explanation of the drawings]

[0054] [Figure 1-1](a) Percentage of surface element concentrations for UT-, TC-, PIII-, and PAC-treated PS microplates. (b) Relative electron paramagnetic resonance (EPR) integrated intensity between UT PS, PIII-, and PAC-treated PS. The measured EPR signal was integrated and normalized by the depth of radical penetration (PIII) or deposition (PAC). (c) Effective depth of PIII treatment and PAC thickness relative to polystyrene obtained from ellipsometry measurements of PAC-treated silicon wafers. (d) Water contact angles for UT-, TC-, PIII-, and PAC-treated PS sheets placed in a 96-well microplate during plasma treatment. (e) Dispersive and polar components of surface free energy for UT-, TC-, PIII-, and PAC-treated PS microplates calculated from the Owens-Wendt-Rabel-Kaelble method. Error bars represent standard error. (f) Average absorbance and autofluorescence with SD for UT (circles), PIII (squares), and PAC (triangles)-treated PS microplates. SD bars are hidden if smaller than the marker size. The left column and y-axis show absorbance at 450 nm, and the right column and y-axis show autofluorescence emission intensity at five excitation / emission wavelengths. [Figure 1-2](a) Percentage of surface element concentrations for UT-, TC-, PIII-, and PAC-treated PS microplates. (b) Relative electron paramagnetic resonance (EPR) integrated intensity between UTPS, PIII-, and PAC-treated PS. The measured EPR signal was integrated and normalized by the depth of radical penetration (PIII) or deposition (PAC). (c) Effective depth of PIII treatment and PAC thickness relative to polystyrene obtained from ellipsometry measurements of PAC-treated silicon wafers. (d) Water contact angles for UT-, TC-, PIII-, and PAC-treated PS sheets placed in a 96-well microplate during plasma treatment. (e) Dispersive and polar components of surface free energy for UT-, TC-, PIII-, and PAC-treated PS microplates calculated from the Owens-Wendt-Rabel-Kaelble method. Error bars represent standard error. (f) Average absorbance and autofluorescence with SD for UT (circles), PIII (squares), and PAC (triangles)-treated PS microplates. SD bars are hidden if smaller than the marker size. The left column and y-axis show absorbance at 450 nm, and the right column and y-axis show autofluorescence emission intensity at five excitation / emission wavelengths. [Figure 1-3](a) Percentage of surface element concentrations for UT-, TC-, PIII-, and PAC-treated PS microplates. (b) Relative electron paramagnetic resonance (EPR) integrated intensity between UTPS, PIII-, and PAC-treated PS. The measured EPR signal was integrated and normalized by the depth of radical penetration (PIII) or deposition (PAC). (c) Effective depth of PIII treatment and PAC thickness relative to polystyrene obtained from ellipsometry measurements of PAC-treated silicon wafers. (d) Water contact angles for UT-, TC-, PIII-, and PAC-treated PS sheets placed in a 96-well microplate during plasma treatment. (e) Dispersive and polar components of surface free energy for UT-, TC-, PIII-, and PAC-treated PS microplates calculated from the Owens-Wendt-Rabel-Kaelble method. Error bars represent standard error. (f) Average absorbance and autofluorescence with SD for UT (circles), PIII (squares), and PAC (triangles)-treated PS microplates. SD bars are hidden if smaller than the marker size. The left column and y-axis show absorbance at 450 nm, and the right column and y-axis show autofluorescence emission intensity at five excitation / emission wavelengths. [Figure 2-1] (a) Schematic diagram showing the oligonucleotide immobilization method and example data plotted on a graph. Fluorescence intensity was measured at steps 1, 3, 5, 7, 8, 9, and 11 and shown on a graph. (b) Distribution map of hybridized DNA on a PAC-treated microplate by a microplate reader. (c) Baseline-subtracted Alexa647 fluorescence intensity of immobilized (pH 4) and hybridized DNA on TC-, PIII-, and PAC-treated microplates. [Figure 2-2] (a) Schematic diagram showing the oligonucleotide immobilization method and example data plotted on a graph. Fluorescence intensity was measured at steps 1, 3, 5, 7, 8, 9, and 11 and shown on a graph. (b) Distribution map of hybridized DNA on a PAC-treated microplate by a microplate reader. (c) Baseline-subtracted Alexa647 fluorescence intensity of immobilized (pH 4) and hybridized DNA on TC-, PIII-, and PAC-treated microplates. [Figure 2-3](a) Schematic diagram showing the oligonucleotide immobilization method and example data plotted on a graph. Fluorescence intensity was measured at steps 1, 3, 5, 7, 8, 9, and 11 and shown on a graph. (b) Distribution map of hybridized DNA on a PAC-treated microplate by a microplate reader. (c) Baseline-subtracted Alexa647 fluorescence intensity of immobilized (pH 4) and hybridized DNA on TC-, PIII-, and PAC-treated microplates. [Figure 3-1] (a) DNA immobilization and hybridization on PIII-treated microplates at pH 3 to 8. (b) DNA immobilization and hybridization on PAC-treated microplates at pH 3 to 8. (c) DNA immobilization and hybridization on PIII-treated microplates 1 week, 1 month, and 3 months after treatment. (d) DNA immobilization and hybridization on PAC-treated microplates 1 week, 1 month, and 3 months after treatment. [Figure 3-2] (a) DNA immobilization and hybridization on PIII-treated microplates at pH 3 to 8. (b) DNA immobilization and hybridization on PAC-treated microplates at pH 3 to 8. (c) DNA immobilization and hybridization on PIII-treated microplates 1 week, 1 month, and 3 months after treatment. (d) DNA immobilization and hybridization on PAC-treated microplates 1 week, 1 month, and 3 months after treatment. [Figure 4] DNA immobilization density on PIII- and PAC-treated microplates. Cy3-labeled DNA (20xA-GCTCTGCAATCAACTTATCCC-Cy3) was immobilized on a plasma-treated microplate, and the Cy3 intensity was measured after a washing step. The trend of immobilized DNA density is similar to that of hybridized DNA. This indicates that the decrease in hybridization density in some samples is not caused by overcrowding of immobilized DNA on the surface. [Figure 5-1]Immobilization of streptavidin on microplates. (a) Cy3 fluorescence intensity of streptavidin-Cy3 immobilized on TC-, PIII-, and PAC-treated microplates. (b) Biotin-binding ability of immobilized streptavidin. (c) Immobilization of streptavidin on PIII-treated microplates at pH 3-9. (d) Immobilization of streptavidin on PAC-treated microplates at pH 3-9. (e) Immobilization of streptavidin on PIII-treated microplates aged 1 week, 1 month, and 3 months. (f) Immobilization of streptavidin on PAC-treated microplates aged 1 week, 1 month, and 3 months. [Figure 5-2] Immobilization of streptavidin on microplates. (a) Cy3 fluorescence intensity of streptavidin-Cy3 immobilized on TC-, PIII-, and PAC-treated microplates. (b) Biotin-binding ability of immobilized streptavidin. (c) Immobilization of streptavidin on PIII-treated microplates at pH 3-9. (d) Immobilization of streptavidin on PAC-treated microplates at pH 3-9. (e) Immobilization of streptavidin on PIII-treated microplates aged 1 week, 1 month, and 3 months. (f) Immobilization of streptavidin on PAC-treated microplates aged 1 week, 1 month, and 3 months. [Figure 5-3] Immobilization of streptavidin on microplates. (a) Cy3 fluorescence intensity of streptavidin-Cy3 immobilized on TC-, PIII-, and PAC-treated microplates. (b) Biotin-binding ability of immobilized streptavidin. (c) Immobilization of streptavidin on PIII-treated microplates at pH 3-9. (d) Immobilization of streptavidin on PAC-treated microplates at pH 3-9. (e) Immobilization of streptavidin on PIII-treated microplates aged 1 week, 1 month, and 3 months. (f) Immobilization of streptavidin on PAC-treated microplates aged 1 week, 1 month, and 3 months. [Figure 6]Three different conditions, each with three mesh sizes, were used for plasma treatment to reduce nanoparticle deposition in cell culture plate wells. [Figure 7] Top view (A), bottom view (B), and dimensioned schematic (C) of the Faraday cage plate holder. [Figure 8] FIG. 1 shows a plasma processing chamber containing a plate to be plasma processed. [Figure 9] Nanoparticle counting using flow cytometry. [Figure 10] SEM images of silicon wafers in a plasma-treated 24-well plate without a mesh (A, B) and in a plasma-treated 24-well plate with a Faraday cage and a 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 placed in various wells and plasma deposited using different processing conditions. [Figure 12] Comparison of thickness of PAC deposited on a 24-well plate without mesh and a 24-well plate with a Faraday cage with a 50 mesh lid. Shading is used to indicate the range of different thicknesses. [Figure 13] Comparison of three plates showing the surface area coverage of beating colonies observed in each well. 24-well plates were not plasma-treated (Untreated), plasma-treated without a mesh (-Mesh), and plasma-treated in a Faraday cage with a 50-mesh lid (+Mesh). All plates were incubated with laminin-521 before seeding. Cells were cultured for up to 41 days. DETAILED DESCRIPTION OF THE INVENTION

[0055] definition In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set forth below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the present invention only, and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0056] As used herein, the term "about" may mean within one or more standard deviations, in accordance with the practice in the art. Alternatively, "about" may mean a range of up to 20%. When a specific value is given in the specification and claims, the meaning of "about" should be assumed to be within an acceptable error range for that specific value.

[0057] In the context of the present invention, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals. Examples of mammals include non-human primates, horses, cows, dogs, and the like.

[0058] In the context of the present invention, the words "comprise", "comprising" and the like are to be interpreted in an inclusive rather than an exclusive sense, i.e. in the sense of "including but not limited to".

[0059] The terms "preferred" and "preferably" refer to embodiments of the invention that may offer certain advantages, under particular circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0060] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about."

[0061] The recitation of numerical ranges using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0062] The term "au" means absorbance units.

[0063] The term "biomolecule" refers to molecules found in living organisms, such as amino acids, lipids, carbohydrates, proteins, polysaccharides, and nucleic acids.

[0064] The terms "culture plate," "microplate," "microtiter plate," "microwell plate," "multiwell plate," and ELISA plate refer to plates that may contain, for example, 6, 12, 24, 48, 96, 384, or 1536 sample wells arranged in a 2:3 rectangular matrix. Each well may hold nanoliters to milliliters of liquid and may be round or square.

[0065] The terms "Petri dish" and "culture dish" refer to a shallow cylindrical dish that can be used to contain growth medium in which cells can be cultured.

[0066] The term "non-planar shape" refers to structures that are not completely prone or confined on a single plane. Such structures are not substantially flat and have more than two dimensions (e.g., walls and a base). Non-planar shapes include U-shaped, V-shaped, and flat-bottom wells, ELISA plates, culture dishes, and Petri dishes.

[0067] As used herein, the term "pulse duration" refers to the time that the pulsed bias is applied in each pulse.

[0068] As used herein, the term "Faraday cage" means a container made from an electrically conductive material.

[0069] Preferred Embodiments of the Invention Although the present invention has been described with reference to the specific embodiments detailed herein, other embodiments may achieve the same or similar results. Variations and modifications of the present invention will be apparent to those skilled in the art, and the present invention is intended to cover all such modifications and equivalents. It is intended.

[0070] The present invention is further illustrated by the following non-limiting examples. [Example]

[0071] Example 1 - Microplate Preparation In this study, TC-treated polystyrene (PS) microplates (CLS3603) (Sigma-Aldrich) were subjected to high-energy plasma treatment and compared with unmodified TC-treated and untreated (UT) PS microplates (CLS3370). All microplates were made of polystyrene (PS). These microplates had wells approximately 6 mm in diameter and 11 mm deep. When the microplates were placed on a PIII holder or PAC stainless steel holder, a 2–3 mm gap existed between the holder and the bottom of the well. To improve contact with the holder, aluminum foil was molded to fill the gap. Before plasma treatment, the microplates were cut into four pieces with a hot wire cutter, which were then mounted in the sample holder of a small prototype plasma reactor and reassembled with tape for DNA and streptavidin immobilization and fluorescence detection.

[0072] Example 2 - PIII treatment The PIII process was performed by generating plasma using 13.56 MHz inductively coupled radio frequency (RF) power (ENI radio frequency power generator) and applying a negative voltage bias to the stainless steel sample holder to accelerate positive ions toward the sample. The pressure inside the chamber was maintained at 5 × 10. -5 The chamber was evacuated to less than 100 Torr, and high-purity nitrogen gas was introduced and maintained at 2 mTorr during treatment. During matching, the RF forward power was 100 W and the reverse power was 12 W. The microplate, with or without aluminum foil, was fixed onto a stainless steel holder with tape, and a stainless steel mesh was positioned 5 cm from the holder and electronically connected to it. Nitrogen ions were accelerated through the mesh toward the stainless steel holder using a 20 kV negative bias pulse with a 20 μs duration at a frequency of 50 Hz. The microplate was treated for 400 or 800 seconds (PIII400 or PIII800).

[0073] Example 3 - PAC Treatment In a separate plasma system, PAC treatment was performed using a 13.56 MHz capacitively coupled RF power (Eni OEM-6) and a negative pulse bias generated by a RUP6 pulse generator (GBS Elektronik GmbH, Dresden, Germany). The microplate was placed on a stainless steel sample holder connected to the RUP6, and aluminum foil was placed under the microplate to eliminate the air gap between the sample holder and the plate and to enhance the accelerating electric field on the plate surface. The chamber was 5 × 10 -5The chamber was degassed to less than 100 Torr. Prior to plasma coating, the microplate was activated with argon ions for 2 minutes at 75 W RF power and an applied bias of 500 V to promote coating adhesion. The argon pressure inside the chamber was maintained at approximately 70-80 mTorr. For PAC deposition, a reactive gas mixture of acetylene, nitrogen, and argon was introduced into the chamber. The gas ratio was controlled by a mass flow controller (Allicat Scientific), and the pressure inside the chamber was maintained at 110 mTorr during coating deposition. Plasma deposition was performed for 10 or 30 minutes at a plasma discharge of 50 W and a negative bias voltage of 500 V. The negative bias from the RUP6 was applied at a frequency of 3 kHz and a pulse duration of 20 μs. For comparison, four different gas ratios were selected: no nitrogen (0% nitrogen), low nitrogen (21% nitrogen), medium nitrogen (71% nitrogen), and high nitrogen (93% nitrogen) (Table 1). To measure the coating thickness, another PAC procedure was performed on a smooth silicon wafer with a native oxide layer.

[0074] [Table 1]

[0075] Upon exposure to air, the radicals on the treated surface react with atmospheric oxygen to form oxygen-containing groups. Because these changes in surface chemistry saturate after one week, the microplates were covered with aluminum foil after each treatment and stored in air at room temperature for at least one week before any subsequent analyses or biomolecule immobilization.

[0076] Example 4 - Chemical Composition of PAC and PIII Modified Microplates The thickness of the PAC coating was calculated using ellipsometry. Silicon wafers were used as substrates for measuring the coating thickness, and coatings were deposited using various PAC recipes, as shown in Table 1. Ellipsometry data were collected at three angles of incidence (65°, 70°, and 75°) using a JA Woollam M2000V spectroscopic ellipsometer. The data were fitted using a Cauchy layer model representing the silicon substrate, a silicon oxide layer (2 nm), and the PAC layer to obtain the film thickness.

[0077] The surface free energy of PIII- and PAC-treated TC-treated 96-well microplates (1 week after treatment) was calculated and compared to untreated polystyrene. Contact angle measurements were performed using two liquid probes (water and diiodomethane) using a Theta Tensiometer (Biolin Scientific). Results are the average of 10 drops for each sample. The Owens-Wendt-Rabel-Kaelble method was used to calculate the surface free energy.

[0078] To measure the radical density of microplates after PIII and PAC treatment, polystyrene film (Goodfellow, 0.19 mm thick) was cut into 40 mm x 5 mm strips and treated with PIII or PAC. Polystyrene film was used as a substitute for microplates that did not fit the measurement instrument. Microwave absorption by unpaired electrons from the samples was measured using an electron paramagnetic resonance (EPR) spectrometer (Bruker EMX X-band). Measurements were performed at room temperature using a microwave power of 2 mW and a frequency of 9.8 MHz. The magnetic field was scanned with a center value of 3523 G and a sweep width of 200 G. Ten scans were performed for each sample. Similar measurements were performed on 2,2-diphenyl-1-picrylhydrazyl (DPPH) powder containing EPR tubes with known radical densities, and the number of unpaired electrons for each sample was calculated.

[0079] The surface chemical changes after PIII and PAC treatment of the microplates were determined by comparing the FTIR-ATR spectra of untreated and treated polystyrene. Polystyrene films were cut into small disks and placed at the bottom of microplate wells for PIII and PAC treatment. One week after treatment, the disks were analyzed using a Hyperion FTIR spectrometer (Bruker) equipped with a microgermanium crystal ATR accessory. Each analysis was performed on a 4 cm -1 For comparison, the spectrum consists of 128 scans at 1000 Hz resolution. of polystyrene is 1490 cm -1 The peak intensity was normalized to that of the original peak.

[0080] The chemical composition of PIII-treated microplates was determined by XPS, comparing treatment times of 400 seconds (PIII-400) and 800 seconds (PIII-800) (Figure 1A). The oxygen content of PIII-treated microplates (6.1–11.1%) was not significantly different from that of TC-treated microplates (8.3%). Similarly, the surface nitrogen content was very low before (0.6%) and after (0.5–0.8%) PIII treatment. This was surprising, since previous studies have shown that nitrogen content (7–12%) increases after 400 seconds of PIII treatment of polystyrene spin-coated glass coverslips (Kosobrodova et al., ACS Applied Materials & Interfaces 2018,10,227-237), and it has been shown that the oxygen content of polystyrene films increases when exposed to air after treatment (Kosobrodova et al., Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 2013, 304, 57-66. 41) The differences in the results observed here may be due to the deep well geometry of the microplate or differences in the type of polystyrene structure (e.g., orientation of phenolic groups) in the microplate compared to the polystyrene film or spin-coated layer.

[0081] Long-lived radicals present in the modified surface layer have been proposed as the primary mechanism underlying the covalent immobilization of biomolecules on PIII surfaces (Bilek et al., Proceedings of the National Academy of Sciences of the United States of America 2011, 108, 14405-14410). The radical density of a sample can be calculated from its EPR intensity by comparing it with that obtained from a standard DPPH sample with known radical density. However, due to differences in shape and volume (DPPH powder was placed in an EPR tube, while PIII and PAC-treated PS strips were attached to an EPR tube), it was not possible to calculate the exact radical density for the samples. Instead, the relative radical densities of the samples were compared by normalizing by the plasma treatment depth, assuming the measurement area was the same for all samples.

[0082] For PIII samples, the processing depth was determined as described in the literature (Gan et al., Nuclear The relative radical density on the treated microplate was found to be significantly increased after 400 and 800 seconds of PIII treatment (0.10 ± 0.001 and 0.24 ± 0.0003, respectively) compared with the UT PS microplate (1.12 × 10-6, Figure 1B) (one-way ANOVA, p < 0.0001). The increase from PIII-400 to PIII-800 was also significant (one-way ANOVA, p < 0.0001).

[0083] Surface wettability is another property known to be affected by PIII treatment. Here, we observed that PIII treatment significantly reduced the water contact angles of PIII-400 (45.5 ± 1.7°) and PIII-800 (37.0 ± 1.9°) treated microplates compared to TC-treated microplates (79.1 ± 0.6°) (one-way ANOVA, p < 0.0001) (Figure 1D). According to the Owens-Wendt-Rabel-Kaelble method, surface energy can be considered to consist of polar and dispersive components. The polar component of the PIII-treated microplate (23.7±0.3 to 30.1±0.3 mJ / m 2 ) was measured using TC-treated microplates (4.8±0.1mJ / m 2 The values ​​of the wettability and polar surface energy components of the PIII-treated microplates were significantly higher than those of the PIII-treated microplates (p<0.0001, one-way ANOVA, p<0.0001) and were found to increase with treatment time (Figure 1E). The values ​​of the wettability and polar surface energy components of the PIII-treated microplates were more strongly correlated with the relative radical densities described above than with the surface nitrogen and oxygen compositions.

[0084] The chemical composition of PAC microplates was determined by XPS, comparing four different gas recipes: no nitrogen, low nitrogen, moderate (mod) nitrogen, and high nitrogen (Figure 1A). Surface nitrogen was found to increase with the nitrogen gas percentage in all PAC recipes. Measurements were for TC (0.6%), no nitrogen PAC (4.0%), low nitrogen PAC (9.7%), moderate nitrogen PAC (13.8%), and high nitrogen PAC (14.6%). Regarding surface oxygen, the no nitrogen PAC-treated microplates had increased surface oxygen (14.9%). All other recipes had similar oxygen contents (8.5–9.4%) to the TC-treated microplates (8.3%). It is important to note that the sampling depth (approximately 10 nm) measured by XPS may include the polystyrene substrate beneath the deposition layer, which reduces the nitrogen percentage.

[0085] There were no significant differences between the relative radical densities of the different PAC-treated samples (Figure 1B, no nitrogen 0.020 ± 0.01, low nitrogen 0.019 ± 0.0008, medium nitrogen 0.016 ± 0.013, high nitrogen 0.021 ± 0.0035). As above, these values ​​were normalized by the thickness of the PAC coating (Figure 1C).

[0086] The water contact angle of the microplates decreased after PAC treatment (Figure 1D). There was no significant difference in water contact angle between low-nitrogen PAC and medium-nitrogen PAC, but the hydrophilicity of the high-nitrogen PAC was significantly higher than that of the no-nitrogen PAC (one-way ANOVA, p<0.0001). Water contact angles ranged from 42.1° to 65.1°. The polar content of the PAC samples also increased with increasing nitrogen content in the PAC recipe.

[0087] Overall, PIII microplates were found to be very hydrophilic, with low surface nitrogen composition and high relative radical density. PAC microplates had high nitrogen composition and moderate relative radical density, indicating greater hydrophilicity than untreated plates. Many measured parameters were found to differ between PIII and PAC-treated microplates. PAC-treated microplates had significantly higher surface nitrogen content than PIII-treated microplates (PIII 0.5-0.8%, PAC 4-14.6%). PIII-treated microplates also had higher relative radical densities (PIII 0.10-0.24, PAC 0.016-0.021), and were more hydrophilic (37.0-45.5°C) compared to all PACs (50.7-65.1°C), except for the high-nitrogen treatment (42.1°C). Relative radical density appeared to correlate better with surface hydrophilicity than surface nitrogen and oxygen composition.

[0088] Example 5 - Optical properties of PIII and PAC modified microplates Because optical reading is the primary method of detection for biochemical plate assays, changes in optical properties can affect the usefulness of plasma-activated microplates. The changes in absorbance and autofluorescence after PIII and PAC treatment of microplates were measured using a PHERAstar FSX (BMG Labtech) and compared with untreated microplates. Absorbance was measured at 450 nm with 22 flashes per well using the top optic. Five optical modules (460 / 510, 485 / 520, 540 / 580, 575 / 620, 635-20 / 680-20) were used to measure fluorescence intensity at five commonly used excitation and emission wavelengths. Each endpoint measurement The top optics was used to scan the center of the well with 10 flashes, the focal height was adjusted for each sample, and the gain was set to 1400.

[0089] The effects of PIII and PAC treatment on absorbance and autofluorescence were characterized (Figure 1F). Absorbance was measured at 450 nm because this wavelength is used in many assays, such as HRP colorimetric assays. The mean absorbance at 450 nm was found to be significantly higher for PIII (0.150–0.207 au) and PAC (0.052–0.056 au) compared with untreated microplates (0.046 ± 0.001 au) (one-way ANOVA, p < 0.0001). The greatest increase in absorbance was observed with PIII-800 (0.207 ± 0.008 au), followed by PIII-400 (0.150 ± 0.010 au). Browning of the transparent microplates with PIII treatment was visible to the naked eye and increased with treatment time. This browning may be due to clustering of carbonized structures in the modified layer, which increase in number and size with increasing treatment time. PIII samples with aluminum had smaller absorbance variations across multiple wells (0.016 au) compared to samples without aluminum (0.030 au), indicating that aluminum modification improved treatment uniformity across the microplate. PAC-treated microplates showed a much smaller increase in absorbance (1.13-1.22-fold) compared to PIII-treated microplates (3.36-4.5-fold), with no significant differences between PAC gas treatments (0.052-0.056 au, one-way ANOVA, p>0.1). Overall, PIII significantly increased absorbance at 450 nm (288%) compared to untreated microplates, while PAC had a relatively more modest increase (17.5%).

[0090] Generally, aromatic polymers have a π to π *The PAC-treated microplates have higher autofluorescence at shorter wavelengths due to their lower transition energy to 460 / 510 and 485 / 520 nm. This is consistent with our observations, where all untreated and treated microplates have higher autofluorescence at shorter wavelengths (460 / 510 and 485 / 520 nm) than at longer wavelengths (540 / 580, 575 / 620, 635 / 680 nm). In addition to this trend, PAC-treated microplates had significantly higher autofluorescence at shorter wavelengths (162–196 nm, one-way ANOVA, p<0.0001) compared to untreated (155–157 nm), and PIII-treated ones had significantly lower autofluorescence (100–144 nm, one-way ANOVA, p<0.0001). With PIII treatment, the aromatic rings are partially amorphized, which explains the lower autofluorescence observed with PIII, as supported by the observation that PIII-800 had lower autofluorescence (100-118) than PIII-400 (126-144). In contrast, at longer wavelengths, the difference in autofluorescence was smaller among all samples. Overall, PAC and PIII treatments altered the autofluorescence of the microplates at shorter wavelengths (510-520 nm), but the changes were smaller at longer wavelengths (580-680 nm). The changes at shorter wavelengths were smaller compared to the total autofluorescence of the polystyrene substrate: an 8% increase for PAC and a 27% decrease for PIII. At longer wavelengths, PAC increased by 9% and PIII decreased by 14% compared to untreated polystyrene.

[0091] Example 6 - Immobilization of DNA oligonucleotides on PIII and PAC modified microplates For DNA binding assay applications, it is important that the immobilized DNA maintains its ability to hybridize with complementary DNA. Therefore, measurements focused on the density of hybridized DNA, which was detected by Alexa-647 fluorophores covalently attached to the hybridized DNA oligonucleotides.

[0092] A linker of 20 additional adenine nucleotides was used to create a 21-nucleotide (nt We designed an ssDNA sequence (IDT DNA) of the linker (Table 3). Under acidic conditions, the amine groups in the adenine nucleotides of the linker were protonated, and therefore, the linker was expected to be preferentially electrostatically attracted to the negatively charged plasma surface compared to the core DNA sequence.

[0093] Wells of untreated, PIII-treated, and PAC-treated 96-well microplates were incubated with 40 μL of 2 μM ssDNA in 10 mM citric acid / sodium citrate buffer at pH 3, 4, 5, or 6 or disodium hydrogen phosphate / sodium dihydrogen phosphate buffer at pH 7 or 8 for 1 hour at room temperature on a shaker. The DNA solution was replaced with 200 μL of 1% BSA in 10 mM PBS (pH 7.4) for 1 hour at room temperature on a shaker to block the remaining active surface. The BSA solution was removed, and the wells were washed three times with 200 μL of 2% SDS with vigorous shaking. After rinsing three times with 200 μL of MilliQ water, DNA hybridization was performed by adding a 21-nt complementary DNA strand (IDT DNA) bearing a 3' Alexa647 fluorophore modification. Complementary DNA was added to hybridization buffer (2 mM magnesium chloride (Sigma-Aldrich), 1x Tris EDTA (Sigma-Aldrich), 1% BSA, and 0.6% SDS) to a final concentration of 0.8 μM. Each well was incubated with 40 μL of 0.8 μM complementary DNA solution on a shaker for 1 hour at room temperature. After removing the solution, the wells were washed three times with 200 μL of 10 mM PBS while vigorously shaking. The wells were then washed three times with 200 μL each of three wash buffers: Wash Buffer 1 (2x saline-sodium citrate (SSC) + 0.6% SDS), Wash Buffer 2 (0.2x SSC + 0.6% SDS), and Wash Buffer 3 (0.1x SSC + 0.5% Tween 20) while vigorously shaking. After rinsing the wells with 200 μL of 10 mM PBS, each well was filled with 40 μL of 10 mM PBS for fluorescence intensity measurements. The fluorescence intensity of Alexa647 modification on complementary DNA was measured using the 635-20 / 680-20 optical module of the PHERAstar FSX. For each measurement, a 10 x 10 matrix of wells with a diameter of 3-5 mm was scanned using the upper optical system with 10 flashes at each scan point. The focal height was adjusted for each sample, and the gain was set to 2000.

[0094] To ensure accurate detection of only DNA hybridized to the covalently immobilized DNA, washing steps were necessary to remove nonspecifically bound DNA. Washing steps were included to remove noncovalently bound ssDNA (step 4, Figure 2A) and nonhybridized ssDNA from the microplate (steps 7–11, Figure 2A). For both the control without immobilized ssDNA (step 2) or the control with the mishybridized DNA sequence (step 6, Figure 2C), washing was found to be effective in reducing the negative control fluorescence signal to baseline levels (t-test, p<0.0001). For example, a 50% reduction from steps 7 to 11 corresponds to the removal of approximately 8.4 × 10 ssDNA from the surface. 10 This is equivalent to removing 100 DNA molecules. Preliminary measurements showed that the density of hybridized DNA was higher in the center of the microplate wells compared to the edges (Figure 2B). The edges of the wells have less efficient fluid mixing due to shadowing and a reduced ratio of sheath area to wall area, and have lower injected ion flow rates, which limit the DNA density near the edges. Therefore, hybridization density measurements were averaged over a 3 mm diameter circle (with a total diameter of 5 mm) in the center of the well.

[0095] DNA hybridization was characterized for all recipes of PIII (PIII-400, PIII-800) and PAC (no nitrogen, low nitrogen, medium nitrogen, and high nitrogen). 10 molecules / cm 2 ), all plasma-treated samples showed higher DNA hybridization density than A large fluorescent signal was observed, which was higher in the PAC-treated microplate than in the PIII-treated microplate (PIII 1.3 × 10 11 molecules / cm 2 , PAC3.4×10 11 molecules / cm 2, one-way ANOVA, p<0.0001) (Figure 2C, Table 2). DNA hybridization was approximately 2-5 times more dense in PAC than in PIII (1.1-1.6 × 10 11 molecules / cm 2 , one-way ANOVA, p<0.0001), the nitrogen-free and low-nitrogen PAC recipes had the highest amount of hybridized DNA overall (4.7–4.8 × 10 11 molecules / cm 2 , one-way ANOVA, p<0.0001). There were no significant differences among the four different PIII surfaces, indicating that aluminum foil and treatment time did not affect DNA immobilization. The effect of pH on DNA immobilization was tested by incubating immobilized ssDNA at pHs between 3 and 8 (step 2, Figure 2A). For almost all PIII- and PAC-treated microplates, the highest hybridization density was observed at pH 3 and 4 (Figure 3A, B). The DNA immobilization capacity of the PIII-treated microplates decreased one month after PIII treatment, but there was no further decrease for up to three months (Figure 3C). All PAC samples maintained the same DNA immobilization capacity for up to three months (Figure 3D). DNA immobilization and hybridization of the nitrogen-free and low-nitrogen PAC samples were further optimized by using freshly prepared hybridization solution and measuring fluorescence intensity from a 3 mm diameter instead of a 5 mm diameter. After further optimization, the average hybridization density for the nitrogen-free and low-nitrogen PAC samples was 5.2 × 10 12 molecule / cm 2 (Figure 4).

[0096] Interestingly, the measured relative radical density was found not to correlate with the amount of DNA hybridized to the surface. The PIII microplate had a relative radical density 5-15 times higher than that of the PAC, but a DNA hybridization capacity 2-5 times lower. This indicates that the low amount of radicals on the PAC surface was sufficient for high-density DNA immobilization.

[0097] [Table 2]

[0098] The PAC surface has a higher DNA density than PIII, which is explained by the higher nitrogen composition that is protonated on the PAC surface, which results in more favorable electrostatic interactions. The highest DNA density was found on the PAC surface, which has a lower surface nitrogen composition. Ta.

[0099] Surface hydrophilicity is another important factor that can affect intermolecular interactions with DNA. Both hydrophilic and hydrophobic surfaces can attract DNA. Hydrophobic surfaces can attract nearby DNA through short-range vdW forces, while hydrophilic surfaces can attract DNA over longer distances through dipole-dipole and hydrogen bonds. However, hydrophilic surfaces tend to form a dense hydration layer, which competes with the intermolecular attraction between the surface and DNA via hydrogen bonds. Therefore, the higher DNA density on the no-nitrogen and low-nitrogen PAC surfaces compared to the other samples can be explained by their relative hydrophobicity compared to the medium- and high-nitrogen PACs and all PIII conditions, which minimizes the formation of a hydration layer and maximizes vdW forces.

[0100] Example 7 - Immobilization of streptavidin on PIII and PAC modified microplates Streptavidin immobilized on plasma-treated surfaces was detected directly by the fluorescence of covalently modified Cy3 streptavidin. In the case of PAC surfaces, it was also detected indirectly by binding to dual-biotin- and Alexa647-modified DNA strands. The former quantifies immobilized protein, while the latter detects only active protein on the surface.

[0101] Wells of PIII-treated, PAC-treated, and untreated microplates were incubated with 40 μL of 10 μg / mL streptavidin-Cy3 (Sigma-Aldrich, P6402) in 10 mM citric acid / sodium citrate buffer (pH 3, 4, 5, or 6) or disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7 or 8) on a shaker at room temperature for 1 hour. The streptavidin solution was replaced with 200 μL of 1% BSA in 10 mM PBS (pH 7.4) for 1 hour on a shaker at room temperature to block the remaining active surface. The BSA solution was removed, and the wells were washed three times with 200 μL of 10 mM PBS. After rinsing, each well was washed three times with 200 μL of 10% Triton in 10 mM PBS. The wells were then rinsed three more times with 200 μL of 10 mM PBS and then incubated with 2 μM 5'-biotin-modified ssDNA (IDT DNA, Table 3) in 10 mM PBS (pH 7.4) on a shaker for 1 hour at room temperature. After biotin-DNA incubation, the wells were incubated with Alexa647-modified complementary DNA as described in the DNA immobilization method. The hybridized surface was washed three times with 200 μL of wash buffer 4 (2×SSC + 0.05% Tween 20) and then rinsed with 10 mM PBS. The immobilized streptavidin-Cy3 and hybridized complementary Alexa647-DNA were detected using a PHERAstar FSX fluorescent plate reader equipped with optical modules 540 / 580 and 635-20 / 680-20, respectively. For each measurement, a 10 × 10 matrix of wells with diameters of 3–5 mm was scanned with 10 flashes at each scan point using the top optics. The focal height was adjusted for each sample, and the gain was set to 1000.

[0102] [Table 3]

[0103] Similar to DNA, streptavidin was also found to bind more on PAC-treated microplates than on PIII-treated microplates (Figure 5A), and all plasma treatment conditions were significantly increased over the untreated control (TC-treated 0.01 × 10 11 molecules / cm 2 , PIII0.7×10 11 molecules / cm 2 , PAC4.9×10 11 molecules / cm 2 , one-way ANOVA, p<0.0001). In contrast to DNA, low-nitrogen PAC had the highest amount of bound streptavidin (2.2 × 10 11 molecules / cm 2 , and 9.0 × 10 for low nitrogen. 11 molecules / cm 2 , and 4.3 × 10 for medium nitrogen. 11 molecules / cm 2 , and 4.0 × 10 for high nitrogen. 11 molecules / cm 2 ), whereas for DNA, both low-nitrogen PAC and nitrogen-free PAC were similarly high. This trend of PAC was reproduced with biotin-DNA binding, indicating that the immobilized protein maintained activity (Figure 5B, nitrogen-free × 10 11 pcs, low nitrogen 12.2 x 10 11 pcs, medium nitrogen 7.3 x 10 11 7.8 x 10 high nitrogen 11 biotin-DNA molecules / cm 2). Calculations indicated that there were an average of 2.14 hybridized DNA-biotin molecules bound to each immobilized streptavidin on the surface. The best PIII surface for streptavidin immobilization was PIII-400 with aluminum, but it had approximately 10-fold less streptavidin than the best PAC condition. The immobilization pH affected the amount of streptavidin immobilized on PIII and PAC microplates. For PIII microplates, optimal immobilization was achieved at pH 5 (Figure 5C). For PAC microplates, optimal streptavidin immobilization was achieved at pH 5 for nitrogen-free PAC and at pH 4–7 for the other PAC recipes (Figure 5D). Streptavidin immobilization significantly decreased after 1 month in all PIII samples (Figure 5E). However, the amount of immobilized streptavidin did not change significantly in PAC samples aged up to 3 months (Figure 5F).

[0104] Example 8 - Comparison of PIII and PAC modified microplates PAC surfaces have more nitrogen and radical density than PIII surfaces, are more hydrophobic, and were found to be stable for over 3 months. Optimal conditions for high-density DNA (PAC, 0% or 21% nitrogen, pH 3-4) and streptavidin (PAC, 21% nitrogen, pH 5-7) binding were obtained. PAC-activated microplates enable high-density covalent immobilization of functional DNA and proteins in a single step without specific linker chemistry.

[0105] Example 9 - Preventing the accumulation of plasma-generated nanoparticles We observed that plasma-generated nanoparticles accumulated in the PAC-coated 24-well plate. To prevent the plasma-generated nanoparticles from falling into the wells, we modified the sample holder configuration. Plasma treatment of the 24-well plate was performed using the following three conditions, as shown in Figure 6. Polymer mesh: square mesh with three sizes (3, 4, and 5 mm spacing) The tubes were modified from tissue culture plate lids using laser cutting and fitted to the well plates during plasma treatment. Conductive mesh: Stainless steel mesh was cut to cover the plate in three 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). The mesh was made of stainless steel wire with square gaps ranging in size from 6 mm to 0.45 mm. The mesh was secured to the top of the plate using a polymer fitting cut from the lid of a tissue culture plate. The stainless steel mesh was not electrically connected to the sample holder. Faraday cage with mesh lid: A Faraday cage with mesh lid consists of three 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, aluminum, etc.) and is sized to accommodate the plate or dish to be coated. The cap, also made of 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., the mesh lid is sandwiched between the cap and the open container). This creates a Faraday cage around the plate or dish. Using the same sample holder and other parameters, three mesh sizes (4 mesh, 10 mesh, and 50 mesh) were investigated for the effects of plasma treatment. The configuration and size of the Faraday cage suitable for securing the plate are shown in Figure 7.

[0106] Plasma treatment was performed using a custom-built three-chamber system (Figure 8). Culture plates (Corning Costar, 24-cell tissue culture plates) were placed on a metal plate or on a sample holder in a Faraday cage with a mesh lid. 5 × 10 -5The system was evacuated until a substrate pressure of 100 Torr was reached. The plasma treatment consisted of two steps: argon plasma surface activation and plasma deposition. Both steps utilized plasma generated by an RF power supply, set at 75 W for surface activation and 50 W for deposition. A 500 V pulse negative bias at 3 kHz and 20 μs duration was applied to the sample holder. During the surface activation step, the argon pressure was adjusted within the range of 70–80 mTorr. During the deposition step, a gas mixture containing acetylene, nitrogen, and argon was maintained at 110 mTorr with flow rates of 1, 3, and 10 sccm, respectively. Surface activation was performed for 2, 5, and 10 minutes, and the duration of the deposition step was fixed at 10 minutes.

[0107] Silicon wafers and glass cover slips placed inside the wells were used to measure PAC thickness and quantify nanoparticles. Spectroscopic ellipsometry (JA Woollam M2000) was used to determine PAC thickness, and nanoparticle counts were performed using an Olympus BMX-10 optical microscope. Because the nanoscale size of plasma particles and contaminant dust makes their identification by optical microscopy difficult, further verification was performed using a flow cytometer and a scanning electron microscope (SEM). Zeiss Sigma HD SEM images were taken of silicon wafers at 1000x and 5000x magnifications using an SEM. To reduce surface charging and enable imaging, the samples were coated with a 5-nm-thick conductive gold layer. An in-lens detector with a working distance ranging from 3 to 5 mm was used in the SEM imaging process. The field emission gun was set at 5 kV to facilitate the imaging process.

[0108] The use of mesh during plasma treatment reduced nanoparticle counts. The use of polymer mesh resulted in a slight reduction in particles. Conductive mesh is more effective at preventing particles than polymer mesh, especially when the mesh spacing size is smaller. A Faraday cage with a 50 mesh lid exhibits particle counts comparable to the untreated control plate (Table 1).

[0109] [Table 4]

[0110] The number of particles in the solution was counted using a Cytek Aurora flow cytometer (USA). 200 ml of Milli-Q water was added to the wells of plasma-treated 24-well plates with and without a mesh, and nanoparticles were removed using four methods with increasing levels of shear force, as described below. · Distiller: Gently add water to remove particles. Pipette: Use a pipette to draw and release water several times to remove particles. Rocker: Add water to the sample and leave on a rocker for 5 minutes to remove particles. · Sonication: High power sonication is applied for 5 minutes to remove particles.

[0111] The nanoparticle-containing solution was then collected and analyzed using flow cytometry (Cytek Aurora, USA). A stop volume of 15 μl was set for each sample, and the number of detected nanoparticles was recorded by FlowJo.

[0112] Figure 9 compares the number of nanoparticles detected in 15 μl of liquid using a flow cytometer and shows that plasma treatment using a Faraday cage with a 50-mesh lid significantly reduces particle counts compared to conventional treatment without a mesh. The high level of nanoparticles in the liquid sample removed from the untreated plate without a mesh was also observed. The density indicates that these nanoparticles are loosely bound to the PAC and can be easily removed by low shear forces such as distillation.

[0113] The nanoparticles formed on the coating were visualized in SEM images (Figure 10). They are present as single particles or clusters of particles on the coating obtained by plasma treatment without a mesh. In contrast, when a Faraday cage with a 50-mesh lid was used, very few nanoparticles were found on the clear, smooth coating.

[0114] The coating thickness was determined by placing a silicon wafer inside the well and measuring the PAC deposited on the silicon surface using spectroscopic ellipsometry. Figure 11 compares the PAC thickness when the silicon wafer was placed at various positions on the plate. Coating thickness is generally thicker when using a stainless steel mesh than when using a polymer mesh, especially as the mesh size decreases. Because the polymer mesh cannot eliminate the charge on the polymer mesh, it is hypothesized that the positive charge increases over time, reducing the electric field generated by the negative bias and therefore the force that draws ions from the plasma to create the coating. This is less of an issue with the stainless steel mesh because the charge is eliminated.

[0115] Figure 12 shows a comprehensive comparison of PAC thickness between treatments without a mesh and treatments using a Faraday cage with a 50-mesh lid. Without a mesh, the PAC thickness was measured to be 1.13 ± 0.35 nm. However, with a mesh, the PAC thickness increased to 1.89 ± 0.54 nm.

[0116] Example 10 - Comparison of cell culture in plasma-treated wells The effect of plasma treatment on the differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs) was investigated in 24-well plates that were untreated (UT), plasma-treated without a mesh (-Mesh), and plasma-treated in a Faraday cage with a 50-mesh lid (+Mesh) and then coated with laminin 521 (LN521).

[0117] Briefly, laminin 521 (LN521) was incubated in PBS (Ca 2+ and Mg 2+ The solution was diluted to a concentration of 10 μg / mL in mTeSR Plus basal medium (containing 5X Supplement) and 200 μL was added to each well of the UT, -Mesh, and +Mesh treatments. After overnight incubation at 4°C, the wells were washed. Human induced pluripotent stem cells (iPSCs) (Gibco Episomal iPSC Line; A18945) cultured on Matrigel in a 6-well plate (Corning Costar) in mTeSR Plus basal medium (STEMCELL, #100-0276) supplemented with Plus 5X Supplement were passaged to approximately 3 × 10 5 The cells were plated at a density of 10 cells / well. The day after seeding (day -1), Plus Fresh mTeSR Plus basal medium containing 5X supplement was added to dilute the ROCK inhibitor. The next day (day 0), CM differentiation medium A (STEMCELL, #05010) was added for 2 days. Differentiation medium B (STEMCELL, #05010) was added on day 2, followed by differentiation medium C (STEMCELL, #05010) on days 4 and 6. Finally, the differentiation medium was replaced with Cardiomyocyte Maintenance Basal Medium (STEMCELL, #05010) on day 8 and continuously replenished every 2 days.

[0118] The progression of iPSCs to beating cardiomyocytes (CMs) was measured using a Nikon microscope equipped with a 4x objective. The amount of beating colonies as a percentage of the visible area in the eyepiece was recorded at nine different locations and averaged to estimate the relative percentage of the total wells containing beating colonies. Each sample was run in triplicate, yielding n = 3 data points, and analyzed using GraphPad (GraphPad Software, version 9.5.1, San Digest). ego, California).

[0119] Figure 13 shows that +Mesh provides a stable surface on which iPSCs can grow and differentiate into CMs. +Mesh outperforms -Mesh, which in turn outperforms UT. This is hypothesized to be due to an increased number of nanoparticles in the UT and -Mesh-treated wells that are laminin-bound and washed away during medium changes, exposing areas without laminin coating. In contrast, +Mesh wells have a uniform PAC coating with covalently bound laminin and fewer nanoparticles.

Claims

1. 1. A method of coating a plastic surface, said method comprising: (a) treating the plastic surface with ions under a pulsed bias; (b) depositing plasma on the plastic surface by plasma-enhanced chemical vapor deposition (PECVD) of a gas mixture comprising acetylene and argon under a pulsed bias; A method in which a mesh is placed on the plastic surface, whereby the plasma passes through the mesh before being deposited on the plastic surface, and the method produces a plasma activated coating (PAC) on the plastic surface, whereby the PAC covalently bonds to biomolecules.

2. The method of claim 1 , wherein the ions are argon ions.

3. The method of claim 1 or 2, wherein the gas mixture further comprises nitrogen.

4. 4. The method of claim 1, wherein the atmospheric proportion of nitrogen in the gas mixture is less than about 35%.

5. 5. The method according to claim 1, wherein the atmospheric proportion of nitrogen in the gas mixture is about 21%.

6. The method of any one of claims 1 to 5, wherein the treatment with ions is carried out at an RF power of about 75 W and a negative pulsed bias voltage of about 500 V for about 2 minutes.

7. The method of any one of claims 1 to 6, wherein the plasma deposition is carried out at a plasma discharge of about 50 W and a negative pulsed bias voltage of about 500 V for about 10 to about 30 minutes.

8. 8. The method of claim 1, wherein the negative pulsed bias is applied at a frequency of about 3 kHz and a pulse duration of about 20 μs.

9. The method of any one of claims 1 to 8, wherein the mesh is formed from a conductive metal.

10. 10. The method of claim 9, wherein the mesh forms the lid of a Faraday cage surrounding the plastic surface.

11. 11. The method of claim 9 or 10, wherein the conductive metal is stainless steel.

12. The method according to any one of claims 1 to 11, wherein the plastic surface has a non-flat topography.

13. The method of any one of claims 1 to 12, wherein the plastic surface is a microplate well or a Petri dish.

14. The method of any one of claims 1 to 13, wherein the plastic is polystyrene.

15. The method according to any one of claims 1 to 14, wherein the biomolecule is a nucleic acid or a protein.

16. The PAC comprises: a surface nitrogen concentration of about 4 to about 14%; a relative radical density of about 0.016 to about 0.021; an average absorbance at 450 nm of about 0.052 to about 0.056 a.u.; a thickness of about 1 nm to about 10 nm; Approximately 2×10 １１ ~Approx. 5×10 １１ molecules / cm ２ hybridizing DNA at a concentration of Approximately 2×10 １１ ~Approx. 9×10 １１ molecules / cm ２ Immobilizing streptavidin at a concentration of 16. The method of any one of claims 1 to 15, having properties selected from one or more of:

17. A plastic surface coated with a plasma activated coating (PAC), said PAC covalently binding biomolecules and comprising: a surface nitrogen concentration of about 4 to about 14%; a relative radical density of about 0.016 to about 0.021; an average absorbance at 450 nm of about 0.052 to about 0.056 a.u.; a thickness of about 1 nm to about 10 nm; Approximately 2×10 １１ ~Approx. 5×10 １１ molecules / cm ２ hybridizing DNA at a concentration of Approximately 2×10 １１ ~Approx. 9×10 １１ molecules / cm ２ Immobilizing streptavidin at a concentration of A plastic surface having properties selected from one or more of:

Images