Patent Description:
In blood, biomolecule, and blood analyte testing, it is desirable to minimize biomolecule adsorption and binding to plastic ware used with these biological substances. Plastic microwell plates, chromatography vials, and other containers, as well as pipettes (sometimes spelled "pipets"), pipette tips, centrifuge tubes, microscope slides, and other types of laboratory ware (also known as labware) used to prepare and transfer samples commonly have hydrophobic surfaces and readily adsorb biomolecules such as proteins, DNA, and RNA. Surfaces of these and other types of laboratory ware components made of polymeric plastic can cause binding of the biomolecule samples. It is thus a desire to provide surfaces for plastic laboratory ware and other articles that contact biological substances, to reduce a wide range of biomolecules from adhering.

<CIT> describes that a portion of an organic polymer article is made hydrophilic by exposing a hydrophobic surface of the article to a depth of about <NUM> to about <NUM> angstroms to atomic oxygen or hydroxyl radicals at a temperature below <NUM> to form a hydrophilic uniform surface layer of hydrophilic hydroxyl groups. The atomic oxygen and hydroxyl radicals are generated by a flowing afterglow microwave discharge, and the surface is outside of a plasma produced by the discharge. According to <CIT>, such membrane having both hydrophilic and hydrophobic surfaces can be used in an immunoassay or in cell culturing; prior to adhering cells, the hydrophilic surface may be grafted with a compatibilizing compound.

In one aspect, the invention pertains to a method for treating a surface of a plastic substrate. The method comprises:.

to form a converted surface having a biomolecule recovery percentage, for an aqueous protein dispersion having a concentration from <NUM> to <NUM>, optionally <NUM> to <NUM>, optionally <NUM> to <NUM>, in contact with the converted surface, greater than <NUM>%.

In a more detailed embodiment, the method includes at least two treatment steps. The conditioning step includes conditioning the surface with remote conditioning plasma of one or more non-polymerizing compounds, forming a conditioned surface. The conversion step includes converting the conditioned surface with remote conversion plasma of water to form a converted surface. The converted surface has a biomolecule recovery percentage greater than the biomolecule recovery percentage of the surface prior to treatment according to the method.

In another aspect, the invention pertains to an article resulting from the method according to the invention.

The invention will be described in conjunction with the following drawings in which like reference numerals designate like elements and in which:.

The following reference characters are used in this description and the accompanying Figures:.

According to the invention, methods are disclosed for reducing biomolecule adhesion to a surface. A method for treating a surface, optionally an entire or partial surface of a substrate or a surface of a material, is provided, most generally comprising treating the surface with conversion plasma of one or more non-polymerizing compounds to form a treated surface.

The term "biomolecule" is used respecting any embodiment to include any nucleotides or peptides, or any combination of them. Nucleotides include oligonucleotides and polynucleotides, also known as nucleic acids, for example deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Peptides include amino acids, oligopeptides, polypeptides, and proteins. Nucleotides and peptides further include modified or derivatized nucleotides and peptides that adhere to a surface that is not treated according to the present invention.

The presently defined biomolecules include to one or more of the following aqueous proteins: mammal serum albumin, for example Bovine Serum Albumin (BSA); Fibrinogen (FBG); Transferrin (TFN), for example blood serotransferrin (or siderophilin, also known as transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); membrane-associated melanotransferrin; Protein A (PrA); Protein G (PrG); Protein A/G; Protein L; Insulin, for example hexameric insulin, monomeric insulin, porcine insulin, human insulin, recombinant insulin and pharmaceutical grades of insulin; pharmaceutical protein; blood or blood component proteins; or any recombinant form, modification, full length precursor, signal peptide, pro-peptide, or mature variant of these proteins; and a combination of two or more of these.

Biomolecule adhesion to a surface is defined for any embodiment as a reduction of the aqueous concentration of a biomolecule dispersed in an aqueous medium stored in contact with the surface. It is not limited by the mechanism of reduction of concentration, whether literally "adhesion," adsorption, or another mechanism.

"Plasma," as referenced in any embodiment, has its conventional meaning in physics of one of the four fundamental states of matter, characterized by extensive ionization of its constituent particles, a generally gaseous form, and incandescence (i.e. it produces a glow discharge, meaning that it emits light).

"Conversion plasma treatment" refers to any plasma treatment that reduces the adhesion of one or more biomolecules to a treated surface.

"Conditioning plasma treatment" refers to any plasma treatment of a surface to prepare the surface for further conversion plasma treatment. "Conditioning plasma treatment" includes a plasma treatment that, in itself, reduces the adhesion of one or more biomolecules to a treated surface, but is followed by conversion plasma treatment that further reduces the adhesion of one or more biomolecules to a treated surface. "Conditioning plasma treatment" also includes a plasma treatment that, in itself, does not reduce the adhesion of one or more biomolecules to a treated surface.

A "remote" conversion plasma treatment, generally speaking, is conversion plasma treatment of a surface located at a "remote" point where the radiant energy density of the plasma, for example in Joules per cm<NUM>, is substantially less than the maximum radiant energy density at any point of the plasma glow discharge (referred to below as the "brightest point"), but the remote surface is close enough to some part of the glow discharge to reduce the adhesion of one or more biomolecules to the treated remote surface. "Remote" is defined in the same manner respecting a remote conditioning plasma treatment, except that the remote surface must be close enough to some part of the glow discharge to condition the surface.

The radiant energy density at the brightest point of the plasma is determined spectrophotometrically by measuring the radiant intensity of the most intense emission line of light in the visible spectrum (<NUM> nanometer (nm) to <NUM> wavelength) at the brightest point. The radiant energy density at the remote point is determined spectrophotometrically by measuring the radiant energy density of the same emission line of light at the remote point. "Remoteness" of a point is quantified by measuring the ratio of the radiant energy density at the remote point to the radiant energy density at the brightest point. The present specification and claims define "remote" quantitatively as a specific range of that ratio. Broadly, the ratio is from <NUM> to less than <NUM>, optionally from <NUM> to <NUM>, optionally about <NUM>, optionally exactly <NUM>. Remote conversion plasma treatment can be carried out where the ratio is zero, even though that indicates no measurable visible light at the remote point, because the dark discharge region or afterglow region of plasma contain energetic species that, although not energetic enough to emit light, are energetic enough to modify the treated surface to reduce the adhesion of one or more biomolecules.

A "non-polymerizing compound" is defined operationally for all embodiments as a compound that does not polymerize on a treated surface or otherwise form an additive coating under the conditions used in a particular plasma treatment of the surface. Examples of compounds that can be used under non-polymerizing conditions are the following: O<NUM>, N<NUM>, air, O<NUM>, N<NUM>O, H<NUM>, H<NUM>O<NUM>, NH<NUM>, Ar, He, Ne, and combinations of any of two or more of the foregoing. These may also include alcohols, organic acids, and polar organic solvents as well as materials that can be polymerized under different plasma conditions from those employed. "Non-polymerizing" includes compounds that react with and bond to a preexisting polymeric surface and locally modify its composition at the surface. The essential characterizing feature of a non-polymerizing coating is that it does not build up thickness (i.e. build up an additive coating) as the treatment time is increased.

A "substrate" is an article or other solid form (such as a granule, bead, or particle).

A "surface" is broadly defined as either an original surface (a "surface" also includes a portion of a surface wherever used in this specification) of a substrate, or a coated or treated surface prepared by any suitable coating or treating method, such as liquid application, condensation from a gas, or chemical vapor deposition, including plasma enhanced chemical vapor deposition carried out under conditions effective to form a coating on the substrate.

A treated surface is defined for all embodiments as a surface that has been plasma treated as described in this specification.

The terms "optionally" and "alternatively" are regarded as having the same meaning in the present specification and claims, and may be used interchangeably.

The "material" in any embodiment can be any material of which a plastic substrate is formed, including a thermoplastic material, optionally a thermoplastic injection moldable material. The substrate according to any embodiment may be made, for example, from material including: an olefin polymer; polypropylene (PP); polyethylene (PE); cyclic olefin copolymer (COC); cyclic olefin polymer (COP); polymethylpentene; polyester; polyethylene terephthalate; polyethylene naphthalate; polybutylene terephthalate (PBT); PVdC (polyvinylidene chloride); polyvinyl chloride (PVC); polycarbonate; polymethylmethacrylate; polylactic acid; polylactic acid; polystyrene; hydrogenated polystyrene; poly(cyclohexylethylene) (PCHE); epoxy resin; nylon; polyurethane polyacrylonitrile; polyacrylonitrile (PAN); an ionomeric resin; or Surlyn® ionomeric resin.

The term "vessel" as used throughout this specification may be any type of article that is adapted to contain or convey a liquid, a gas, a solid, or any two or more of these. One example of a vessel is an article with at least one opening (e.g., one, two or more, depending on the application) and a wall defining an interior contacting surface.

A wide variety of different surfaces can be treated according to any embodiment. One example of a surface is a vessel lumen surface, where the vessel is, for example, a vial, a bottle, a jar, a syringe, a cartridge, a blister package, or an ampoule. For more examples, the surface of the material can be a fluid surface of an article of labware, for example a microplate, a centrifuge tube, a pipette tip, a well plate, a microwell plate, an ELISA plate, a microtiter plate, a <NUM>-well plate, a <NUM>-well plate, a centrifuge tube, a chromatography vial, an evacuated blood collection tube, or a specimen tube.

The treated surface of any embodiment can be a coating or layer of PECVD deposited SiOxCyHz or SiNxCyHz, in which x is from about <NUM> to about <NUM> as measured by X-ray photoelectron spectroscopy (XPS), y is from about <NUM> to about <NUM> as measured by XPS, and z is from about <NUM> to about <NUM> as measured by Rutherford backscattering spectrometry (RBS). Another example of the surface to be treated is a barrier coating or layer of SiOx, in which x is from about <NUM> to about <NUM> as measured by XPS, or an oxide or nitride of an organometallic precursor that is a compound of a metal element from Group III and/or Group IV of the Periodic Table, e.g. in Group III: Boron, Aluminum, Gallium, Indium, Thallium, Scandium, Yttrium, or Lanthanum, (Aluminum and Boron being preferred), and in Group IV: Silicon, Germanium, Tin, Lead, Titanium, Zirconium, Hafnium, or Thorium (Silicon and Tin being preferred).

A gas or gases employed to treat a surface can be an inert gas or a reactive gas, and can be any of the following: O<NUM>, N<NUM>, air, O<NUM>, N<NUM>O, NO<NUM>, N<NUM>O<NUM>, H<NUM>, H<NUM>O<NUM>, H<NUM>O, NH<NUM>, Ar, He, Ne, Xe, Kr, a nitrogen-containing gas, other non-polymerizing gases, gas combinations including an Ar/O<NUM> mix, an N<NUM>/O<NUM> mix following a pre-treatment conditioning step with Ar, a volatile and polar organic compound, the combination of a C<NUM>-C<NUM> hydrocarbon and oxygen; the combination of a Ci-C<NUM> hydrocarbon and nitrogen; a silicon-containing gas; or a combination of two or more of these. The treatment employs a non-polymerizing gas as defined in this specification.

A volatile and polar organic compound can be, for example water, for example tap water, distilled water, or deionized water; an alcohol, for example a C<NUM>-C<NUM> alcohol, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, s-butanol, t-butanol; a glycol, for example ethylene glycol, propylene glycol, butylene glycol, polyethylene glycol, and others; glycerine, a C<NUM>-C<NUM> linear or cyclic ether, for example dimethyl ether, diethyl ether, dipropyl ether, dibutyl ether, glyme (CH<NUM>OCH<NUM>CH<NUM>OCH<NUM>); cyclic ethers of formula -CH<NUM>CH<NUM>On- such as diethylene oxide, triethylene oxide, and tetraethylene oxide; cyclic amines; cyclic esters (lactones), for example acetolactone, propiolactone, butyrolactone, valerolactone, and caprolactone; a C<NUM>-C<NUM> aldehyde, for example formaldehyde, acetaldehyde, propionaldehyde, or butyraldehyde; a C<NUM>-C<NUM> ketone, for example acetone, diethylketone, dipropylketone, or dibutylketone; a C<NUM>-C<NUM> carboxylic acid, for example formic acid, acetic acid, propionic acid, or butyric acid; ammonia, a C<NUM>-C<NUM> amine, for example methylamine, dimethylamine, ethylamine, diethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine, nonylamine, decylamine, undecylamine, or dodecylamine; hydrogen fluoride, hydrogen chloride, a C<NUM>-C<NUM> epoxide, for example ethylene oxide or propylene oxide; or a combination of any two or more of these.

A C<NUM>-C<NUM> hydrocarbon can be methane, ethane, ethylene, acetylene, n-propane, i-propane, propene, propyne; n-butane, i-butane, t-butane, butane, <NUM>-butyne, <NUM>-butyne, or a combination of any two or more of these.

A silicon-containing gas can be a silane, an organosilicon precursor, or a combination of any two or more of these. The silicon-containing gas can be an acyclic or cyclic, substituted or unsubstituted silane, optionally comprising, consisting essentially of, or consisting of any one or more of: Si<NUM>-Si<NUM> substituted or unsubstituted silanes, for example silane, disilane, trisilane, or tetrasilane; hydrocarbon or halogen substituted Si<NUM>-Si<NUM> silanes, for example tetramethylsilane (TetraMS), tetraethyl silane, tetrapropylsilane, tetrabutylsilane, trimethylsilane (TriMS), triethyl silane, tripropylsilane, tributylsilane, trimethoxysilane, a fluorinated silane such as hexafluorodisilane, a cyclic silane such as octamethylcyclotetrasilane or tetramethylcyclotetrasilane, or a combination of any two or more of these. The silicon-containing gas can be a linear siloxane, a monocyclic siloxane, a polycyclic siloxane, a polysilsesquioxane, an alkyl trimethoxysilane, a linear silazane, a monocyclic silazane, a polycyclic silazane, a polysilsesquiazane, or a combination of any two or more of these, for example hexamethyldisiloxane (HMDSO), tetramethyldisiloxane (TMDSO), octamethylcyclotetrasiloxane (OMCTS), tetramethyldisilazane, hexamethyldisilazane, octamethyltrisilazane, octamethylcyclotetrasilazane, tetramethylcyclotetrasilazane, or a combination of any two or more of these.

The electrical power used to excite the plasma used in plasma treatment in any embodiment, can be, for example, from <NUM> to <NUM> Watts, optionally from <NUM> to <NUM> Watts, optionally from <NUM> to <NUM> Watts, optionally <NUM> to <NUM> Watts, optionally from <NUM> to <NUM> Watts, optionally from <NUM> to <NUM> Watts, optionally from <NUM> to <NUM> Watts, optionally from <NUM> to <NUM> Watts, optionally from <NUM> to <NUM> Watts.

The frequency of the electrical power used to excite the plasma used in plasma treatment, in any embodiment, can be any type of energy that will ignite plasma in the plasma zone. For example, it can be direct current (DC) or alternating current (electromagnetic energy) having a frequency from <NUM> to <NUM>. Electromagnetic energy in this range generally includes radio frequency (RF) energy and microwave energy, more particularly characterized as extremely low frequency (ELF) of <NUM> to <NUM>, super low frequency (SLF) of <NUM> to <NUM>, voice or ultra-low frequency (VF or ULF) of <NUM> to <NUM>, very low frequency (VLF) of <NUM> to <NUM>, low frequency (LF) of <NUM> to <NUM>, medium frequency (MF) of <NUM> to <NUM>, high frequency (HF) of <NUM> to <NUM>, very high frequency (VHF) of <NUM> to <NUM>, ultra-high frequency (UHF) of <NUM> to <NUM>, super high frequency (SHF) of <NUM> to <NUM>, extremely high frequency (EHF) of <NUM> to <NUM>, or any combination of two or more of these frequencies. For example, high frequency energy, commonly <NUM>, is useful RF energy, and ultra-high frequency energy, commonly <NUM>, is useful microwave energy, as two examples of commonly used frequencies.

The plasma exciting energy, in any embodiment, can either be continuous during a treatment step or pulsed multiple times during the treatment step. If pulsed, it can alternately pulse on for times ranging from one millisecond to one second, and then off for times ranging from one millisecond to one second, in a regular or varying sequence during plasma treatment. One complete duty cycle (one "on" period plus one "off" period) can be <NUM> to <NUM> milliseconds (ms), optionally <NUM> to <NUM> milliseconds (ms), optionally <NUM> to <NUM>, optionally <NUM> to <NUM>, optionally <NUM> to <NUM> long.

Optionally in any embodiment, the relation between the power on and power off portions of the duty cycle can be, for example, power on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, optionally on <NUM>-<NUM> percent of the time, and power off for the remaining time of each duty cycle.

The plasma pulsing described in Mark J. Kushner, Pulsed Plasma-Pulsed Injection Sources For Remote Plasma Activated Chemical Vapor Deposition, J. <NUM>, <NUM> (<NUM>), can optionally be used.

The flow rate of process gas during plasma treatment according to any embodiment can be from <NUM> to <NUM> sccm (standard cubic centimeters per minute), optionally <NUM> to <NUM> sccm, optionally from <NUM> to <NUM> sccm, optionally <NUM>-<NUM> sccm, optionally <NUM>-<NUM> sccm, optionally <NUM>-<NUM> sccm.

Optionally in any embodiment, the plasma chamber is reduced to a base pressure from <NUM> milliTorr (mTorr, <NUM> Pascal) to <NUM> Torr (<NUM>,<NUM> Pascal) before feeding gases. Optionally the feed gas pressure in any embodiment can range from <NUM> to <NUM>,<NUM> mTorr (<NUM> to <NUM> Pascal), optionally from <NUM> mTorr to <NUM> Torr (<NUM> to <NUM> Pascal), optionally from <NUM> to <NUM> mTorr (<NUM> to <NUM> Pascal), optionally from <NUM> to <NUM> milliTorr (<NUM> to <NUM> Pascal).

The treatment volume in which the plasma is generated in any embodiment can be, for example, from <NUM> to <NUM> liters, preferably <NUM> liters to <NUM> liters.

The plasma treatment time in any embodiment can be, for example, from <NUM> to <NUM> seconds, optionally <NUM> to <NUM> sec. , optionally <NUM> to <NUM> sec. , optionally <NUM> to <NUM> sec. , optionally <NUM> to <NUM> sec. , optionally from <NUM> to <NUM> seconds.

The number of plasma treatment steps can vary, in any embodiment. For example one plasma treatment can be used; optionally two or more plasma treatments can be used, employing the same or different conditions.

In any embodiment, the plasma treatment apparatus employed can be any suitable apparatus, for example that of <FIG>, <FIG>, <FIG>, or <FIG> described in this specification, as several examples. Plasma treatment apparatus of the type that employs the lumen of the vessel to be treated as a vacuum chamber, shown for example in <CIT>, <FIG>, can also be used in any embodiment.

The plasma treatment process of any embodiment optionally can be combined with treatment using an ionized gas. The ionized gas can be, as some examples, any of the gases identified as suitable for plasma treatment. The ionized gas can be delivered in any suitable manner. For example, it can be delivered from an ionizing blow-off gun or other ionized gas source. A convenient gas delivery pressure is from <NUM>-<NUM> psi (pounds per square inch) (<NUM> to <NUM> kPa, kiloPascals) (gauge or, optionally, absolute pressure), optionally <NUM> psi (<NUM> kPa). The water content of the ionized gas can be from <NUM> to <NUM>%. The polar-treated surface with ionized gas can be carried out for any suitable treatment time, for example from <NUM>-<NUM> seconds, optionally for <NUM> seconds.

After the plasma treatment(s) of any embodiment, the treated surface, for example a vessel lumen surface, can be contacted with an aqueous protein. Some examples of suitable proteins are: mammal serum albumin, for example Bovine Serum Albumin (BSA); Fibrinogen (FBG); Transferrin (TFN), for example blood serotransferrin (or siderophilin, also known as transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); and membrane-associated melanotransferrin; Protein A (PrA); Protein G (PrG); Protein A/G; Protein L; Insulin, for example hexameric insulin, monomeric insulin, porcine insulin, human insulin, recombinant insulin and pharmaceutical grades of insulin; Pharmaceutical protein; blood or blood component proteins; or any recombinant form, modification, full length precursor, signal peptide, propeptide, or mature variant of these proteins; or a combination of two or more of these.

Optionally, the treated surface has a protein recovery percentage greater than the protein recovery percentage of the unconditioned and unconverted surface for at least one of Bovine Serum Albumin (BSA); Fibrinogen (FBG); Transferrin (TFN), for example blood serotransferrin (or siderophilin, also known as transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); and membrane-associated melanotransferrin; Protein A (PrA); Protein G (PrG); Protein A/G; Protein L; Insulin, for example hexameric insulin, monomeric insulin, porcine insulin, human insulin, recombinant insulin and pharmaceutical grades of insulin; pharmaceutical protein; blood or blood component proteins; or any recombinant form, modification, full length precursor, signal peptide, propeptide, or mature variant of these proteins.

A vessel having a substrate according to the more detailed embodiment may be made, for example, from any of the plastic materials defined above. For applications in which clear and glass-like polymers are desired (e.g., for syringes and vials), a cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polymethylmethacrylate, polyethylene terephthalate or polycarbonate may be preferred. Also contemplated are linear polyolefins such as polypropylene and aromatic polyolefins such as polystyrene. Such substrates may be manufactured, e.g., by injection molding or injection stretch blow molding (which is also classified as injection molding in this disclosure), to very tight and precise tolerances (generally much tighter than achievable with glass).

A vessel can be a sample tube, e.g. for collecting or storing biological fluids like blood or urine, a syringe (or a part thereof, for example a syringe barrel) for storing or delivering a biologically active compound or composition, e.g., a medicament or pharmaceutical composition, a vial for storing biological materials or biologically active compounds or compositions, a pipe, e.g., a catheter for transporting biological materials or biologically active compounds or compositions, or a cuvette for holding fluids, e.g., for holding biological materials or biologically active compounds or compositions. Other examples of vessels include well or non-well slides or plates, for example titer plates or microtiter plates (a. microplates). Other examples of vessels include measuring and delivery devices such as pipettes, pipette tips, Erlenmeyer flasks, beakers, and graduated cylinders. The specific vessels described herein with respect to an actual reduction to practice of an embodiment are polypropylene <NUM>-well microplates and beakers. However, a skilled artisan would understand that the methods and equipment set-up described herein can be modified and adapted, consistent with the present invention, to accommodate and treat optional vessels.

The surface of the vessel may be made from the substrate material itself, e.g., any of the thermoplastic resins listed above. Optionally, the surface may be a pH protective coating or layer of PECVD deposited SiOxCyHz or SiNxCyHz, in which x is from about <NUM> to about <NUM> as measured by X-ray photoelectron spectroscopy (XPS), y is from about <NUM> to about <NUM> as measured by XPS, and z is from about <NUM> to about <NUM> as measured by Rutherford backscattering spectrometry (RBS). Another example of the surface is a barrier coating or layer of PECVD deposited SiOx, in which x is from about <NUM> to about <NUM> as measured by XPS, or an oxide or nitride of an organometallic precursor that is a compound of a metal element from Group III and/or Group IV of the Periodic Table, e.g. in Group III: Boron, Aluminum, Gallium, Indium, Thallium, Scandium, Yttrium, or Lanthanum (Aluminum and Boron being preferred), and in Group IV: Silicon, Germanium, Tin, Lead, Titanium, Zirconium, Hafnium, or Thorium (Silicon and Tin being preferred). Methods and equipment for depositing these coatings or layers are described in detail in <CIT>.

Methods according to the more detailed embodiment employ the use of remote conversion plasma treatment. Unlike direct plasma processing, in the case of remote conversion plasma, neither ions nor electrons of plasma contact the article surface. Neutral species, typically having lower energy, are present in the plasma afterglow, which are sufficiently energetic to react with the article surface, without sputtering or other higher energy chemical reactions induced by ions and electrons. The result of remote conversion plasma is a gentle surface modification without the high energy effects of "direct" plasmas.

Methods according to the more detailed embodiment employ non-polymerizing gases, such as O<NUM>, N<NUM>, air, O<NUM>, N<NUM>O, H<NUM>, H<NUM>O<NUM>, NH<NUM>, Ar, He, Ne, other non-polymerizing gases, and combinations of any of two or more of the foregoing. These may also include non-polymerizing alcohols, non-polymerizing organic acids and non-polymerizing polar organic solvents. Experiments have been carried out in which the conditioning step (non-polymerizing compound step) used Ar, N<NUM>, Ar/O<NUM> mix, or N<NUM>/O<NUM> mix and a pre-treatment conditioning step with Ar. These and other non-polymerizing gases do not necessarily deposit a coating. Rather, they react with the surface to modify the surface, e.g., to form a treated surface, in which the treated surface has a biomolecule recovery percentage greater than the biomolecule recovery percentage of the unconditioned and unconverted surface. For example, the surface reactions may result in new chemical functional groups on the surface, including carbonyl, carboxyl, hydroxyl, nitrile, amide, amine. It is contemplated that these polar chemical groups increase the surface energy and hydrophilicity of otherwise hydrophobic polymers that an unconditioned and unconverted surface may typically comprise. While hydrophobic surfaces are generally good binding surfaces for biomolecules, hydrophilic surfaces, which attract water molecules, facilitate the blocking of biomolecules binding to that surface. While the invention is not limited according to this theory of operation, it is contemplated that this mechanism prevents biomolecule binding to surfaces.

Optionally, methods according to the more detailed embodiment may be used to reduce the propensity of a substrate surface to cause biomolecules to adhere thereto. Preferably, the methods will reduce biomolecule adhesion across a wide spectrum of biomolecules, including one or more of the following aqueous proteins: mammal serum albumin, for example Bovine Serum Albumin (BSA); Fibrinogen (FBG); Transferrin (TFN), for example blood serotransferrin (or siderophilin, also known as transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); and membrane-associated melanotransferrin; Protein A (PrA); Protein G (PrG); Protein A/G; Protein L; Insulin, for example hexameric insulin, monomeric insulin, porcine insulin, human insulin, recombinant insulin and pharmaceutical grades of insulin; Pharmaceutical protein; blood or blood component proteins; or any recombinant form, modification, full length precursor, signal peptide, pro-peptide, or mature variant of these proteins; and a combination of two or more of these.

<FIG> is a schematic generic view of a remote conversion plasma treatment apparatus <NUM> having common features with each more particular embodiment of <FIG>, <FIG>, <FIG>, and <FIG> for carrying out remote conversion plasma treatment according to the invention. Plasma gas from a fluid source <NUM> capable of supporting the generation of plasma in the plasma zone <NUM> having a boundary <NUM> (plasma is defined here as a visible glow discharge) is introduced via a fluid inlet <NUM> to a plasma zone <NUM>, and plasma energy from a plasma energy source <NUM> is provided to the plasma zone <NUM> to generate plasma having a boundary <NUM> in the plasma zone <NUM>.

The plasma energy of the more detailed embodiment broadly can be any type of energy that will ignite plasma in the plasma zone <NUM>. For example, it can be direct current (DC) or alternating current (electromagnetic energy) having a frequency from <NUM> to <NUM>. Electromagnetic energy in this range generally includes radio frequency (RF) energy and microwave energy, more particularly characterized as extremely low frequency (ELF) of <NUM> to <NUM>, super low frequency (SLF) of <NUM> to <NUM>, voice or ultra-low frequency (VF or ULF) of <NUM> to <NUM>, very low frequency (VLF) of <NUM> to <NUM>, low frequency (LF) of <NUM> to <NUM>, medium frequency (MF) of <NUM> to <NUM>, high frequency (HF) of <NUM> to <NUM>, very high frequency (VHF) of <NUM> to <NUM>, ultra-high frequency (UHF) of <NUM> to <NUM>, super high frequency (SHF) of <NUM> to <NUM>, extremely high frequency (EHF) of <NUM> to <NUM>, or any combination of two or more of these frequencies. For example, high frequency energy, commonly <NUM>, is useful RF energy, and ultra-high frequency energy, commonly <NUM>, is useful microwave energy, as two examples of commonly used frequencies.

The nature of the optimal applicator <NUM> is determined by the frequency and power level of the energy, as is well known. If the plasma is excited by radio waves, for example, the applicator <NUM> can be an electrode, while if the plasma is excited by microwave energy, for example, the applicator <NUM> can be a waveguide.

An afterglow region <NUM> is located outside but near the plasma boundary <NUM>, and contains treatment gas <NUM>. The afterglow region <NUM> can be the entire treatment volume <NUM> outside the plasma boundary <NUM> and within the reaction chamber wall <NUM> and lid <NUM>, or the afterglow region <NUM> can be a subset of the treatment volume <NUM>, depending on the dimensions of and conditions maintained in the treatment volume. The treatment gas <NUM> in the afterglow region <NUM> is not ionized sufficiently to form plasma, but it is sufficiently energetic to be capable of modifying a surface that it contacts, more so than the same gas composition at the same temperature and pressure in the absence of the plasma.

It will be understood by a skilled person that some gas compositions are sufficiently chemically reactive that they will modify a substrate in the apparatus <NUM> when plasma is absent. The test for whether a region of, or adjacent to, remote conversion plasma treatment apparatus is within the afterglow, for given equipment, plasma, gas feed, and pressure or vacuum conditions producing a visible glow discharge outside the region, is whether a substrate located in the region under the given equipment, plasma, gas feed, and pressure is modified compared to a substrate exposed to the same equipment, gas feed and pressure or vacuum conditions, when no plasma is present in the plasma zone as the result of the absence of or insufficiency of the plasma energy <NUM>.

Remote conversion plasma treatment is carried out by providing plasma in the plasma zone <NUM>, which generates an afterglow in the afterglow region or remote conversion plasma (two terms for the same region) <NUM>, which contacts and modifies a substrate placed at least partially in the afterglow region <NUM>.

As one option in the remote conversion plasma treatment apparatus, the plasma gas enters the plasma zone, is excited to form plasma, then continues downstream to the afterglow region <NUM> where it has less energy, is then defined as treatment gas <NUM>, and contacts the substrate. In other words, at least a portion of the gas flows through the plasma zone <NUM>, is energized to form plasma, and continues to the afterglow region <NUM>, becoming more energetic in the plasma zone <NUM> and less energetic by the time it enters the afterglow region <NUM> (but still energized compared to the gas before entering the plasma zone <NUM>). Where this option is adopted, the plasma and the afterglow region <NUM> are in gas communication and at least some of the same gas is fed through both zones. Optionally, as where plasma is not generated in the entire cross-section of flowing gas, some of the gas may bypass the plasma by staying outside the boundary <NUM> of the plasma zone <NUM> and still flow through the afterglow region <NUM>, while other gas flows through both the plasma zone <NUM> and the afterglow region <NUM>.

As another option in the remote conversion plasma treatment, the plasma gas can be different molecules from the treatment gas <NUM> (though the plasma gas and treatment gas may either have identical compositions or different compositions), and the plasma gas remains in or is fed through only the plasma zone <NUM> and not the afterglow region <NUM>, while the treatment gas is energized by the plasma gas but is separate from the plasma gas and while in the afterglow region <NUM> is not energized sufficiently to form plasma.

The nature of the applicator <NUM> can vary depending on the application conditions, for example the power level and frequency of the plasma energy <NUM>. For example, the applicator can be configured as an electrode, antenna, or waveguide.

Optionally, a shield <NUM> may be placed between the plasma and at least a portion of the substrate <NUM> in the treatment area to prevent the plasma from contacting or coming undesirably close to the substrate <NUM> or unevenly affecting the substrate <NUM>. For one example, the optional shield <NUM> in <FIG> can be perforated to allow gas flow through it, particularly flow of the neutral species forming the afterglow, but the shield <NUM> is configured or equipped, suitably for the choice of plasma-forming energy, to prevent the plasma from penetrating the shield. For example, the perforations may be sized or the shield can be electrically biased such that the plasma-forming energy or the plasma cannot pass through it. This arrangement has the advantage that, if the plasma zone has a substantial area intersecting with the shield, the substantial area optionally is flattened so the plasma boundary <NUM> has a "flat spot" <NUM>, illustrated in <FIG>, which can be placed parallel to the surface of the substrate to be treated so they are equidistant over a substantial area, instead of the plasma terminating in a tapered tail that extends much closer to one portion of the substrate <NUM> than to other parts of the substrate <NUM> not aligned with the tail, illustrated in <FIG>.

Another shield option is that the shield can be made such that it passes neither gas nor plasma, serving as an obstruction of the direct path between some or all of the plasma and some or all of the treatment area. The obstruction can fill less than all of the gas cross-section flowing from the plasma zone <NUM> to the afterglow region <NUM>, so non-ionized gas can flow around the shield and reach the afterglow region <NUM> by a circuitous path, while plasma cannot either circumvent or pass through it.

Yet another shield option is that the substrate <NUM> to be treated can be positioned in the apparatus during treatment such that one portion of a substrate <NUM> that can withstand contact with plasma is exposed to the plasma, shielding from the plasma another portion of the substrate <NUM> or another substrate receiving remote conversion plasma treatment.

Still another shield option is that the gas flow path through the plasma and treatment area can be sharply bent, for example turning a <NUM> degree corner between the plasma and treatment area, so the wall of the apparatus itself shields the treatment area from line-of-sight relation to the plasma under certain treatment conditions.

The substrate orientation in the treatment volume can vary, and the substrate, applicator, gas and vacuum sources can optionally be arranged to provide either substantially even or uneven exposure to remote conversion plasma across a substrate.

Another option is that the substrate itself can serve as the reactor wall or a portion of the reactor wall, so treatment gas <NUM> introduced into reactor treats the portion of the substrate serving as the reactor wall.

Another option is the introduction of a second non-polymerizing gas, functioning as diluent gas, into the reactor, in addition to the non-polymerizing compound or water vapor which is the active agent of the treatment gas <NUM>. Diluent gases are defined as gases introduced at the fluid inlet <NUM> that do not materially interact with the substrate <NUM> to the extent they find their way into the treatment gas <NUM>, given the treatment apparatus and conditions applied. Diluent gases can either participate or not participate in formation of the plasma. The diluent gas can be introduced through the inlet <NUM> or elsewhere in the reactor. Diluent gases can be added at a rate from <NUM>% to <NUM>,<NUM>% by volume, optionally <NUM>% to <NUM>% by volume, optionally <NUM>% to <NUM>% by volume, of the rate of addition of the non-polymerizing compound or water vapor.

As another option, some or all of the non-polymerizing compound or water vapor can be added to the treatment volume <NUM> in such a manner as to bypass the plasma zone <NUM> en route to the treatment gas <NUM>.

<FIG> shows another version of the apparatus of <FIG>. The apparatus again can be used for carrying out the remote conversion plasma treatment according to the more detailed embodiment. The chamber of this apparatus comprises a treatment volume <NUM> defined and enclosed by a reaction chamber wall <NUM>, which optionally is not electrically conductive. The treatment volume <NUM> is supplied with a fluid source <NUM> (in this instance, a tubular fluid inlet <NUM> projecting axially into the treatment volume <NUM>, however other fluid sources are contemplated, e.g., "shower head" type fluid sources). Optionally, the treatment volume <NUM> can be defined by a treatment chamber wall <NUM> or by the lumen within a vessel or other article to be treated. Feed gases are fed into the treatment volume <NUM>. The plasma reaction chamber comprises as an optional feature a vacuum source <NUM> for at least partially evacuating the treatment volume <NUM> compared to ambient pressure, for use when plasma treating at reduced pressure, although plasma treating under suitable conditions at ambient atmospheric pressure or at a pressure higher than ambient atmospheric pressure is also contemplated.

The plasma reaction chamber also comprises an optional outer applicator <NUM>, here in the form of an electrode surrounding at least a portion of the plasma reaction chamber. A radio frequency (RF) plasma energy source <NUM> is coupled to the reaction chamber by an applicator <NUM> and provides power that excites the gases to form plasma. The plasma forms a visible glow discharge <NUM> that optionally is limited to a close proximity to the fluid source <NUM>.

Microplates <NUM> optionally can be oriented such that the surfaces of the microplates <NUM> on which treatment is desired (the surface that is configured and intended to contact/hold a biomolecule-containing solution) face the fluid source <NUM>. However, the surfaces to be treated can also or instead face away from the fluid source <NUM>, as shown in <FIG>. In addition, in the illustrated arrangement the microplate <NUM> is shielded with a shield <NUM> to block the microplate <NUM> from being in the direct "line of sight" of (i.e. having an unobstructed path to) the fluid source <NUM>. As an example, the respective surfaces of the microplates <NUM> can be positioned a horizontal distance of approximately <NUM> inches (<NUM>) from the fluid source, although operation is contemplated with placement of the microplate <NUM> surfaces at a horizontal distance of from ½ to <NUM> inches (<NUM> to <NUM>), optionally <NUM> to <NUM> inches (<NUM> to <NUM>) from the fluid source. In this manner, the process relies on remote conversion plasma (as opposed to direct plasma) to treat the microplates' <NUM> surfaces. In this example, the system has a capacity of <NUM> parts (microplates) per batch at a total process time of eight minutes per batch.

<FIG> shows another version of the apparatus of <FIG>. The process used to treat the microplates in <FIG> uses a radio-frequency (RF) plasma system. The system has a gas delivery input, a vacuum pump and RF power supply with matching network. The microplates are shown oriented with the front surfaces containing the wells <NUM> facing away from and shielded from the plasma along the perimeter of the chamber.

These details are illustrated in <FIG>, where there is shown another exemplary setup having all the elements of the apparatus of <FIG> for use in a plasma reaction chamber for carrying out remote conversion plasma treatment according to the more detailed embodiment. In this case, the chamber comprised a treatment volume <NUM> defined and enclosed by a reaction chamber wall <NUM> having a fluid source <NUM> (in this instance, a tubular fluid inlet <NUM> projecting axially into the treatment volume <NUM>, however other fluid sources are contemplated, e.g., "shower head" type fluid sources). The reaction chamber wall <NUM> was provided with a removable lid <NUM> that is openable to allow substrates to be inserted or removed and sealable to contain the process and, optionally, evacuate the treatment volume. The fluid source <NUM> was made of metallic material, electrically grounded, and also functioned as an applicator, in the form of an inner electrode. As is well known, the plasma optionally can be generated without an inner electrode.

Feed gases were fed into the treatment volume <NUM>. The plasma reaction chamber comprised an optional feature of a vacuum source <NUM> for at least partially evacuating the treatment volume <NUM>. The plasma reaction chamber wall <NUM> also functioned as an applicator <NUM> in the form of an outer applicator or electrode surrounding at least a portion of the plasma reaction chamber. A plasma energy source <NUM>, in this instance a radio frequency (RF) source, was coupled to applicators <NUM> defined by the reaction chamber wall <NUM> and the fluid source <NUM> to provide power that excited the gases to form plasma. The plasma zone <NUM> formed a visible glow discharge that was limited by the plasma boundary <NUM> in close proximity to the fluid source <NUM>. The afterglow region also known as a remote conversion plasma region <NUM> is the region radially or axially outside the boundary <NUM> of the visible glow discharge and extending beyond the substrates treated.

Microplates <NUM> having front surfaces <NUM> and back surfaces <NUM> were oriented such that the wells <NUM> on the front surfaces of the microplates <NUM> on which treatment was desired (the front surface that is configured and intended to contact/hold a biomolecule-containing solution) faced away from the fluid source <NUM> and the back surfaces <NUM> faced toward the fluid source <NUM>. The front surfaces <NUM> of the microplates <NUM> were shielded by their own back surfaces <NUM> to block the microplate front surfaces <NUM> from being in the direct "line of sight" of the fluid source <NUM>. In this manner, the process relied on remote conversion plasma (as opposed to direct plasma) to treat the surfaces of the wells <NUM>.

<FIG> shows another version of the apparatus of <FIG>, having corresponding features. The version of <FIG> provides a "shower head" fluid inlet <NUM> and a plate electrode as the applicator <NUM> that provide more uniform generation and application of treatment gas <NUM> over a wider area of the substrate <NUM>.

<FIG> shows another version of the apparatus of <FIG>, having corresponding features. The version of <FIG> provides microwave plasma energy <NUM> delivered through an applicator <NUM> configured as a waveguide. In this version the plasma zone <NUM> and substrate support <NUM> are provided in separate vessels connected by a conduit.

Optionally, the treated surface has a biomolecule recovery percentage greater than the biomolecule recovery percentage of the unconditioned and unconverted surface for at least one of Bovine Serum Albumin (BSA); Fibrinogen (FBG); Transferrin (TFN), for example blood serotransferrin (or siderophilin, also known as transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); and membrane-associated melanotransferrin; Protein A (PrA); Protein G (PrG); Protein A/G; Protein L; Insulin, for example hexameric insulin, monomeric insulin, porcine insulin, human insulin, recombinant insulin and pharmaceutical grades of insulin; pharmaceutical protein; blood or blood component proteins; or any recombinant form, modification, full length precursor, signal peptide, propeptide, mature variant of these proteins and a combination of two or more of these.

In one embodiment, a plasma treatment process comprises, consists essentially of, or consists of the following two steps using remote conversion plasma: (<NUM>) an oxygen plasma step (or more generically, a non-polymerizing compound plasma step) followed by (<NUM>) a water vapor plasma step. It should be understood that additional steps prior to, between or after the aforementioned steps may be added and remain within the scope of the more detailed embodiment. Further, it should also be understood that the oxygen plasma step may utilize optional gases in addition to oxygen, including nitrogen or any non-polymerizing gases listed in this specification.

Optional process parameter ranges for the conditioning step (non-polymerizing compound plasma step) and conversion step (water vapor plasma step) of the more detailed embodiment are set forth in Table <NUM>.

Optionally, no pretreatment step is required prior to the non-polymerizing gas plasma step.

Optionally, the remote conversion plasma used to treat a substrate surface may be RF generated plasma. Optionally, plasma enhanced chemical vapor deposition (PECVD) or other plasma processes may be used consistent with the more detailed embodiment.

Optionally, the treatment volume in a plasma reaction chamber may be from <NUM> to <NUM> liters, preferably <NUM> liters to <NUM> liters for certain applications. Optionally, the treatment volume may be generally cylindrical, although other shapes and configurations are also contemplated.

In an aspect of the substrate in any embodiment the converted and optionally conditioned surface has a biomolecule recovery percentage of at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>%, optionally at least <NUM>% optionally at least <NUM>%.

In an aspect of the substrate in any embodiment the converted and optionally conditioned surface is a vessel lumen surface.

In an aspect of the substrate in any embodiment the biomolecule recovery percentage is determined for at least one of: mammal serum albumin; Bovine Serum Albumin (BSA); Fibrinogen (FBG); Transferrin (TFN); egg white ovotransferrin (conalbumin); membrane-associated melanotransferrin; Protein A (PrA); Protein G (PrG); Protein A/G; Protein L; Insulin; Pharmaceutical protein; blood or blood component proteins; and any recombinant form, modification, full length precursor, signal peptide, propeptide, or mature variant of these proteins.

In an aspect of the substrate in any embodiment the converted and optionally conditioned surface comprises thermoplastic material, for example a thermoplastic resin, for example an injection-molded thermoplastic resin.

In an aspect of the substrate in any embodiment the converted and optionally conditioned surface comprises a hydrocarbon, for example an olefin polymer, polypropylene (PP), polyethylene (PE), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polymethylpentene, polystyrene, hydrogenated polystyrene, polycyclohexylethylene (PCHE), or combinations of two or more of these. The converted and optionally conditioned surface optionally comprises a heteroatom-substituted hydrocarbon polymer, for example a polyester, polyethylene terephthalate, polyethylene naphthalate, polybutylene terephthalate (PBT), polyvinylidene chloride (PVdC), polyvinyl chloride (PVC), polycarbonate, polylactic acid, epoxy resin, nylon, polyurethane polyacrylonitrile, polyacrylonitrile (PAN), an ionomeric resin, Surlyn® ionomeric resin, or any combination, composite or blend of any two or more of the above materials.

In an aspect of the substrate in any embodiment the converted and optionally conditioned surface is a coating or layer of PECVD deposited SiOxCyHz or SiNxCyHz, in which x is from about <NUM> to about <NUM> as measured by X-ray photoelectron spectroscopy (XPS), y is from about <NUM> to about <NUM> as measured by XPS, and z is from about <NUM> to about <NUM> as measured by Rutherford backscattering spectrometry (RBS).

In an aspect of the substrate in any embodiment, the converted and optionally conditioned surface is a barrier coating or layer of SiOx, in which x is from about <NUM> to about <NUM> as measured by XPS, or an oxide or nitride of an organometallic precursor that is a compound of a metal element from Group III and/or Group IV of the Periodic Table, e.g. in Group III: Boron, Aluminum, Gallium, Indium, Thallium, Scandium, Yttrium, or Lanthanum (Aluminum and Boron being preferred), and in Group IV: Silicon, Germanium, Tin, Lead, Titanium, Zirconium, Hafnium, or Thorium (Silicon and Tin being preferred).

In an aspect of the substrate in any embodiment the converted and optionally conditioned surface is a fluid surface of an article of labware. For example, the converted and optionally conditioned surface can be a fluid surface of a microplate, a centrifuge tube, a pipette tip, a well plate, a microwell plate, an ELISA plate, a microtiter plate, a <NUM>-well plate, a <NUM>-well plate, a vial, a bottle, a jar, a syringe, a cartridge, a blister package, an ampoule, an evacuated blood collection tube, a specimen tube, a centrifuge tube, or a chromatography vial.

In an aspect of any embodiment the converted and optionally conditioned surface is a vessel lumen surface.

In an aspect of any embodiment the converted and optionally conditioned surface is in contact with an aqueous protein. In an aspect of any embodiment the aqueous protein comprises: mammal serum albumin, for example Bovine Serum Albumin (BSA); Fibrinogen (FBG); Transferrin (TFN), for example blood serotransferrin (or siderophilin, also known as transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); and membrane-associated melanotransferrin; Protein A (PrA); Protein G (PrG); Protein A/G; Protein L; Insulin, for example hexameric insulin, monomeric insulin, porcine insulin, human insulin, recombinant insulin and pharmaceutical grades of insulin; Pharmaceutical protein; blood or blood component proteins; or any recombinant form, modification, full length precursor, signal peptide, propeptide, or mature variant of these proteins; or a combination of two or more of these.

In an aspect of any embodiment the converted and optionally conditioned surface has a protein recovery percentage greater than the protein recovery percentage of the unconditioned and unconverted surface for at least one of Bovine Serum Albumin (BSA); Fibrinogen (FBG); Transferrin (TFN), for example blood serotransferrin (or siderophilin, also known as transferrin); lactotransferrin (lactoferrin); milk transferrin; egg white ovotransferrin (conalbumin); and membrane-associated melanotransferrin; Protein A (PrA); Protein G (PrG); Protein A/G; Protein L; Insulin, for example hexameric insulin, monomeric insulin, porcine insulin, human insulin, recombinant insulin and pharmaceutical grades of insulin; Pharmaceutical protein; blood or blood component proteins; or any recombinant form, modification, full length precursor, signal peptide, propeptide, or mature variant of these proteins.

In an aspect of any embodiment the converted and optionally conditioned surface has a protein recovery percentage greater than the protein recovery percentage of the unconditioned and unconverted surface for Bovine Serum Albumin having an atomic mass of <NUM>,<NUM> Daltons (BSA) on NUNC® <NUM>-well round bottom plates, following the protocol in the present specification.

In an aspect of any embodiment the converted and optionally conditioned surface has a protein recovery percentage at <NUM> hours on NUNC® <NUM>-well round bottom plates greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally up to <NUM>% for BSA, following the protocol in the present specification.

In an aspect of any embodiment the converted and optionally conditioned surface has a protein recovery percentage at <NUM> hours greater than the protein recovery percentage of the unconditioned and unconverted surface for Fibrinogen having an atomic mass of <NUM>,<NUM> Daltons (FBG) on NUNC® <NUM>-well round bottom plates, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on NUNC® <NUM>-well round bottom plates greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally up to <NUM>% for FBG, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on NUNC® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Transferrin having an atomic mass of <NUM>,<NUM> Daltons (TFN), following the protocol in the present specification.

In an aspect of in any embodiment the converted surface has a protein recovery percentage at <NUM> hours on NUNC® <NUM>-well round bottom plates greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally up to <NUM>% for TFN, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on NUNC® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Protein A having an atomic mass of <NUM>,<NUM> Daltons (PrA), following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on NUNC® <NUM>-well round bottom plates greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally up to <NUM>% for PrA, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on NUNC® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Protein G having an atomic mass of <NUM>,<NUM> Daltons (PrG), following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on NUNC® <NUM>-well round bottom plates greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally greater than <NUM>%, optionally up to <NUM>% for PrG, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours greater than the protein recovery percentage of the unconditioned and unconverted surface for Bovine Serum Albumin having an atomic mass of <NUM>,<NUM> Daltons (BSA) on Eppendorf LoBind® low-protein-binding <NUM>-well round bottom plates, following the protocol in the present specification. Eppendorf LoBind® is a trademark of Eppendorf AG, Hamburg, Germany.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on Eppendorf LoBind® <NUM>-well round bottom plates greater than <NUM>% for BSA, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours greater than the protein recovery percentage of the unconditioned and unconverted surface for Fibrinogen having an atomic mass of <NUM>,<NUM> Daltons (FBG) on Eppendorf LoBind® <NUM>-well round bottom plates, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on Eppendorf LoBind® <NUM>-well round bottom plates greater than <NUM>% for FBG, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on Eppendorf LoBind® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Transferrin having an atomic mass of <NUM>,<NUM> Daltons (TFN), following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on Eppendorf LoBind® <NUM>-well round bottom plates greater than <NUM>% for TFN, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on Eppendorf LoBind® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Protein A having an atomic mass of <NUM>,<NUM> Daltons (PrA), following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on Eppendorf LoBind® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Protein G having an atomic mass of <NUM>,<NUM> Daltons (PrG), following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on Eppendorf LoBind® <NUM>-well round bottom plates greater than <NUM>% for PrG, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours greater than the protein recovery percentage of the unconditioned and unconverted surface for Bovine Serum Albumin having an atomic mass of <NUM>,<NUM> Daltons (BSA) on GREINER® <NUM>-well round bottom plates, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on GREINER® <NUM>-well round bottom plates greater than <NUM>%, optionally up to <NUM>%, for BSA, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours greater than the protein recovery percentage of the unconditioned and unconverted surface for Fibrinogen having an atomic mass of <NUM>,<NUM> Daltons (FBG) on GREINER® <NUM>-well round bottom plates, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on GREINER® <NUM>-well round bottom plates greater than <NUM>%, optionally up to <NUM>%, for FBG, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on GREINER® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Transferrin having an atomic mass of <NUM>,<NUM> Daltons (TFN), following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on GREINER® <NUM>-well round bottom plates greater than <NUM>%, optionally up to <NUM>%, for TFN, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on GREINER® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Protein A having an atomic mass of <NUM>,<NUM> Daltons (PrA), following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on GREINER® <NUM>-well round bottom plates greater than <NUM>%, optionally up to <NUM>%, for PrA, following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on GREINER® <NUM>-well round bottom plates greater than the protein recovery percentage of the unconditioned and unconverted surface for Protein G having an atomic mass of <NUM>,<NUM> Daltons (PrG), following the protocol in the present specification.

In an aspect of any embodiment the converted surface has a protein recovery percentage at <NUM> hours on GREINER® <NUM>-well round bottom plates greater than <NUM>%, optionally up to <NUM>%, for PrG, following the protocol in the present specification.

In an aspect of any embodiment the surface consists essentially of polypropylene, optionally polypropylene homopolymer.

In an aspect of any embodiment the converted polypropylene surface has a protein recovery percentage greater than the protein recovery percentage of the unconditioned and unconverted polypropylene surface for Bovine Serum Albumin having an atomic mass of <NUM>,<NUM> Daltons (BSA), following the protocol in the present specification.

In an aspect of any embodiment the converted polypropylene surface has a protein recovery percentage greater than the protein recovery percentage of the unconditioned and unconverted polypropylene surface for Fibrinogen having an atomic mass of <NUM>,<NUM> Daltons (FBG), following the protocol in the present specification.

In an aspect of any embodiment the converted polypropylene surface has a protein recovery percentage greater than the protein recovery percentage of the unconditioned and unconverted polypropylene surface for Transferrin having an atomic mass of <NUM>,<NUM> Daltons (TFN), following the protocol in the present specification.

In an aspect of any embodiment the converted polypropylene surface has a protein recovery percentage greater than the protein recovery percentage of the unconditioned and unconverted polypropylene surface for Protein A having an atomic mass of <NUM>,<NUM> Daltons (PrA), following the protocol in the present specification.

In an aspect of any embodiment the converted polypropylene surface has a protein recovery percentage greater than the protein recovery percentage of the unconditioned and unconverted polypropylene surface for Protein G having an atomic mass of <NUM>,<NUM> Daltons (PrG), following the protocol in the present specification.

Various aspects will be illustrated in more detail with reference to the following Examples, but it should be understood that the more detailed embodiment is not deemed to be limited thereto.

The following protocol was used to test the plates in all embodiments, except as otherwise indicated in the examples:.

Purpose: The purpose of this experiment was to determine the amount of protein binding over time to a surface coated microtiter plate (a microtiter plate is also referred to in this disclosure as a "microplate;" both terms have identical meaning in this disclosure).

Materials: BIOTEK® Synergy H1 Microplate Reader and BIOTEK Gen5® Software, MILLIPORE® MILLI-Q® Water System (sold by Merck KGAA, Darmstadt, Germany), MILLIPORE® Direct Detect Spectrometer, ALEXA FLUOR® <NUM> Labeled Proteins (Bovine Serum Albumin (BSA), Fibrinogen (FBG), Transferrin (TFN), Protein A (PrA) and Protein G (PrG), sold by Molecular Probes, Inc. , Eugene, Oregon USA), 10X Phosphate Buffered Saline (PBS), NUNC® Black <NUM>-well Optical Bottom Plates, <NUM> Plastic Bottle, <NUM>-<NUM> Glass Beakers, Aluminum Foil, <NUM>-<NUM> Pipette, <NUM>-<NUM>µL Pipette, <NUM>-<NUM>µL Pipette, <NUM>-<NUM>µL Multichannel Pipette.

The selected proteins, one or more of those listed above, were tested on a single surface coated microplate. Each protein was received as a fluorescently labeled powder, labeled with ALEXA FLUOR® <NUM>:.

Once received, each vial of protein was wrapped in aluminum foil for extra protection from light and labeled accordingly, then placed into the freezer for storage.

A solution of <NUM> X PBS (phosphate buffer solution) was made from a stock solution of 10X PBS: <NUM> of 10X PBS was added to a plastic <NUM> bottle, followed by <NUM> of distilled water from the MILLIPORE® Q-pod, forming 1X PBS. Using a <NUM>-<NUM>µL pipette, <NUM>µL of 1X PBS was pipetted into each vial of protein separately, to create protein solutions. Each vial was then inverted and vortexed to thoroughly mix the solution.

Each protein was then tested on the MILLIPORE® Direct to get an accurate protein concentration. Using a <NUM>-<NUM>µL pipette, a <NUM>µL sample of PBS was placed on the first spot of the Direct Detect reading card and marked as a blank in the software. A <NUM>µL sample of the first protein was then placed onto the remaining <NUM> spots and marked as samples. After the card was read, an average of the <NUM> protein concentrations was recorded in mg/mL. This was repeated for the remaining four proteins. The protein solutions were then placed into the refrigerator for storage.

A standard curve was prepared with 1X PBS for each protein. The standard curve started at <NUM> and a serial 2x dilution was performed to obtain the other tested concentrations, for example one or more of <NUM>, <NUM>, <NUM> and <NUM>. Further dilutions to <NUM> were also prepared in some instances. The <NUM> solution prepared from the standard curve was used for testing.

Once the dilutions for all tested proteins were done, the standard curve for each protein was prepared and tested as follows. <NUM><NUM>-mL glass beakers were set into rows of <NUM>. Each beaker was wrapped in aluminum foil and labeled with the name of the protein the curve corresponded to and the concentration of the solution in the beaker. Row <NUM> was the standard curve for BSA; row <NUM>, FBG; row <NUM>, TFN; row <NUM>, PrA; row <NUM>, PrG. Therefore the first row was labeled as follows: BSA <NUM>, BSA <NUM>, BSA <NUM>, BSA <NUM>, BSA <NUM>.

After a standard curve was made, it was tested using the microplate reader, then the next standard curve was made and tested, and so on.

The BIOTEK® Synergy H1 microplate reader and BIOTEK Gen5® software were used for analysis.

After the first standard curve was prepared, it was ready to be tested on the Synergy H1. Using a <NUM>-<NUM>µL multichannel pipette, <NUM>µL of 1X PBS was pipetted into wells A1-A4 of a black optical bottom microplate. Then, <NUM>µL of the <NUM> solution was pipetted into wells B1-B4, <NUM>µL of <NUM> solution was pipetted into wells C1-C4, <NUM>µL of <NUM> solution was pipetted into wells D1-D4, <NUM>µL of <NUM> solution was pipetted into wells E1-E4, <NUM>µL of <NUM> solution was pipetted into wells F1-F4, and <NUM>µL of <NUM> solution was pipetted into wells G1-G4. A similar procedure was used to fill the wells with other dilutions of the protein solution.

Once the microplate was filled with solution, it was wrapped in aluminum foil and the sections and time points were labeled.

After <NUM> hours, using a <NUM>-<NUM>µL multichannel pipette and poking through the aluminum foil, <NUM>µL of BSA solution was pipetted from the wells in the <NUM> hr column (column <NUM>) and placed into a black optical bottom microplate. The black microplate was placed into the microplate tray. The other four proteins were then read the same way by opening their corresponding experiments. The same thing was done after <NUM> hours, <NUM> hours and <NUM> hours. After the <NUM> hr read, "Plate --t Export" was then selected from the menu bar. An excel spreadsheet will appear and can then be saved in the desired location with the desired name.

Using the data produced by the BIOTEK Gen5® software, the <NUM> solution concentrations from both the standard curve and SPL1 were averaged. The concentrations in the <NUM> wells at <NUM> hr were averaged. This was then done for <NUM> hr, <NUM> hr and <NUM> hr also. The average concentration at each time point was then divided by the average concentration of The <NUM> solution from the beginning and multiplied by <NUM> to get a percent recovery at each time point: <MAT>.

Polypropylene <NUM>-well microplates were plasma treated according to the more detailed embodiment. The process used to treat the microplates used a radio-frequency (RF) plasma system. The system had a gas delivery input, a vacuum pump and RF power supply with matching network. The microplates were oriented facing away from and shielded from the plasma along the perimeter of the chamber. These details are illustrated in <FIG>. The shielding resulted in remote plasma treatment in which the ratio between the radiant density at the remote points on the surfaces of the microplates and the brightest point of the plasma discharge was less than <NUM>.

The two step remote conversion plasma process used according to this example is summarized in Table <NUM>:.

The biomolecule binding resistance resulting from this remote conversion plasma process on the surface of the converted microplates was analyzed by carrying out the Testing of All Embodiments. The percent recovery is the percentage of the original concentration of the protein remaining in solution, i.e., which did not bind to the surface of a microplate.

In this testing, samples of three different types of microplates were tested for percent recovery. The samples included: (<NUM>) unconditioned and unconverted polypropylene microplates ("Untreated" samples); (<NUM>) polypropylene microplates molded by SiO2 Medical Products and converted according to the more detailed embodiment described in Example <NUM> of this specification ("SiO2" samples); and (<NUM>) Eppendorf LoBind® microplates ("EPPENDORF" samples). The bar chart in <FIG> shows the results of this comparative testing. As <FIG> illustrates, the SiO2 plates had a <NUM>% increase in biomolecule recovery compared to the Untreated Samples and an <NUM>-<NUM>% increase in biomolecule recovery compared to the Eppendorf LoBind samples.

Accordingly, remote conversion plasma treatment according to the more detailed embodiment has been demonstrated to result in lower biomolecule adhesion (or the inverse, higher biomolecule recovery) than other known methods. In fact, the comparative data of the SiO2 plates and the Eppendorf LoBind samples were particularly surprising, since Eppendorf LoBind labware has been considered the industry standard in protein resistant labware. The SiO2 plates' <NUM>-<NUM>% increase in efficacy compared to the EPPENDORF samples represents a marked improvement compared to the state of the art.

In this example, the SiO2 plates of Example <NUM> were compared to the same microplates that were converted with same process steps and conditions, except (and this is an important exception), the second samples were treated with direct plasma instead of remote conversion plasma (the "Direct Plasma" samples). Surprisingly, as shown in <FIG>, the Direct Plasma samples had a biomolecule recovery percentage after <NUM> hours of <NUM>%, while the SiO2 plates (which were converted under the same conditions/process steps except by remote conversion plasma) had a biomolecule recovery percentage after <NUM> hours of <NUM>%. This remarkable step change demonstrates the unexpected improvement resulting solely from use of remote conversion plasma in place of direct plasma.

In this example, the SiO2 plates of Example <NUM> were compared to the same microplates that were treated with only the conditioning step of the method of the more detailed embodiment (i.e., the non-polymerizing compound plasma step or conditioning plasma treatment) without the conversion step (water vapor plasma step or conversion plasma treatment) ("Step <NUM> Only" samples). As shown in <FIG>, Step <NUM> Only samples had a biomolecule recovery percentage after <NUM> hours at about <NUM> (the aging of all protein samples in this specification is at <NUM> unless otherwise indicated) of <NUM>%, while the SiO2 plates (which were processed under the same conditions/process steps except also converted by remote conversion plasma) had a biomolecule recovery percentage after <NUM> hours of <NUM>%. Accordingly, using both steps of the method according to the more detailed embodiment results in significantly improved biomolecule recovery percentage than using only the conditioning step alone.

A further contemplated optional advantage of the more detailed embodiment is that it provides high levels of resistance to biomolecule adhesion without a countervailing high extractables profile. For example, Eppendorf LoBind® labware is resistant to biomolecule adhesion by virtue of a chemical additive, which has a propensity to extract from the substrate and into a solution in contact with the substrate. By contrast, the more detailed embodiment does not rely on chemical additives mixed into a polymer substrate to give the substrate its biomolecule adhesion resistant properties. Moreover, processes according to the more detailed embodiment do not result in or otherwise cause compounds or particles to extract from a converted substrate. Applicant has further determined that the pH protective coating or layer described in this disclosure does not result in or otherwise cause compounds or particles to extract from a converted surface.

Accordingly, in one optional aspect, the more detailed embodiment described herein relates to a method for treating a surface, also referred to as a material or workpiece, to form a converted surface having a biomolecule recovery percentage greater than the biomolecule recovery percentage of the surface prior to conversion treatment, and in which any conditioning or conversion treatment does not materially increase the extractables profile of the substrate. Applicants contemplate that this would bear out in actual comparative tests between the unconditioned and unconverted surface and the converted surface.

A test similar to Example <NUM> was carried out to compare the biomolecule recovery from unconditioned and unconverted polypropylene (UTPP) laboratory beakers, remote conversion plasma converted polypropylene (TPP) laboratory beakers according to the more detailed embodiment, and unconditioned and unconverted glass laboratory beakers. The biomolecules used were <NUM> dispersions of lyophilized BSA, FBG, TFN, PrA, and PrG.

In a first trial, the biomolecule dispersion was made up in the beaker and aspirated several times to mix it. The biomolecule recovery was measured in relative fluorescence units (RFU). The initial RFU reading (<NUM>) was taken to establish a <NUM>% recovery baseline, then the biomolecule dispersion in the beaker was stirred for <NUM> with a pipet tip, after which it was allowed to remain on the laboratory bench undisturbed for the remainder of the test. The biomolecule recovery was measured initially, and then a sample was drawn and measured for percentage biomolecule recovery at each <NUM>-minute interval. The results are shown in Table <NUM>.

A second trial, with results shown in Table <NUM>, was carried out in the same manner as the first trial except that glass beakers, not converted according to the more detailed embodiment, were used as the substrate.

<FIG> plots the TFN results in the Tables above, showing plots <NUM> for the unconditioned and unconverted polypropylene beaker, <NUM> for the converted polypropylene beaker, and <NUM> for glass. As <FIG> shows, the converted polypropylene beaker provided the highest biomolecule recovery after <NUM> to <NUM> minutes, glass produced a lower biomolecule recovery after <NUM> to <NUM> minutes, and the unconditioned and unconverted polypropylene beaker provided the lowest biomolecule recovery at all times after the initial measurement.

A test similar to Example <NUM> was carried out to compare the biomolecule recovery from multiwell polypropylene plates of two types, versus protein concentration, after <NUM> hours of contact between the protein and the plate. "SiO2" plates were molded from polypropylene and plasma converted according to Example <NUM>. "CA" (Competitor A) plates were commercial competitive polypropylene plates provided with a coating to provide reduced non-specific protein binding.

The results are provided in Table <NUM> and <FIG> showing that essentially all the protein of each type was recovered from the converted SiO2 plates at all tested concentrations, so the recovery was independent of concentration. In contrast, the protein recovery from the CA plates depended strongly on the concentration, particularly at lower concentrations.

A test similar to Example <NUM> was carried out to compare the biomolecule recovery from converted "SiO2" plates and "CA" plates of the types described in Example <NUM>. The biomolecules used were <NUM> or <NUM> dispersions of lyophilized BSA, FBG, TFN, PrA, and PrG.

The conditions and results are shown in Table <NUM>. For the BSA, PrA, PrG, and TFN proteins, the converted SiO2 plates provided substantially superior protein recovery, compared to the CA plates. For the FBG protein, the converted SiO2 plates provided better protein recovery than the CA plates.

A test similar to Example <NUM> was carried out to compare <NUM>-well, <NUM>µL SiO2 and CA plates. The conditions and results are shown in Table <NUM>. For the BSA, PrA, PrG, and TFN proteins, as well as the <NUM> concentration of FBG, the converted SiO2 plates provided substantially superior protein recovery, compared to the CA plates. The <NUM> concentration of FBG was anomalous.

A test similar to Example <NUM> was carried out to compare <NUM>-well, <NUM>µL converted SiO2 and CA plates. The conditions and results are shown in Table <NUM>. For the BSA, PrA, and PrG proteins, the converted SiO2 plates provided substantially superior protein recovery, compared to the CA plates. The FBG proteins did not demonstrate substantially superior protein recovery.

A test similar to Example <NUM> was carried out to compare <NUM>-well <NUM>µL (converted SiO2) vs <NUM>µL (CA) shallow plates. The conditions and results are shown in Table <NUM>. For the BSA, PrA, and PrG proteins, the converted SiO2 plates provided substantially superior protein recovery, compared to the CA plates. The FBG proteins did not demonstrate substantially superior protein recovery.

A test similar to Example <NUM> was carried out to compare the SiO2 converted plates of the more detailed embodiment to polypropylene plates treated with StabilBlot® BSA Blocker, a commercial treatment used to reduce BSA protein adhesion, sold by SurModics, Inc. , Eden Prairie, MN, USA. The conditions and results are shown in Table <NUM>, where converted SiO2 is the plate according to Example <NUM>, Plate A is a polypropylene plate treated with <NUM>% BSA blocker for one hour and Plate B is a polypropylene plate treated with <NUM>% BSA blocker for one hour. Except for FBG protein, the present invention again provided superior results compared to the BSA blocker plates.

A test similar to Example <NUM> was carried out to compare the protein recovery rates of SiO2 converted plates in accordance with Example <NUM> over longer periods of time - from <NUM> to <NUM> months. The conditions and results are shown in Table <NUM>, which illustrates that roughly uniform resistance to protein adhesion was observed for all of the proteins over a substantial period.

The uniformity of binding among the different wells of a single plate was tested using two <NUM>-well plates with deep (<NUM>µL) wells, a converted SiO2 plate prepared according to Example <NUM> except testing <NUM> PrA protein after two hours in all <NUM> wells, and the other a Competitor A plate, again testing <NUM> PrA protein after two hours in all <NUM> wells. The protein recovery from each well on one plate was measured, then averaged, ranged (determining the highest and lowest recovery rates among the <NUM> wells), and a standard deviation was calculated. For the converted SiO2 plate, the mean recovery was <NUM>%, the range of recoveries was <NUM>%, and the standard deviation was <NUM>%. For the CA plate, the mean recovery was <NUM>%, the range of recoveries was <NUM>%, and the standard deviation was <NUM>%.

The same test as in the preceding paragraph was also carried out using <NUM>-well plates with <NUM>µL wells. For the converted SiO2 plate, the mean recovery was <NUM>%, the range of recoveries was <NUM>%, and the standard deviation was <NUM>%. For the CA plate, the mean recovery was <NUM>%, the range of recoveries was <NUM>%, and the standard deviation was <NUM>%.

This testing indicated that the conversion treatment of Example <NUM> allows at least as uniform a recovery rate among the different wells as the protein resisting coating of the CA plate. This suggests that the SiO2 plasma treatment is very uniform across the plate.

This example was carried out to compare the protein recovery from multiwell polypropylene plates of two types versus protein concentration, after <NUM> hours of contact between the protein and the plate. SiO2 plates were molded from polypropylene and plasma converted according to Example <NUM>. "EPP" plates were commercial competitive polypropylene Eppendorf LoBind® plates. The testing protocol is the same as in Example <NUM>, except that the smallest protein concentrations -- <NUM> -- were much lower than those in Example <NUM>.

The results are shown in Table <NUM> and <FIG>. In fact, the comparative data of the converted SiO2 plates (plots <NUM>, <NUM>, and <NUM>) and the Eppendorf LoBind® plates (i.e. "EPP" plates, plots <NUM>, <NUM>, and <NUM>) were particularly surprising, since Eppendorf LoBind® has been considered the industry standard in protein resistant labware. For all three types of proteins tested in the example (i.e. BSA, PrA and PrG), the protein recovery was substantially constantly high regardless of the concentration for converted SiO2 plates. However, for "EPP" plates, the protein recovery was dramatically lowered at low concentration. Especially at ultra-low concentration (e.g. from <NUM> to less than <NUM>), the protein recoveries for the SiO2 converted plates were far superior to the "EPP" plate.

For the PrG protein as shown by data marked with asterisks in Table <NUM>, the <NUM> SiO2 converted plate data point was regarded as anomalous, since the true protein recovery of the SiO2 converted plate cannot exceed <NUM>% plus the error limit assigned to the data point. The <NUM> EPP Plate PrG data point also was regarded as anomalous, since it deviates substantially from the trend of the other data points. These anomalous data points are not shown in <FIG>.

This testing was carried out on a <NUM>-well microplate to evaluate if the present conversion treatment adds extractables to the solution in contact with the substrate. The microplate was molded from polypropylene and converted with plasma according to Example <NUM>.

<NUM>µL isopropanol (IPA) was added to a total of <NUM> wells in the <NUM>-well microplate. After the addition, the plate was covered firmly with a glass plate and stored at room temperature for <NUM> hours. Following extraction, the contents of the <NUM> wells were combined in one individual vial, capped, and inverted to mix. Individual aliquots were transferred to autosampler vials for GC-MS analyses.

The GC-MS (gas chromatography - mass spectrometry) analysis conditions are shown in Table <NUM> and a resulting plot, annotated with the eight peak assignments made, is shown in <FIG> and the peak assignments are described in Table <NUM>.

<FIG> show the GC-MS plots, measured in the same way as <FIG>, characterizing extracted organic species for an isopropanol blank (<FIG>) vs. the converted SiO2 low protein binding treated microplates according to Example <NUM> (<FIG>).

An LC-MS (liquid chromatography - mass spectroscopy) method was used to analyze the organic extractables and evaluate if the present conversion treatment adds organic extractables to the solution in contact with the substrate. Extraction procedures are the same as in Example <NUM>.

Analyses were conducted with an Agilent G6530A Q-TOF mass spectrometer and extracts were run in both positive and negative APCI modes. The LC-MS conditions for positive APCI are shown in Table <NUM> and the LC-MS conditions for negative APCI are shown in Table <NUM>.

<FIG> shows the comparison of the LC-MS isopropanol extracted ion chromatogram (positive APCI mode) of the SiO2 low protein binding converted plates (lower plot) vs that of the isopropanol blank (upper plot).

<FIG> shows the comparison of the LC-MS isopropanol extracted ion chromatogram (negative APCI mode) of the SiO2 low protein binding converted plates (lower plot) vs that of the isopropanol blank (upper plot).

The only unmatched peak for SiO2 converted plates is at m/z <NUM> which is consistent with Irganox® <NUM> in the unconditioned and unconverted SiO2 plates' isopropanol extract (<FIG>, lower plot), vs. an isopropanol blank (upper plot). Therefore, this extracted compound was not added by the present low protein binding treatment. It came from the resin, as it also was extracted from unconditioned and unconverted SiO2 plates.

An ICP-MS method was used to compare the inorganic extractable level of three types of <NUM>-well microplates. The three types of microplates are unconditioned and unconverted commercial Labcyte polypropylene microplates (Labcyte), unconditioned and unconverted commercial Porvair polypropylene microplates (Porvair) and SiO2 low binding plasma converted microplates, molded by SiO2 Medical Products, Inc. from polypropylene and converted with plasma according to Example <NUM>.

The wells in the microplates were filled with a <NUM>% v/v nitric acid (HNO<NUM>) solution in de-ionized (DI) water, covered with a glass plate, and allowed to extract at room temperature for <NUM> hours. Then approximately <NUM> of the solution were transferred into autosampler tubes and analyzed by ICP-MS using an Agilent 7700x spectrometer and the conditions are shown in Table <NUM>.

The results are shown in Table <NUM>. The results show that nitric acid extracts of converted SiO2 plates have low levels of inorganics, near equivalent to that of unconditioned and unconverted Labcyte and Porvair plates. Therefore SiO2 Medical Products low protein binding conversion treatment does not add inorganic extractables.

Claim 1:
A method for treating a surface of a plastic substrate, the method comprising:
optionally, a conditioning plasma treatment carried out by treating a surface of a plastic substrate with conditioning plasma of one or more non-polymerizing compounds generated at a remote point from the surface, where the ratio of the radiant energy density at the remote point to the radiant energy density at the brightest point of the conditioning plasma is less than <NUM>, optionally less than <NUM>, forming a conditioned surface; and
a conversion plasma treatment carried out by treating the conditioned surface (if the optional step is performed) or unconditioned surface (if the optional step is omitted) with conversion plasma of water vapor, the conversion plasma being generated at a remote point from the conditioned surface, where the ratio of the radiant energy density at the remote point of conversion plasma treatment to the radiant energy density at the brightest point of the conversion plasma is less than <NUM>, optionally less than <NUM>, wherein the radiant energy density at the brightest point of the plasma is determined spectrophotometrically by measuring the radiant intensity of the most intense emission line of light in the spectrum from <NUM> to <NUM> wavelength at the brightest point, and the radiant energy density at the remote point is determined spectrophotometrically by measuring the radiant energy density of the same emission line of light at the remote point,
to form a converted surface having a biomolecule recovery percentage, for an aqueous protein dispersion having a concentration from <NUM> to <NUM>, optionally <NUM> to <NUM>, optionally <NUM> to <NUM>, in contact with the converted surface, greater than <NUM>%, wherein the biomolecule recovery percentage is determined according to the method "Testing of all embodiments" described in the description.