Improved method for plasma immobilization of biomolecule to substrate via linking molecule

Abstract

To provide a plasma technique, including a method for introducing biological molecules into a substrate.SOLUTION: The present invention relates to a two-step method for immobilization of a biomolecule through a linking molecule on a sample surface of a substrate by generating and maintaining a non-thermal atmospheric pressure plasma at a temperature between room temperature and 60°C. A preferred plasma temperature is room temperature. The method includes a first step and a second step, which are sequentially executed. In the first step of the method, the linking molecule is deposited onto the sample surface through exposing the sample surface to a first plasma jet and the linking molecule, thereby generating a linking layer onto the sample surface. In a second sequential step of the method, the biomolecule is deposited onto the linking layer through exposing the linking layer to a second plasma jet and the biomolecule.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to plasma techniques, and in particular involves the inclusion of biological molecules in plasma deposited layers. [Background technology]

[0002] WO 2017 / 136334 A1 discloses a method for depositing biomolecules, pharmaceuticals, and other therapeutic active agents on a surface. The method includes a plasma device having one or more electrodes, a gas supply inlet, a plasma outlet exposed to ambient pressure, and an ignition system operably connected to the electrodes to provide a non-thermal equilibrium plasma inside the plasma chamber. A particle delivery system can be used to introduce the active agent as a dry powder into or downstream of the plasma, and deposit the plasma-treated active agent to produce a coating on the surface. The coating can retain the activity of the active agent. The present invention attempts to solve the problem of eliminating any wet chemistry steps to obtain at least two layers of biological coatings on a substrate using non-thermal atmospheric plasma. However, this method is not suitable for many substrate-biomolecule combinations due to the harsh plasma conditions to which the biomolecules are exposed.

[0003] WO 2005 / 106477 discloses a method for immobilizing biomolecules on a surface by generating and maintaining an atmospheric pressure plasma. Immobilization is achieved by introducing a sample into the space between two electrodes, where a plasma is generated by applying an alternating voltage to the electrodes, and a coating is deposited. The atmosphere that is mixed contains an aerosol containing reactive precursors and an aerosol containing biomolecules, both of which are deposited and immobilized during the deposition step. This method is also not suitable for many combinations of substrate and biomolecule. Another drawback of this method is the efficiency of immobilization of biomolecules. Simultaneous deposition of both linking molecules and biomolecules can result in steric hindrance, limiting the amount of biomolecules that can be deposited. Furthermore, deposition of a first linking layer can increase the amount of biomolecules immobilized on the surface.

[0004] The document US 2007 / 118211 A1 discloses a method for preparing a drug-eluting medical device, which comprises the application to a stent of a polymer having functional groups capable of chemically binding biological molecules, characterized in that said application is carried out in a single step by a cold plasma method. Moreover, the present invention also relates to the medical device obtained therefrom.

[0005] WO 2017 / 136334 A1 discloses a plasma system for depositing biomolecules, pharmaceuticals, and other therapeutic active agents on a surface. The system can include a plasma device having one or more electrodes, a gas supply inlet, a plasma outlet exposed to ambient pressure, and an ignition system operably connected to the electrodes to provide a non-thermal equilibrium plasma inside the plasma chamber. A particle delivery system can be used to introduce the active agent as a dry powder into or downstream of the plasma and deposit the plasma-treated active agent to produce a coating on the surface. The coating can retain the activity of the active agent.

[0006] The document US 2009 / 298251 A1 discloses an aerosol spray device and a method of forming a film using the aerosol spray device. The aerosol spray device according to an embodiment of the present invention vaporizes a liquefied gas to form a capillary. The device includes a carrier gas injection section that generates rear gas and pressurizes the carrier gas, an aerosol formation section that mixes the carrier gas with powder to form an aerosol, and a film formation section that sprays the aerosol under normal pressure so that a film is formed on the surface of the substrate. This device can perform coating processing without limiting the type or size of powder, and can simplify the process by forming a film under normal temperature and pressure environments, and can control the film thickness over a wide range in a short time.

[0007] The document US 2008 / 118734 A1 discloses a method for forming an active material containing coating on a substrate, the method comprising the steps of: i) introducing one or more gaseous or atomized liquid and / or solid coating-forming materials that undergo chemical bond forming reactions within the plasma environment, and one or more active materials that do not substantially undergo chemical bond forming reactions within the plasma environment, into an atmospheric or low pressure non-thermal equilibrium plasma discharge and / or an excited gas flow resulting therefrom; and ii) exposing a substrate to the resulting mixture of atomized coating-forming materials and at least one active material that is deposited on the substrate surface to form a coating, the substrate being not a wipe, cloth, or sponge for household care or hair removal care, or a water-soluble household cleaning unit dose product. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to overcome at least some of the problems and drawbacks mentioned above.The object of the present invention is to provide a method which eliminates these drawbacks. [Means for solving the problem]

[0009] Summary of the Invention In a first aspect, the present invention provides a two-step method for immobilizing biomolecules via linking molecules on a sample surface of a substrate by generating and maintaining a non-thermal atmospheric pressure plasma as claimed in claim 1.

[0010] The method according to the invention allows a much wider selection of substrate and biomolecule combinations. In particular, combinations of relatively inert substrates with substrates to which biomolecules have proven difficult to bind, as well as biomolecules that are plasma-sensitive, can be advantageously immobilized by the invention. This can be done by selecting the appropriate linking layer and the corresponding plasma conditions of both steps.

[0011] The presence of the tie layer allows for more efficient immobilization of biomolecules, either in terms of the amount of biomolecules bound, the ability to control the orientation of the immobilization of the biomolecules, or both. Furthermore, immobilization via the tie layer by using plasma technology results in a simple two-step method, without the need for time-consuming and complicated multi-step wet chemistry processes. This also eliminates the use of expensive and environmentally harmful solvents.

[0012] In a second aspect, the present invention provides an apparatus suitable for the two-step immobilization of biomolecules onto a sample surface of a substrate via a linking molecule as claimed in claim 10 or 11.

[0013] The device is advantageously adapted for the two-step process according to the invention. The device is suitable for normal atmospheric non-thermal plasmas. However, the device is particularly suitable for plasma-sensitive biomolecules.

[0014] The device is adapted to generate a relatively uniform plasma along the surface of the substrate due to the nozzle. Furthermore, the nozzle creates a slight overpressure relative to the environment, which largely excludes oxygen, as the oxygen inactivates the ionized molecules before said molecules reach the surface. A much more uniform plasma is produced without any photocatalytic degradation. This also allows for gentler plasma conditions along the jet while still retaining enough energy to deposit molecules on a surface or a bonding layer.

[0015] In a third aspect, the present invention discloses a device obtainable using a two-step immobilization of biomolecules to a sample surface of a substrate via a linking molecule, as claimed in claim 13.

[0016] The broader combinations of substrates and biomolecules that can be combined by the methods of the present invention allow for new or improved applications or products. These applications can be found in, but are not limited to, the medical, chemical, environmental, food, and materials industries. Specific products include, but are not limited to, biosensors, lab-on-a-chip, biomimetic materials, solar cells, non-fouling surfaces, antibacterial coatings, templates for both in vitro or in vivo growth and functional tissues, bio-derived crystal morphology, conductive coatings, or biocatalytic applications.

[0017] Advantageously, the present invention provides a solution to the industrialization limitations caused by the use of wet chemical steps in the manufacture of medical devices, wound care products, biosensors, labs-on-a-chip, non-contaminating surfaces and antimicrobial coatings. One particular example includes the immobilization of biomolecules on glass, quartz or silicon wafers for research and diagnostics. Another is the immobilization of biomolecules, especially those with antibacterial or antifungal properties, on textiles for wound dressings. [Brief description of the drawings]

[0018] [Figure 1] 1 shows the interactions between reagents in a sandwich ELISA. [Diagram 2] A comparison of the functionality of plasma deposited antibodies to those deposited by conventional wet chemical methods is shown. [Diagram 3]The relationship between blank and positive samples is shown (Ab+AA, no incubation, 4 slm, 9 seconds). [Figure 4] Shown is the relationship between blank samples and positive samples (Ab only, no incubation, 2 slm, different exposure times) and between control samples without capture antibody and positive samples. [Diagram 5] The relationship between empty sample and positive sample is shown (Ab only, no incubation, different aspiration flows, 10 seconds). [Figure 6] FIG. 1 shows a comparison of the effect of different incubation methods on the functionality of antibodies after immobilization using atmospheric plasma (Ab+AA). [Figure 7] FIG. 1 shows a comparison of the effect of different incubation methods on the functionality of antibodies after immobilization using atmospheric plasma (Ab only). [Figure 8] A comparison of the functionality of immobilized antibodies with and without simultaneous plasma polymerization of acrylic acid is shown. [Figure 9] 1 shows the effect of aspiration flow on ELISA endpoints. [Figure 10] The effect of exposure time on the ELISA endpoint (without incubation) is shown. [Figure 11] FIG. 1 shows the effect of exposure time on the ELISA endpoint (wet incubation). [Figure 12] The effect of exposure time on the ELISA endpoint (1 slm, no incubation, Ab+AA) is shown. [Figure 13] FIG. 1 shows the effect of exposure time on the ELISA endpoint (4 slm, no incubation, Ab+AA). [Figure 14] 1 shows a front view of an embodiment of a device according to the invention; [Figure 15a] 1 shows a longitudinal view of an embodiment of a shield according to the invention. [Figure 15b] 1 shows a side view of an embodiment of a shield according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention relates to a two-step method and apparatus for immobilizing biomolecules via linking molecules on a sample surface of a substrate via non-thermal atmospheric pressure plasma. The present invention is summarized in the corresponding section above. In the following, the present invention is described in detail, preferred embodiments are discussed, and the present invention is illustrated by examples.

[0020] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, definitions of terms are included to better understand the teachings of the present invention.

[0021] As used herein, the following terms have the following meanings. As used herein, "a," "an," and "the" refer to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more compartments.

[0022] "About" as used herein to refer to a measurable value such as a parameter, amount, temporal duration, and the like, is meant to encompass variations of up to + / -20%, preferably up to + / -10%, more preferably up to + / -5%, even more preferably up to + / -1%, and even more preferably up to + / -0.1%, etc., of the specified value, such variations being suitable for practice with the disclosed invention, but it is also to be understood that the value to which the modifier "about" refers is itself specifically disclosed.

[0023] As used herein, the terms "comprise", "comprising", and "comprises" and "comprised" are used interchangeably. "Of" is synonymous with "include," "including," "includes," or "contain," "containing," or "contains" and is an inclusive or open-ended term specifying the presence of what follows, e.g., a component, and does not exclude or preclude the presence of additional, unrecited components, features, elements, materials, or steps that are known in the art or disclosed therein.

[0024] Moreover, the terms first, second, third, etc. in the specification and claims are used to distinguish between similar elements, unless otherwise specified, and are not necessarily intended to describe a sequential or chronological order. The terms so used are interchangeable under appropriate circumstances, and it is to be understood that the embodiments of the invention described herein may operate in sequences other than those described or illustrated herein.

[0025] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0026] The term "one or more" or "at least one," such as one or more or at least one member of a group of members, will itself be clear with further illustration, but the term specifically encompasses reference to any one of said members, or any two or more of said members, for example > 3, > 4, > 5, > 6 or > 7 of any of said members, etc., up to all of said members.

[0027] As used herein, "sample surface" means a portion or the entire surface of a substrate. Preferably, the sample surface is at least 1 μm 2 The surface of the

[0028] As used herein, "tie layer" means a layer that includes tie molecules that can undergo polymerization or remain as monomers on the surface upon deposition.

[0029] As used herein, "plasma jet" refers to the plasma jet and / or the afterglow of the plasma jet. For example, a coating precursor injected into the "plasma jet" inside a shield can refer to a coating precursor injected into the plasma jet and / or the afterglow of the plasma jet inside a shield.

[0030] As used herein, "moiety" refers to a part of a molecule that is named such that it may also be found within other types of molecules. A moiety as defined herein includes at least one functional group or at least one smaller moiety or a combination thereof.

[0031] As used herein, "biomolecule" refers to molecules or ions present in living organisms, particularly those essential to some typical biological processes, as well as any molecule that is biologically and pharmacologically active. This includes synthetic chemicals that exhibit biological or pharmacological activity even if they do not occur "in nature."

[0032] A "biologically or pharma- ceutical active molecule" as described herein relates to a molecule that has a biological or pharmacological activity on a living organism, i.e., the molecule relates to an effect on a biological material that is either beneficial or detrimental.

[0033] In a first aspect, the present invention provides a two-step method for immobilizing biomolecules via linking molecules on a sample surface of a substrate by generating a non-thermal atmospheric pressure plasma and maintaining it at a temperature between room temperature and 60°C. The preferred plasma temperature is room temperature. Room temperature is understood to be between 5°C and 25°C, more preferably between 15°C and 25°C, and most preferably about 20°C. The method comprises a first step and a second step carried out consecutively. In the first step of the method, the linking molecules are deposited on the sample surface by exposing the sample surface to a first plasma jet and the linking molecules, producing a linking layer on the sample surface. In the second consecutive step of the method, the biomolecules are deposited on the linking layer by exposing the linking layer to a second plasma jet and the biomolecules.

[0034] Tie layers are frequently required to improve adhesion between the surface of a substrate and biomolecules. The two-step process is advantageous because it allows the use of different plasma conditions for the generation of the tie layer and for the immobilization of biomolecules. This is particularly advantageous when using surfaces that require harsh plasma treatment or when immobilizing biomolecules that are sensitive to plasma.

[0035] As used herein, "atmospheric pressure" indicates that the pressure is approximately the same as that of the surrounding environment. This term distinguishes the present plasma technology from low and high pressure plasma technologies that require the reaction vessel to maintain a substantial pressure differential with the environment. Thus, those skilled in the art of plasma technology will understand that "atmospheric pressure" as used herein should not be interpreted as "atm", a unit of pressure defined as 101325 Pa. Preferably, the plasma maintains a pressure between 0.6 and 5.0 bar, more preferably, the plasma maintains a pressure between 0.7 and 4.5 bar, more preferably, the plasma maintains a pressure between 0.8 and 3.0 bar, more preferably, the plasma maintains a pressure between 0.9 and 2.5 bar, more preferably, the plasma maintains a pressure between 1.0 bar and 2.5 bar, and most preferably, the plasma maintains a pressure above 1.0 bar and below 2.5 bar. Most preferably, the plasma maintains a slightly higher pressure compared to the pressure of the environment. This allows for a large amount of pressure to be transferred from the environment to the plasma. This is particularly advantageous in combination with the device according to the invention, since it significantly reduces the oxygen influx into the plasma. The result is a more stable and homogeneous plasma. The increased homogeneity between the plasma jet and the surface of the substrate allows the use of milder plasma jet conditions while retaining sufficient activity to generate a linking layer or immobilize biomolecules. This is particularly advantageous for plasma-sensitive molecules. Most biologically active molecules, such as proteins, are plasma-sensitive.

[0036] In a preferred embodiment, the plasma gas used to generate both said plasma jets is selected from the group comprising helium, argon, nitrogen, air, carbon dioxide, ammonium or combinations thereof. It is possible to use two different plasma gases for each step of the process. More preferably, argon is used as the plasma gas to generate the second plasma jet. Argon provides a low aggressive climate and maintains the integrity of epitopes. This allows plasma sensitive molecules, such as many biomolecules that would otherwise denature in the presence of plasma, to be immobilized on the surface while retaining their biological functionality. More preferably, argon is used as the plasma gas to generate both said plasma jets. This advantageously allows both plasma jets to be generated using the same plasma gas. This also allows the use of a plasma device with a single plasma jet and multiple emitting precursor channels, which has lower capital costs and lower operating costs. Preferably, the multiple emitting precursor channels are located upstream of the jet outlet, more preferably within the plasma jet generator. In an embodiment, the device comprises 2, 3, 4, 5 or more emitting precursor channels. Depending on the process for which the device is intended, the number of emitting precursor channels can be selected taking into account the number of different precursors used in the process, and more preferably the number of emitting precursor channels is at least the number of different precursors.

[0037] In a preferred embodiment, the first plasma jet is generated with a first electrode power and the second plasma jet is generated with a second electrode power, the first electrode power being higher than the second electrode power. The lower second electrode power allows the generation of a less energetic plasma, which is favorable for immobilizing plasma-sensitive biomolecules, while the more energetically ordered plasma is beneficial for depositing a connection layer.

[0038] It is understood that the plasma jet is generated using electrodes operating at electrode powers. A first plasma jet is generated at a first electrode power. A second plasma jet is generated at a second electrode power. As used herein, "electrode power" is defined as a function of the area of ​​the plasma exit of the nozzle. This more accurately represents the input of power per surface area of ​​the affected substrate.

[0039] In a preferred embodiment, the first electrode power is at least 1.0 W / cm 2 , preferably at least 1.1 W / cm 2 , more preferably at least 1.2 W / cm 2 , more preferably at least 1.3 W / cm 2 , more preferably at least 1.4 W / cm 2 , more preferably about 1.5 W / cm 2 These electrode outputs are suitable for a wide range of combinations of linking molecules and substrates, especially relatively inert substrate surfaces.

[0040] In a preferred embodiment, the second electrode power is up to 1.5 W / cm 2 , preferably up to 1.4 W / cm 2 , more preferably up to 1.3 W / cm 2 , more preferably up to 1.2 W / cm 2 , more preferably up to 1.1 W / cm 2 , more preferably up to 1.0 W / cm 2 , more preferably up to 0.9 W / cm 2 , more preferably up to 0.8 W / cm 2 , more preferably about 0.7 W / cm 2 It is preferable to keep the second electrode power as low as possible to limit biomolecule fragmentation or any other effects that may result in reduced biomolecule activity.

[0041] In a preferred embodiment, the first electrode power is at least 1.0 W / cm 2 and the second electrode power is up to 1.5 W / cm 2and more preferably the first electrode power is at least 1.1 W / cm 2 and the second electrode power is up to 1.4 W / cm 2 and more preferably the first electrode power is at least 1.2 W / cm 2 and the second electrode output is up to 1.3 W / cm 2 and more preferably the first electrode power is at least 1.2 W / cm 2 and the second electrode power is up to 1.2 W / cm 2 and more preferably the first electrode power is at least 1.3 W / cm 2 and the second electrode output is up to 1.1 W / cm 2 and more preferably the first electrode power is at least 1.4 W / cm 2 and the second electrode output is up to 1.0 W / cm 2 and more preferably the first electrode power is at least 1.5 W / cm 2 and the second electrode output is up to 1.0 W / cm 2 and more preferably the first electrode power is at least 1.6 W / cm 2 and the second electrode power is up to 0.9 W / cm 2 and more preferably the first electrode power is at least 1.7 W / cm 2 and the second electrode power is up to 0.8 W / cm 2 and most preferably the first electrode power is about 1.8 W / cm 2 and the second electrode power is about 0.7 W / cm 2 It is.

[0042] In a preferred embodiment, the substrate is selected from the group including biological materials, composite materials, crystalline solids, textiles, metals, plastics, polymers, ceramics, glass or combinations thereof. Preferably, the sample surface is selected from the group consisting of glass, quartz, plastics, polymers, textiles, silicon, skin, tissue or combinations thereof. Glass, quartz and silicon are particularly advantageous in research and diagnostic environments. They are reusable, relatively cheap, inert and already used as industry standards.

[0043] Tissue, textiles and skin are particularly advantageous for medical and diagnostic products used on biological materials such as wound dressings. Immobilization of biologically active molecules on surfaces allows for more targeted use of biomolecules. Biomolecules can be prevented from dispersing or absorbing after application to biological materials. Biomolecules can be removed by removing the surface.

[0044] In a preferred embodiment, the linking molecule is a molecule comprising at least one moiety. Preferably, the linking molecule comprises two moieties. In a preferred embodiment, at least one moiety is suitable for attachment of the linking layer to a biomolecule. A well-chosen moiety allows for better, easier and stronger attachment of the biomolecule to the linking layer. This advantageously allows for the use of more biomolecules, especially sensitive molecules. This also allows for targeting and manipulating the orientation of the biomolecules in the linking layer. A favorable orientation increases the availability of the biomolecules to the biological material. In another preferred embodiment, the moiety is suitable for binding of the linking molecule to the sample. This improves the strength of the bond between the linking layer and the surface of the substrate. In another preferred embodiment, the moiety is suitable for polymerization of the linking molecule. This allows the linking layer to form a polymer network that improves the strength of the bond between the surface of the substrate and the biomolecule. It can also improve the chemical and mechanical properties of the surface, for example increasing the hardness and decreasing the reactivity. In a further preferred embodiment, the linking molecule comprises at least two moieties, one suitable for binding the linking molecule to the surface of the substrate and at least one suitable for binding the biomolecule to the linking layer. In the most preferred embodiments, these moieties are not positioned next to each other to minimize the occurrence of steric hindrance.

[0045] In a preferred embodiment, the at least one moiety is an alkane, alkene, alkyne, benzene derivative, haloalkane, fluoroalkane, chloroalkane, bromoalkane, iodoalkane, alcohol, ketone, aldehyde, acyl halide, carbonate, carboxylate, carboxylic acid, ester, methoxy, hydroperoxide, peroxide, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, heterocycle, orthocarbonate ester, amide, amine, imine, imide, azide, The moiety is selected from the group comprising azo compounds, cyanates, nitrates, nitrites, nitro compounds, nitroso compounds, oximes, pyridine derivatives, thiols, thioethers, disulfides, sulfoxides, sulfones, sulfinic acids, sulfonic acids, sulfonic acid esters, thiocyanates, thioketones, thials, thioesters, phosphines, phosphonic acids, phosphates, phosphodiesters, boronic acids, boronic acid esters, borinic acids, borinic acid esters, or combinations thereof. Preferably, the moiety is selected from the group comprising carboxylic acids, thiols, hydroxyls, amines, or trialkoxysilanes. These moieties are particularly well suited for binding plasma-sensitive biomolecules, in particular antibodies. This allows the immobilization of these molecules to substrates that are otherwise difficult to adhere to, such as glass, quartz, plastics, polymers, textiles, silicon, skin, tissue, or combinations thereof.

[0046] In a preferred embodiment, the biomolecule is a biologically or pharma- ceutical active molecule. In a preferred embodiment, the biomolecule is selected from the group consisting of proteins, polynucleotides, sugars, lipids, growth factors, hormones and bioactive substances or combinations thereof. Preferably, said biomolecule is an antibody or comprises antibacterial and / or antifungal activity. Immobilizing biologically or pharmacologic active molecules is advantageous in research and diagnostics where a controlled environment is desired. It is also advantageous for the application of targeted drugs. Surface-bound biological or pharmacologic activity can be applied in a very controlled manner at a specific time and at a specific place. Thus, the present invention is particularly suitable for use in the manufacture of diagnostic devices or wound healing products. Most preferably, the biomolecule is an antibody. Antibodies are generally very plasma sensitive. The present invention is particularly advantageous for immobilizing plasma sensitive molecules.

[0047] In a preferred embodiment, the biomolecule is administered in a dissolved state. In a more preferred embodiment, the biomolecule is dissolved in an aqueous solvent. In another preferred embodiment, the biomolecule is inserted into the plasma jet as an aerosol. In a further preferred embodiment, the biomolecule is inserted into the plasma jet as an aqueous aerosol. Without being bound by theory, it is believed that this allows the biomolecule to be protected by an aqueous shell upon insertion into the plasma jet, which helps to preserve its biological activity. The administration of the biomolecule via an aqueous aerosol also promotes a uniform distribution of the biomolecule, since the aerosol limits clustering. This improves the availability of the biomolecule on the surface of the substrate.

[0048] In one embodiment, the step of providing a linking layer includes multiple steps of creating multiple layers that function as linking layers. For example, a first linker can improve the adhesion of the linker to the surface of the substrate. A second linker can be linked to the first linker to improve the immobilization of biomolecules.

[0049] In another embodiment, the step of immobilizing the biomolecule on the linking layer comprises multiple steps. For example, several layers of biomolecules can be deposited as multiple layers. Alternatively, different biomolecules can be distributed on the surface of the substrate. This can be advantageous to produce multiple or synergistic effects.

[0050] In one embodiment, the bond between the linking layer and the surface of the substrate is not permanent. In another embodiment, the bond between the linking layer and the biomolecule is not permanent. Non-permanent immobilization of the biomolecule allows for controlled release of the biomolecule at the target location.

[0051] In a second aspect, the present invention provides an apparatus suitable for two-step immobilization of biomolecules on a sample surface of a substrate via a linking molecule. The apparatus comprises a plasma jet generator with a jet outlet. The apparatus further comprises an adapter and a replaceable shield. A seal. The shield includes a jet inlet, a nozzle outlet, and a sidewall extending from the jet inlet to the nozzle outlet. The adapter is configured to removably attach the shield to the plasma jet generator, thereby communicatively coupling the jet outlet and the jet inlet.

[0052] As used herein, "communicatively coupled" refers to a collection of flows, i.e., flows of fluid, gas and / or plasma. Thus, the communicatively coupled jet outlet of the plasma jet generator and the jet inlet of the shield are configured such that the plasma jet exiting said jet outlet enters the shield via the jet inlet.

[0053] In a preferred embodiment, the jet outlet of the plasma jet generator comprises an opening and the jet inlet of the shield comprises an opening, whereby the opening of the jet inlet is larger than the opening of the jet outlet. This is advantageous because the expansion leads to a decrease in velocity and an increase in pressure, thereby assisting in the creation of an overpressure inside the shield relative to the environment. This is further advantageous because a sudden expansion can cause turbulence and / or recirculation and thus mixing of the components present in the corresponding part of the shield. Preferably, the shield comprises a length between the jet inlet and the nozzle outlet, and said at least one precursor inlet is communicatively coupled to the inside of the shield within a distance of the jet inlet equal to at most 50% of said length, preferably at most 40% of said length, more preferably at most 30% of said length. This is advantageous because the inflow of precursors occurs in an area where recirculation also occurs. This is further advantageous because the inflow of precursors occurs in a direction that is not substantially parallel to the inflow of the plasma jet at the jet inlet, preferably essentially perpendicular, preferably essentially parallel to the length, thereby further increasing the turbulence.

[0054] In a preferred embodiment, the nozzle comprises homogenising means. Preferably, the shield comprises said homogenising means, preferably internally. The homogenising means may comprise a flow disruption element. The flow disruption element may comprise a plurality of inclined surfaces. The flow disruption element may comprise a plurality of layers, each layer comprising a number of inclined surfaces. The flow disruption element may comprise a surface that comprises an angle of at least 20° and at most 70° relative to the length of the shield.

[0055] Preferably, the shield is monolithic. The shield may be manufactured by injection molding. The shield may be manufactured by 3D printing. Preferably, the shield comprises an insulating material, more preferably a plastic. The nozzle outlet of the shield comprises an edge. The nozzle outlet of the shield may include a flat edge, i.e. the nozzle outlet is flat. The nozzle outlet of the shield may include a non-planar edge, i.e. the nozzle outlet is non-planar. This allows for in-line coating of non-planar surfaces, thereby maintaining a short distance between each portion of the edge and the surface.

[0056] In a preferred embodiment, the nozzle is adapted for cooling. Preferably, the side wall of the shield comprises a channel for passing a cooling fluid. The channel is preferably located at a distance from the nozzle outlet of at most 60%, more preferably at most 50%, even more preferably at most 45% of the length of the shield.

[0057] In a preferred embodiment, the side wall of the shield comprises at least one precursor inlet, preferably at least two precursor inlets, for example two, three, four or more precursor inlets. The precursor inlet may comprise a tubular hollow body comprising a first outer end communicatively coupled to the inside of the shield and a second outer end for communicatively coupling to a precursor source. The tubular body may be cylindrical. The tubular body may comprise one or more bends. The coating precursor may be injected through said at least one precursor inlet of the plasma jet of the shield.

[0058] In a further preferred embodiment, one precursor inlet is a linking molecule precursor inlet and another precursor inlet is a biomolecule precursor inlet. In a further preferred embodiment, said biomolecule precursor inlet is suitable for aerosolizing said biomolecule precursor, which is preferably a biomolecule dissolved in an aqueous medium. This device is particularly advantageous since a single plasma jet can be used for both steps of a two-step method. This device can be used for a two-step process in which the same plasma gas is used. This allows the process to be carried out more quickly and at lower capital costs than using two separate plasma jets.

[0059] In a preferred embodiment, the apparatus includes a coupled plasma jet generator including a coupled jet outlet. Additionally, the apparatus includes a biomolecular plasma jet generator including a biomolecular jet outlet. Additionally, the apparatus includes an adapter and a replaceable shield. The adapter is configured to removably attach the shield to the coupled plasma jet generator, the biomolecular plasma jet generator, or both, such that the adapter selectively and communicatively couples the coupled jet outlet, the biomolecular jet outlet, or both, to the jet inlet.

[0060] In a third aspect, the present invention discloses a device that can be obtained using a two-step immobilization of biomolecules to the sample surface of a substrate via a linking molecule.

[0061] In a preferred embodiment, the product is a linker-assisted antibody immobilized on a glass, quartz or silicon wafer suitable for diagnostic purposes.

[0062] In a preferred embodiment, the product is a wound-covering patch comprising a textile patch functionalized with a biomolecule having antibacterial properties, antifungal properties, or both.

[0063] The present invention is further illustrated by the following non-limiting examples which further illustrate the invention and are not intended, nor should they be construed, to limit the scope of the invention.

[0064] The invention will now be described in more detail with reference to non-limiting examples. Working Example method The following buffers were used in this experiment: Blocking buffer was made up of 25 mL PBS (Dulbecco's Phosphate Buffered Saline, Gibco) and 0.25 g BSA (Bovine Serum Albumin, Sigma-Aldrich) and shaken to ensure everything was dissolved; Storage buffer containing 25 mL Type I ultrapure water (Synergy UV Milli-Q Water, Millipore), 2.5 g mannitol (D-mannitol ≥ 98%, Sigma-Aldrich) and 0.5 g sucrose (D(+)-sucrose 99.7%, Acros Organics); Wash buffer containing 150 mL PBS and 3 μl Tween 80 (Sigma-Aldrich). The viscosity of the Tween 80 was noted; PTA buffer containing 150 mL PBS, 0.15 g BSA, and 3 μl Tween 80. The masses of the components were determined using an analytical balance.

[0065] For this experiment, all nozzles were specifically 3D printed. An antibody solution (48 μg / ml) was prepared by adding 10 mL of PBS to a glass bottle (PlasmaSpot®) and pipetting 196 μl of goat-derived anti-human IgE antibody (Novex, Invitrogen, USA) (Ab) into it. The solution was mixed by pipetting up and down. To increase the volume, the glass beads were filled with water until they reached the same height as the liquid. Then, 3 mm of fluorine (Merck) was added.

[0066] The bottles containing the solutions were fitted with ice packs secured with tape to maintain the temperature of the solutions at 4°C during deposition and were then stored in a refrigerator. Two clean injectors were wetted by running the "cleaning atomizer" program for 5 min using an aspiration flow of 4 slm. Deionized (DI) water was used for the antibody injector (Ab injector) and acrylic acid (CAS 79-10-7, abcr) (AA) was used for the acrylic acid injector (AA injector). Glass slides were cleaned by wiping with a Nonwoven Wipe (TX 609, TechniCloth®, Texwipe, USA) moistened with ethanol.

[0067] The slides were then argon plasma etched using a PlasmaSpot® instrument and the "Spot Argon" program. The PlasmaSpot® head movement was set to have uniform deposition across the entire 75 mm x 25 mm surface. Etching was completed using a large circular nozzle, 0 slm aspirate gas, 0 slm dilution gas, 100 W, 80 slm argon (Air Liquide) and two passes of the head. Once etching was complete, a PlasmaSpot® bottle containing 30 mL of AA was attached to the AA injector. The head movement remained set as before. A linker layer of AA was applied using a large circular nozzle, 1 slm aspirate gas, 4 slm dilution gas, 100 W, 80 slm argon and two passes of the head over each well. A glass ProPlate® (Grace Bio Labs) was assembled.

[0068] Irrelevant variables were identified: inlet gas, dilution gas, electrode power, electrode frequency, gas used for plasma, concentration of antibody solution, nozzle geometry, nozzle-substrate separation, chemicals used as reactive precursors, linker monolayer, substrate, and substrate pre-deposition etch treatment. All of these variables were kept constant for this study.

[0069] The variables that were changed in this study were aspiration flow, dilution flow, exposure time, presence and type of incubation, and whether or not AA was co-deposited. The PlasmaSpot® head movement was disabled and the nozzle was attached. The AA bottle was left attached to the AA injector and parameters were kept the same as in the pre-deposition method (1 slm aspiration gas, 4 slm dilution gas). A glass PlasmaSpot® bottle containing 10 mL of pre-prepared anti-IgE+PBS solution was attached to the Ab injector and the aspiration and dilution flows were set according to the table below.

[0070] Four different combinations of suction and dilution flows were tested: the first combination includes a suction flow of 1 slm and a dilution flow of 4 slm; the second combination includes a sink flow of 2 slm and a dilution flow of 3 slm; the third combination includes a suction flow of 3 slm and a dilution flow of 2 slm; and the fourth combination includes a suction flow of 4 slm and a dilution flow of 1 slm.

[0071] The electrode power was set to 60 W, the frequency was kept constant at 38 kHz, and the argon plasma gas flow rate was set to 80 slm. For Ab-only conditions, only the Ab injector was used and the AA injector was disabled, and for Ab+AA conditions, both injectors were activated. The nozzle was inserted 2 mm into the well to be treated. Exposure times of 5 to 40 seconds were used, increasing in 5 second increments. After each deposition, the head was moved to remove the nozzle from the well and insert the nozzle into the next one. Each condition was repeated with three wells. Once all wells had been treated, the plate was removed and the appropriate incubation procedure was followed.

[0072] Three different incubation conditions were tested: wet incubation, dry incubation and no incubation. Pipette (Multipipette M4 1 μl Wet incubations were created by adding 175 μl of PBS buffer to each well using a 2.5 mL PBS dispenser (~10 mL, Eppendorf) and a tip (Combitips advanced 2.5 mL PCR clean, Eppendorf) and the plate was stored at 4 °C for 24 h. The wells were then emptied by tapping with NwW until no further residue was apparent. 175 μl of PBS and 1% BSA blocking buffer were pipetted into each well. The plate was then incubated at room temperature (RT) for 2 h, after which the wells were emptied by tapping with NwW. Dry incubations were created by storing the plate at 4 °C for 24 h. 175 μl of PBS and 1% BSA blocking buffer were pipetted into each well. The plate was then incubated at RT for 2 h, and the wells were emptied by tapping with NwW. No incubation conditions were created by pipetting 175 μl of PBS and 1% BSA blocking buffer into each well. The plate was incubated at RT for 2 h. The wells were then emptied by tapping with NwW.

[0073] After incubation, the wells were washed by pipetting 175 μl of PBS and 0.002% Tween 80 solution into them and emptying them with NwW. This process was repeated four times. 175 μL / well of D-mannitol (100 g / L) and sucrose (20 g / L) storage buffer was added to each well and the plate was incubated at room temperature for 3 min. The wells were then emptied by tapping with NwW. Finally, the plate was wrapped in tin foil and stored in a freezer at -20°C.

[0074] The plates were removed from the freezer. The plates were washed 4 times by pipetting 150 μl of PBS and 0.002% Tween 80 into the wells, then emptied by tapping NwW. 175 μL / well of 10 ng / ml IgE solution in PTA (Immunoglobulin E Myeloma kappa, Athens Research & Technology) was added and the plates were incubated for 1 hour at room temperature. One of the triplicate wells was always left empty to act as a blank. Each well was washed 4 times with PBS + 0.002% Tween 80, then emptied by tapping NwW. 175 μL / well of detection antibody, 1 μg / ml biotin-anti-IgE conjugate in PTA (goat anti-human IgE antibody, biotin-linked, cross-adsorbed, Invitrogen) was added to each well. The plates were incubated for 2 hours at room temperature. Each well was washed 4 times with PBS+ and 0.002% Tween 80, then emptied by tapping with NwW. 175 μL / well of split enzyme, 100 ng / ml streptavidin-HRP (streptavidin horseradish peroxidase conjugate, Thermo Fisher) in PTA solution was added to each well. The plate was then left for 1 hour at room temperature. Each well was washed 4 times with PBS+ and 0.002% Tween 80, then emptied by tapping with NwW. 100 μL / well of tetramethylbenzidine (TMB) (1-Step Ultra TMB-ELISA, Thermo Scientific) was added. Note: For the second experiment, 150 μl of TMB was added per well. Immediately after the substrate addition, the plate was inserted into the spectrophotometer. The spectrophotometer was shaken before starting and the standard was set as "Coaster Corning plate black clear bottom". The absorbance of light at wavelengths of 370 nm and 652 nm was measured every 5 min for 1 h after addition of TMB.

[0075] The examples and their reference numbers are shown in the table below: Examples 7 and 8 had an exposure time of 0 seconds and therefore no aspiration or incubation time.

[0076] [Table 1]

[0077] result Optimal conditions for antibody deposition were identified using MPG's atmospheric plasma technology by manipulating exposure times, aspiration and dilution flows, incubation procedures and deposition methods to optimize functionality as demonstrated by sandwich ELISA (Figure 1). The kinetics of the reaction can be estimated by the absorption curves shown below. These indicate the affinity of the capture antibody for the target antibody and provide a measure of the functionality retained by the capture antibody. All endpoints were measured at 60 min, as the reaction stabilized and reached a plateau of absorption after approximately 40 min.

[0078] The capture antibody (goat IgG anti-human IgE) was shown to retain its functionality, as shown in Figure 2. The graph shows the best results from the run compared to a reference curve provided by KU Leuven, obtained using standards derived from a conventional wet chemical deposition method on polystyrene microtiter plates. The reaction kinetics of each reaction were different, with only one run producing a signal indicating a faster reaction than the reference (Example 2: "Ab only, 4 slm, 10 sec, wet incubation"). It should also be noted that the reactions from the second run ("Ab only, 4 slm, 10 sec, wet incubation", "Ab only, 4 slm, 10 sec, no incubation" and "Ab only, 2 slm, 20 sec, no incubation") produced a stronger signal that saturated faster. This can be explained by the increased volume of TMB added in the second run (150 μl instead of 100 μl). As all endpoints were between 3 and 4 arbitrary units (arb.u), they can all be considered comparable to the criteria. This indicates that the atmospheric pressure plasma deposition method is comparable to current methods in terms of functionality retention and that exposure to low-energy plasma does not result in denaturation due to structural changes.

[0079] The specificity of the antibodies was determined by testing blank samples for each condition tested. All samples were emptied by not adding target IgE in the ELISA protocol, except for those where no IgE was deposited. Figures 3-5 show a comparison between the empty condition and the positive samples, which indicates that no non-selective binding occurred and therefore the signal generated is due to the binding of the capture antibody to the target antibody.

[0080] The signal generated by the empty condition is well below that generated by the respective positive sample, both when Ab and AA were deposited simultaneously (Figure 3), and when Ab was deposited alone (Figure 4). This is also the case when no capture antibody was deposited as can be seen in Figure 4. In this test, no capture antibody was present and therefore the target IgE could only interact with the carboxyl groups of the polyacrylic acid linker layer. Since the signal generated is very low and comparable to that generated by a trial lacking target IgE, it can be concluded that no cross-reaction occurs between the polyacrylic acid layer and the target IgE or other elements of the assay. Figure 5 shows that there is also no increase in cross-reaction when the aspiration flow is increased from 1 slm to 4 slm, indicating that this factor does not affect the specificity of the Abs being measured.

[0081] This indicates that atmospheric pressure plasma deposition can create a specific bioassay and that the process does not disturb the conformation of the antibody and therefore does not affect its specificity.

[0082] The effect of incubation was measured by comparing the effect on the reaction rate following three different incubation protocols while keeping all other parameters the same. Since the duration of incubation is critical for wet chemistry methods, determining its necessity for atmospheric plasma deposition represents another way that can save time, making it easier to industrialize the process.

[0083] Different incubation steps were completed for runs with and without AA codeposition, as shown in Figures 6 and 7. In both cases, the data show that the incubation step does not significantly increase the absorbance endpoint values, suggesting that an incubation step is not necessary when using atmospheric plasma to deposit the antibody. The curves in Figure 6 show that the kinetics of all reactions were similar, however, the kinetics of the wet incubation run was faster, as shown in Figure 7. This can be explained by the fact that the "only Ab, 4 slm, 10 sec, wet incubation" (Figure 7) was completed in the second run and therefore had more TBM which saturated the HRP faster, resulting in a faster change in absorbance and faster saturation.

[0084] Heyse et al. (2011) claimed that codeposition, defined as the simultaneous plasma polymerization of chemicals and deposition of proteins, allows the proteins to be trapped in a polymer mesh, increasing the strength of the deposition. To test this theory, Ab was deposited with and without AA, keeping all other parameters the same.

[0085] As seen in Figure 8, the data contradicts Heyse et al.'s theory, since the absorption and reaction rates for the trials without AA codeposition are higher. This suggests that either antibody immobilization is still successful without the addition of a covalent precursor, or that codeposition results in steric hindrance of the antibody binding site, reducing IgE-target conjugation.

[0086] It stands to reason that the deposition conditions will affect the amount of capture Ab immobilized in the well. This will affect the amount of IgE that binds to the capture Ab and therefore the absorbance measured. To investigate this, we varied several deposition parameters to test for ideal deposition conditions. Row and identified.

[0087] Figure 9 shows the endpoint of the reaction plotted against the aspiration flow used for deposition for samples using only AB deposition with a 10 second exposure time and no incubation. One would have thought that a higher aspiration flow would result in more antibody deposited and therefore a stronger signal generated in the ELISA, but this is not the case here. The endpoint only increases up to 3 slm and then decreases. This may be due to steric hindrance caused by excess antibody.

[0088] Figure 10 shows the absorbance after 60 minutes as a function of exposure time (seconds) with a constant suction of 2 slm without incubation. Figure 11 shows the absorbance after 60 minutes as a function of exposure time (seconds) with a constant suction of 2 slm for wet incubation. The exposure time is also thought to correlate with the amount of antibody deposited, and therefore the signal generated in the ELISA will be stronger. Again, this was found not to be the case, as can be seen in Figures 10 and 11. Here, the endpoint signal is shown to increase but then plateau between 10 and 20 seconds (Figures 11 and 10, respectively), indicating that the presence of relatively more antibody does not necessarily lead to a more effective test. Figures 12 and 13 show that at a certain point, the signal begins to decrease with longer exposure times (23 seconds in Figure 13 and 40 seconds in Figure 12). From this, it can be determined that shorter exposure times can be used with similar effect, further reducing the time required to immobilize the antibody.

[0089] Antibody immobilization using atmospheric plasma technology has been shown to be a viable method since antibody functionality was maintained at the same level as with conventional wet chemical methods. It was also shown that the specificity of the antibody active sites was maintained since no cross-reaction or non-specific binding was observed to occur in blank sample conditions. It was concluded that the incubation step is obsolete for this antibody immobilization method since wet or dry incubation does not significantly increase the ELISA signal. Furthermore, deposition of Abs without AA was found to increase the ELISA signal due to steric hindrance generated by the mesh of AA formed in the codeposition. The optimal parameters for deposition were also investigated and it was concluded that antibody functionality was hindered if the aspirate flow and exposure time were of excessively high order.

Claims

1. A two-step method for immobilizing biomolecules via linking molecules on a sample surface of a substrate by generating a non-thermal atmospheric pressure plasma and maintaining it at a temperature between room temperature and 60° C., preferably at room temperature, comprising: - in a first step, said linking molecules are deposited on said sample surface by exposing said sample surface to a first plasma jet and said linking molecules, producing a linking layer on said sample surface; - subsequently, in a second step, depositing said biomolecules on said connection layer by exposing said connection layer to a second plasma jet and said biomolecules; The method includes the steps of: The first plasma jet is generated with a first electrode power and the second plasma jet is generated with a second electrode power, the first electrode power being at least 1.0 W / cm ２ and the second electrode output is a maximum of 1.5 W / cm ２ That is, the method.

2. 2. The method of claim 1, wherein the plasma jet can be generated using a plasma gas selected from the group comprising helium, argon, nitrogen, air, carbon dioxide, ammonium or combinations thereof, preferably argon is used as plasma gas for generating the second plasma jet.

3. 3. The method of claim 1, wherein the first plasma jet is generated with a first electrode power and the second plasma jet is generated with a second electrode power, the first electrode power being higher than the second electrode power.

4. The method according to any of claims 1 to 3, wherein preferably the substrate is selected from the group comprising biological materials, composite materials, crystalline solids, textiles, metals, plastics, polymers, ceramics, glass or combinations thereof, more preferably the sample surface is selected from the group consisting of glass, quartz, plastics, polymers, textiles, silicon, skin or tissue.

5. 5. The method of any of claims 1 to 4, wherein the linking molecule is a molecule comprising at least one moiety, preferably the linking molecule comprises two moieties, more preferably the two moieties are not arranged alongside each other.

6. At least one moiety is an alkane, alkene, alkyne, benzene derivative, haloalkane, fluoroalkane, chloroalkane, bromoalkane, iodoalkane, alcohol, ketone, aldehyde, acyl halide, carbonate, carboxylate, carboxylic acid, ester, methoxy, hydroperoxide, peroxide, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, heterocycle, orthocarbonate ester, amide, amine, imine, imide, azide, azo compound, cyanate, nitrate, nitrite, nitro compound, 6. The method of any of claims 1 to 5, wherein the moiety is selected from the group comprising: a carboxylic acid, a thiol, a hydroxyl, an amine, a trialkoxysilane, a nitroso compound, an oxime, a pyridine derivative, a thiol, a thioether, a disulfide, a sulfoxide, a sulfone, a sulfinic acid, a sulfonic acid, a sulfonate ester, a thiocyanate, a thioketone, a thial, a thioester, a phosphine, a phosphonic acid, a phosphate, a phosphodyester, a boronic acid, a boronic ester, a borinic acid, a borinic acid ester or a combination thereof, preferably the moiety is selected from the group comprising: a carboxylic acid, a thiol, a hydroxyl, an amine or a trialkoxysilane.

7. The method according to any one of claims 1 to 6, wherein the biomolecule is a biologically or pharma- ceutical active molecule.

8. The method according to any of claims 1 to 7, wherein said biomolecule is selected from the group comprising proteins, polynucleotides, sugars, lipids, growth factors, hormones and bioactive substances or combinations thereof, preferably said biomolecule is an antibody or comprises antibacterial and / or antifungal activity, most preferably said biomolecule is an antibody.

9. The method according to any of the preceding claims, wherein the biomolecules are administered to the second plasma jet in an aqueous aerosol.

10. a plasma jet generator (1) equipped with a jet outlet (12), - a nozzle comprising an adapter (3) and an exchangeable shield (2), said shield comprising a jet inlet (22), a nozzle outlet (24) and a side wall (21) extending from said jet inlet to said nozzle outlet. Equipped with the adapter is configured to removably attach the shield to the plasma jet generator, thereby communicatively coupling the jet outlet and the jet inlet; - a coupled plasma jet generator with a coupled jet outlet; - a biomolecular plasma jet generator including a biomolecular jet outlet; Equipped with 10. An apparatus suitable for accomplishing the method of any of claims 1 to 9, wherein the adapter is configured to removably attach the shield to the coupled plasma jet generator, the biomolecular plasma jet generator, or both, thereby selectively communicatively coupling the coupled jet outlet, the biomolecular jet outlet, or both, with the jet inlet.

11. a plasma jet generator (1) equipped with a jet outlet (12), a nozzle comprising an adapter (3) and an exchangeable shield (2), said shield comprising a jet inlet (22), a nozzle outlet (24) and a side wall (21) extending from said jet inlet to said nozzle outlet; Equipped with 10. Apparatus suitable for accomplishing the method of any of claims 1 to 9, wherein the adapter is configured to removably attach the shield to the plasma jet generator, thereby communicatively coupling the jet outlet and the jet inlet, and comprises a plurality of emitting precursor channels.

12. A product obtainable using the method according to any one of claims 1 to 9.

13. 13. The article of manufacture of claim 12 which is a linker-assisted antibody immobilized on a glass, quartz or silicon wafer suitable for diagnostic purposes.

14. 13. The article of manufacture of claim 12, which is a patch for covering a wound, comprising a textile patch functionalized with a biomolecule having antibacterial properties, antifungal properties, or both.

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