JP2025528189A - Methods for covalently attaching biomolecules to plastics - Google Patents

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

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

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

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

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.

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%.

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.

Plasma immersion ion implantation (PIII) has previously been used to immobilize biomolecules on plastics (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. Although this method is fast and reproducible and the reactivity of these surfaces is maintained over time, it is not suitable for treating microplate wells because shadowing caused by the well walls leads to non-uniform reactivity of the plastic surface.

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 depositing sprayed proteins such as collagen directly 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.

Therefore, a rapid and reproducible method for covalently binding biomolecules to microplates over long periods of time is needed.

The object of the present invention is to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Surprisingly, it has been discovered that plasma-enhanced chemical vapor deposition (PECVD) using a carbon-containing gas mixture under pulsed bias produces plasma-activated coatings (PACs) on plastic surfaces that 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 range of reactivity is significantly improved compared to other plasma activation methods (e.g., PIII), making it suitable for use in Petri dishes and microplate wells.

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 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 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, the method producing a plasma activated coating (PAC) on the plastic surface, and the PAC covalently bonding to biomolecules.

In one embodiment, the ions are derived from a noble gas.

In one embodiment, the ions are argon ions.

In one embodiment, the gas mixture further comprises nitrogen.

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

In one embodiment, the atmospheric percentage of nitrogen in the gas mixture is approximately 21%.

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

In one embodiment, the ion treatment is performed at an RF power of about 50 to about 100 W and a negative pulsed bias voltage of about 300 to about 700 V for about 1 to about 15 minutes.

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

In one embodiment, plasma deposition is performed 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.

In one embodiment, plasma deposition is performed 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.

In one embodiment, plasma deposition is performed 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.

In one embodiment, the gas mixture is maintained at approximately 110 mTorr.

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.

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

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

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

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

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

In one embodiment, the conductive metal is stainless steel.

In one embodiment, the plastic surface has a non-flat shape.

In one embodiment, the plastic surface is a microplate well or a Petri dish.

In one embodiment, the plastic is polystyrene.

In one embodiment, the biomolecule is a nucleic acid.

In one embodiment, the biomolecule is a protein.

In one embodiment, the protein is streptavidin or laminin.

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

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

In one embodiment, the PAC has an average absorbance at 450 nm of about 0.052 to about 0.056 a.u.

In one embodiment, the PAC has a thickness of approximately 20 nm.

In one embodiment, the PAC hybridizes DNA at a concentration of about 2×10 ¹¹ to about 5×10 ¹¹ molecules/cm ² .

In one embodiment, the PAC immobilizes streptavidin at a concentration of about 2×10 ¹¹ to about 9×10 ¹¹ molecules/cm ² .

In one embodiment, the Faraday cage with 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.

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.

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%.

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.

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 a.u.

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.

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.

In another aspect, the present invention provides a plastic surface coated with a plasma activated coating (PAC), wherein the PAC hybridizes DNA at a concentration of about 2 x ¹⁰ to about 5 x ¹⁰ molecules/cm ^.

In another aspect, the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC has immobilized streptavidin at a concentration of about 2×10 ¹¹ to about 9×10 ¹¹ molecules/cm ² .

In another aspect, the present invention provides a plastic surface coated with a plasma-activated coating (PAC), wherein the PAC is bound to laminin.

(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 the surface free energy of UT-, TC-, PIII-, and PAC-treated PS microplates calculated from the Owens-Wendt-Rabel-Kaelble method. Error bars represent standard error. (f) Mean 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. (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 the surface free energy of UT-, TC-, PIII-, and PAC-treated PS microplates calculated from the Owens-Wendt-Rabel-Kaelble method. Error bars represent standard error. (f) Mean 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. (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 the surface free energy of UT-, TC-, PIII-, and PAC-treated PS microplates calculated from the Owens-Wendt-Rabel-Kaelble method. Error bars represent standard error. (f) Mean 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. (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 Alexa 647 fluorescence intensity of immobilized (pH 4) and hybridized DNA on TC-, PIII-, and PAC-treated microplates. (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 Alexa 647 fluorescence intensity of immobilized (pH 4) and hybridized DNA on TC-, PIII-, and PAC-treated microplates. (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 Alexa 647 fluorescence intensity of immobilized (pH 4) and hybridized DNA on TC-, PIII-, and PAC-treated microplates. (a) DNA immobilization and hybridization on PIII-treated microplates at pH 3-8. (b) DNA immobilization and hybridization on PAC-treated microplates at pH 3-8. (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. (a) DNA immobilization and hybridization on PIII-treated microplates at pH 3-8. (b) DNA immobilization and hybridization on PAC-treated microplates at pH 3-8. (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. DNA immobilization density on PIII- and PAC-treated microplates. Cy3-labeled DNA (20xA-GCTCTGCAATCAACTTATCCC-Cy3) was immobilized on plasma-treated microplates, and the Cy3 intensity was measured after a washing step. The trend of the immobilized DNA density is similar to that of the hybridized DNA density. This indicates that the decrease in hybridization density in some samples is not caused by overcrowding of immobilized DNA on the surface. 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. 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. 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. Three different conditions, each with three mesh sizes, used for plasma treatment to reduce nanoparticle deposition in cell culture plate wells. Top view (A), bottom view (B), and dimensioned schematic (C) of the Faraday cage plate holder. FIG. 1 shows a plasma processing chamber containing a plate to be plasma processed. Nanoparticle counting using flow cytometry. SEM images of silicon wafers in a plasma-treated 24-well plate without a mesh (A, B) and with a Faraday cage with a 50-mesh lid (C, D). Images were taken at 1000x magnification (A, C) and 5000x magnification (B, D). PAC thickness measured on silicon wafers placed in various wells and plasma deposited using different processing conditions. 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. Comparison of three plates showing the surface area coverage of beating colonies observed in each well. 24-well plates were 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.

DEFINITIONS 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.

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

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, such as non-human primates, horses, cows, dogs, etc.

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

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.

Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the word "about."

Reciting numerical ranges using endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

The term "a.u." stands for absorbance units.

The term "biomolecule" means a molecule found in living organisms, such as amino acids, lipids, carbohydrates, proteins, polysaccharides, and nucleic acids.

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.

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.

The term "non-planar shape" refers to structures that are not completely flat 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.

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

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

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 obvious to those skilled in the art, and the present invention is intended to cover all such modifications and equivalents.

The present invention is further illustrated by the following non-limiting examples.

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 a PAC stainless steel holder, a 2-3 mm gap existed between the holder and the bottom of the wells. 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 miniature prototype plasma reactor and reassembled with tape for DNA and streptavidin immobilization and fluorescence detection.

Example 2 - PIII Treatment. PIII treatment was performed by generating a plasma using 13.56 MHz inductively coupled radio frequency (RF) power (ENI radio frequency power generator) and applying a negative voltage bias to a stainless steel sample holder to accelerate positive ions toward the sample. The pressure inside the chamber was evacuated to less than 5 × ^10-5 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. A microplate with or without aluminum foil was taped onto the stainless steel holder, 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 of 20 μs duration at a frequency of 50 Hz. Microplates were treated for 400 or 800 seconds (PIII400 or PIII800).

Example 3 - PAC Treatment In a separate plasma system, PAC treatment was performed using 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 an air gap between the sample holder and the plate and enhance the accelerating electric field on the plate surface. The chamber was evacuated to less than 5 × ¹⁰ Torr. Prior to plasma coating, the microplate was activated with argon ions for 2 minutes at 75 W RF power and 500 V applied bias 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 during coating deposition was maintained at 110 mTorr. Plasma deposition was performed for 10 or 30 minutes with a plasma discharge of 50 W and a negative bias voltage of 500 V. The negative bias from 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, a separate PAC process was performed on smooth silicon wafers with a native oxide layer.

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

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 with various PAC recipes as shown in Table 1. Ellipsometry data were collected at three angles of incidence (65°, 70°, and 75°) using a J. A. Woollam M2000V spectroscopic ellipsometer. The data were fitted using a model consisting of a Cauchy layer representing the silicon substrate, a silicon oxide layer (2 nm), and the PAC layer to obtain the film thickness.

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. Surface free energy was calculated using the Owens-Wendt-Rabel-Kaelble method.

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. The polystyrene film was used as a substitute for microplates that did not fit into 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 measured for each sample. Similar measurements were applied to 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.

Surface chemical changes following PIII and PAC treatment of microplates were determined by comparing 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 consisted of 128 scans at 4 ^cm resolution. For comparison, the spectra were normalized to the peak intensity of polystyrene at 1490 ^cm .

The chemical composition of PIII-treated microplates was determined by XPS and compared after 400 seconds (PIII-400) and 800 seconds (PIII-800) of treatment (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 microplates or differences in the type of polystyrene structure (e.g., orientation of phenolic groups) of the microplates compared to polystyrene films or spin-coated layers.

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 the radicals in the sample can be calculated from the EPR intensity by comparing it with the EPR intensity 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 the 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 them by the plasma treatment depth, assuming the measurement area was the same for all samples.

For PIII samples, the treatment depth was determined as described in the literature (Gan et al., Nuclear
The relative radical density on the treated microplates 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 to 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).

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 with TC-treated microplates (79.1 ± 0.6°) (one-way ANOVA, p < 0.0001) (Fig. 1D). According to the Owens-Wendt-Rabel-Kaelble method, surface energy is considered to consist of polar and dispersive components. The polar component of PIII-treated microplates (23.7 ± 0.3 to 30.1 ± 0.3 mJ/ ^m ) was significantly higher than that of TC-treated microplates ( ^4.8 ± 0.1 mJ/m , one-way ANOVA, p < 0.0001) and increased with treatment time (Fig. 1E). The wettability and polar surface energy component values of PIII-treated microplates were more strongly correlated with the relative radical densities described above than were the surface nitrogen and oxygen compositions.

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 for all PAC recipes. Measured values were 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 measured by XPS (approximately 10 nm) may include the polystyrene substrate beneath the deposition layer, which reduces the nitrogen percentage.

There were no significant differences in relative radical densities for 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). These values were normalized by the PAC coating thickness, as described above (Figure 1C).

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 and medium-nitrogen PACs, but the hydrophilicity of the high-nitrogen PACs was significantly higher than that of the no-nitrogen PACs (one-way ANOVA, p<0.0001). The 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.

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, making them more hydrophilic 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%), and PIII-treated microplates had higher relative radical density (PIII 0.10-0.24, PAC 0.016-0.021), and were more hydrophilic (37.0-45.5°) compared to all PACs (50.7-65.1°), except for the high-nitrogen treatment (42.1°). Relative radical density appeared to correlate more closely with surface hydrophilicity than surface nitrogen and oxygen composition.

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 to untreated microplates. Absorbance was measured at 450 nm with 22 flashes per well using top optics. Fluorescence intensity was measured at five commonly used excitation and emission wavelengths using five optical modules (460/510, 485/520, 540/580, 575/620, 635-20/680-20). For each endpoint measurement, the center of the well was scanned with 10 flashes using the top optics; the focal height was adjusted for each sample; and the gain was set to 1400.

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 the HRP colorimetric assay. The mean absorbance at 450 nm was found to be significantly higher with PIII (0.150-0.207 a.u.) and PAC (0.052-0.056 a.u.) compared with untreated microplates (0.046 ± 0.001 a.u.) (one-way ANOVA, p<0.0001). The greatest increase in absorbance was observed with PIII-800 (0.207 ± 0.008 a.u.), followed by PIII-400 (0.150 ± 0.010 a.u.). 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 wells (0.016 a.u.) compared to samples without aluminum (0.030 a.u.), indicating that aluminum modification improved treatment uniformity across the microplate. PAC-treated microplates experienced much smaller increases 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 a.u., 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%).

In general, aromatic polymers have higher autofluorescence at shorter wavelengths due to lower π to π ^* transition energy. This is consistent with the observations herein, 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 small 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.

Example 6 - Immobilization of DNA Oligonucleotides on PIII and PAC Modified Microplates In 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, as detected by Alexa-647 fluorophores covalently attached to hybridized DNA oligonucleotides.

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.

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 a 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 the 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 measurement. 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 10x10 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.

To ensure accurate detection of only DNA hybridized to the covalently immobilized DNA, wash steps were necessary to remove nonspecifically bound DNA. Wash 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 controls without immobilized ssDNA (step 2) or with added mishybridizing DNA sequences (step 6, Figure 2C), washes were found to be effective in reducing the fluorescent signal of the negative controls to baseline levels (t-test, p<0.0001). For example, a 50% reduction from steps 7 to 11 equates to the removal of approximately 8.4 × ¹⁰ DNA molecules from the surface. Preliminary measurements indicated 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 and lower injected ion flux due to shadowing and a reduced ratio of sheath area to wall area, which limits 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.

DNA hybridization was characterized for all recipes of PIII (PIII-400, PIII-800) and PAC (no nitrogen, low nitrogen, medium nitrogen, and high nitrogen). Compared with the negative control (<3.1 × ¹⁰ molecules/ ^cm ), larger fluorescent signals, indicating higher DNA hybridization density, were observed in all plasma-treated samples, and were higher in PAC-treated microplates than in PIII-treated microplates (PIII 1.3 × ¹⁰ molecules/ ^cm , PAC 3.4 × ¹⁰ molecules/ ^cm , one-way ANOVA, p < 0.0001) (Figure 2C, Table 2). DNA hybridization was approximately 2-5 times denser on PAC compared to PIII (1.1-1.6 x ¹⁰ molecules/ ^cm² , one-way ANOVA, p<0.0001), with the nitrogen-free and low-nitrogen PAC recipes having the highest overall amounts of hybridized DNA (4.7-4.8 x ¹⁰ molecules/ ^cm² , one-way ANOVA, p<0.0001). There were no significant differences among the four different PIII surfaces, indicating that aluminum foil and treatment time do not affect DNA immobilization. The effect of pH on DNA immobilization was tested by incubating immobilized ssDNA at pH 3-8 (step 2, Figure 2A). For almost all PIII- and PAC-treated microplates, the highest hybridization densities were observed at pH 3 and 4 (Figures 3A and 3B). The DNA immobilization capacity of the PIII-treated microplates decreased one month after PIII treatment, but did not decrease further 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 no-nitrogen 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 no-nitrogen and low-nitrogen PAC samples increased to 5.2 × 10 ¹² molecules/cm ² (Figure 4).

Interestingly, the measured relative radical density was found not to correlate with the amount of DNA hybridized to the surface. PIII microplates had a relative radical density 5-15 times higher than 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.

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, resulting in more favorable electrostatic interactions. The highest DNA density was found on the PAC surface with the lower surface nitrogen composition.

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 ranges through dipole-dipole and hydrogen bonding. However, hydrophilic surfaces tend to form a dense hydration layer, which competes with the intermolecular attraction between the surface and DNA via hydrogen bonding. 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 hydration layer formation and maximizes vdW forces.

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-modified Alexa647-modified DNA strands. The former quantifies immobilized protein, while the latter detects only active protein on the surface.

PIII-treated, PAC-treated, and untreated microplate wells 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 for 1 hour at room temperature. The streptavidin 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 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 (2x 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 10x10 matrix of 3-5 mm well diameters was scanned using the top optics with 10 flashes at each scan point. The focal height was adjusted for each sample and the gain was set to 1000.

Similar to DNA, streptavidin was also found to bind more on PAC-treated microplates than on PIII-treated microplates (Figure 5A), with all plasma treatment conditions showing a significant increase over untreated controls (TC-treated ^0.01x1011 molecules/ ^cm2 , PIII ^0.7x1011 molecules/ ^cm2 , PAC ^4.9x1011 molecules/ ^cm2 ; one-way ANOVA, p<0.0001). In contrast to DNA, low-nitrogen PAC had the highest amount of bound streptavidin (no nitrogen ^2.2x1011 molecules/ ^cm2 , low nitrogen ^9.0x1011 molecules/ ^cm2 , medium nitrogen ^4.3x1011 molecules/ ^cm2 , high nitrogen ^4.0x1011 molecules/ ^cm2 ), although DNA was similarly high for both low- and no-nitrogen PACs. This PAC trend was reproduced with biotin-DNA conjugation, indicating that the immobilized protein maintained activity (Figure 5B; no nitrogen × 10 ¹¹ , low nitrogen 12.2 × 10 ¹¹ , medium nitrogen 7.3 × 10 ¹¹ , and high nitrogen 7.8 × 10 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 compared to the best PAC condition. The pH of immobilization affected the amount of streptavidin immobilized on PIII and PAC microplates. For PIII microplates, optimal immobilization was achieved at pH 5 (Figure 5C). In 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 up to 3 months old (Figure 5F).

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 over three 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 allow for high-density covalent immobilization of functional DNA and proteins in a single step without specific linker chemistry.

Example 9 - Prevention of Accumulation of Plasma-Generated Nanoparticles Plasma-generated nanoparticles were observed to accumulate in PAC-coated 24-well plates. To prevent the plasma-generated nanoparticles from falling into the wells, the sample holder configuration was modified. Plasma treatment of the 24-well plate was performed using the following three conditions, as shown in Figure 6.
Polymer mesh: Square meshes with three sizes (3, 4, and 5 mm spacing) were modified from tissue culture plate lids using laser cutting and fitted into 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 polymer fittings cut from tissue culture plate lids. 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 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 were investigated for the effects of plasma treatment: 4 mesh, 10 mesh, and 50 mesh. The configuration and size of the Faraday cage suitable for securing the plate is shown in Figure 7.

Plasma treatment was performed using a custom-built three-chamber system (Figure 8). Culture plates (Corning Costar, 24-well tissue culture plates) were placed on a metal plate or on a sample holder in a Faraday cage with a mesh lid. The system was evacuated until a substrate pressure of 5 × ^10-5 Torr was reached. Plasma treatment consisted of two steps: surface activation with argon plasma 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.

Silicon wafers and glass cover slips placed inside the wells were used to measure the thickness of the PAC and quantify the nanoparticles. Spectroscopic ellipsometry (J.A. Woollam M2000) was used to determine the thickness of the PAC, and an Olympus BMX-10 optical microscope was used to count the number of nanoparticles. 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 conductive gold layer with a thickness of 5 nm. The SEM imaging process used an in-lens detector with a working distance ranging from 3 to 5 mm. The field emission gun was set at 5 kV to facilitate the imaging process.

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 an untreated control plate (Table 1).

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 a plasma-treated 24-well plate, both with and without a mesh, and nanoparticles were removed using four methods with increasing levels of shear force, as described below.
Distiller: Add water gently 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.

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.

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 density of nanoparticles in the liquid sample removed from the untreated plate without a mesh indicates that these nanoparticles are loosely bound to the PAC and can be easily removed by low shear forces, such as distillation.

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 with 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.

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 greater using stainless steel mesh compared to polymer mesh, especially as the mesh size decreases. It is hypothesized that because the polymer mesh cannot eliminate the charge present on it, 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 stainless steel mesh, as the charge is eliminated.

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 the mesh, the PAC thickness increased to 1.89 ± 0.54 nm.

Example 10 - Comparison of Cell Culture in Plasma-Treated Wells The effect of plasma treatment on differentiation of induced pluripotent stem cells (iPSCs) to 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).

Briefly, laminin 521 (LN521) was diluted to a concentration of 10 μg/mL in PBS (containing Ca ²⁺ and Mg ²⁺ ), 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 and plated at a density of approximately 3 × 10 ⁵ cells/well. The day after seeding (day -1), Plus 5X Supplement was added to the mTeSR Plus basal medium (STEMCELL, #100-0276).
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.

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 well 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 Diego, CA).
The data were plotted on a computer program (Ego, California).

Figure 13 shows that +Mesh provides a stable surface on which iPSCs can grow and differentiate into CMs. +Mesh is superior to -Mesh, which in turn is superior to UT. This is hypothesized to be due to an increased number of laminin-bound nanoparticles in UT and -Mesh-treated wells, which are 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 (17)

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. The method of claim 1, wherein the ions are argon ions. The method of claim 1 or 2, wherein the gas mixture further contains nitrogen. The method of any one of claims 1 to 3, wherein the atmospheric proportion of nitrogen in the gas mixture is less than about 35%. The method of any one of claims 1 to 4, wherein the atmospheric proportion of nitrogen in the gas mixture is approximately 21%. The method of any one of claims 1 to 5, wherein the treatment with ions is performed for about 2 minutes at an RF power of about 75 W and a negative pulse bias voltage of about 500 V. The method of any one of claims 1 to 6, wherein the plasma deposition is carried out for about 10 to about 30 minutes with a plasma discharge of about 50 W and a negative pulse bias voltage of about 500 V. The method of any one of claims 1 to 7, wherein the negative pulse bias is applied at a frequency of approximately 3 kHz and a pulse duration of approximately 20 μs. The method of any one of claims 1 to 8, wherein the mesh is formed from a conductive metal. The method of claim 9, wherein the mesh forms the lid of a Faraday cage surrounding the plastic surface. The method of claim 9 or 10, wherein the conductive metal is stainless steel. The method of any one of claims 1 to 11, wherein the plastic surface has a non-flat shape. The method of any one of claims 1 to 12, wherein the plastic surface is a microplate well or a Petri dish. The method of any one of claims 1 to 13, wherein the plastic is polystyrene. The method of any one of claims 1 to 14, wherein the biomolecule is a nucleic acid or a protein. 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;
16. The method of any one of claims 1 to 15, having properties selected from one or ^more of: hybridizing DNA at a concentration of about 2 x 10 ¹¹ to about 5 x 10 ¹¹ molecules/cm ² ; and immobilizing streptavidin at a concentration of about 2 x 10 11 to about 9 x 10 ¹¹ molecules/cm ² . 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;
A plastic surface having properties selected from one or more of: hybridizing DNA at a concentration of about ^2x1011 to about ^5x1011 molecules/ ^cm2 ; and immobilizing streptavidin at a concentration of about ^2x1011 to about ^9x1011 molecules/ ^cm2 .