Patent Application
20250215175
The invention is within the field of silicone chemistry and specifically involves surface modification of silicone to introduce amino groups that can be further reacted with desired substituents.
Silicones, also known as polysiloxanes, are a class of synthetic polymers that consist of an “inorganic” backbone of alternating silicon and oxygen atoms with two organic side groups linked to the silicon atom. The most common type is PDMS (polydimethylsiloxane; formula I). Formula I shows a depiction of the basic PDMS structure (note that the terminal Si atoms may in some cases, have one or more-OH groups instead of methyl groups). Silicones are both chemically and thermally stable and widely used in insulators, packaging, building materials, cosmetics, drug delivery systems, and medical devices. Silicones are available in many different forms, including elastomers, gels, lubricants, foams, and adhesives. Liquid Silicone rubbers and elastomers can be created from straight-chain polymers by crosslinking. Commercial silicone materials will also typically contain various additives affecting the properties such as color, rheology, adhesiveness etc. Commercial silicone elastomers are commonly reinforced with mineral additives fillers such as silica, calcium carbonate, montmorillonite, carbon black, zinc oxide, titanium dioxide, glass, and graphene, etc. (as well as other materials improve handling such as boric acid and polyhydroxyl alcohol (see e.g. U.S. Pat. No. 4,252,709). Medical-grade silicones are biocompatible and bioinert and, therefore, a common material in medical implants, including prostheses, artificial heart valves, and various catheters.
Although flexibility and other properties favor the medical use of silicone, the hydrophobic surface of silicone is prone to bacterial and fungal colonization and biofilm formation. Fungal or bacterial biofilm growth on the surface of silicone can be an issue for medical applications inside body, as an example, Catheter-Associated Urinary Tract Infections (CAUTI)(Siddiq & Darouiche, NATURE REVIEWS UROLOGY 2012 9 305 DOI10.1038/nrurol.2012.68) that are the most common nosocomial infections causing a significant health burden. Microbial contamination of silicone surfaces can also be a risk factor for applications in the food sector.
Surface modification: The surface of silicone can be chemically modified to increase the resistance to bacterial colonization and biofilm formation. The most common approach is to introduce some hydrophilic moieties such as polyethylene glycol (PEG) to reduce the hydrophobicity and repel bacteria, slowing the colonization and biofilm formation. An alternative approach is to load the polymer matrix with a lipophilic disinfectant or antimicrobial drug that can leach from the material to kill infectious microorganisms (Kottmann et al. CHEMISTRY-AN ASIAN JOURNAL 2017 12 1168 DOI10.1002/asia.201700244). The disadvantage of this latter approach is that efficacy will be reduced over time as the antimicrobial agent is lost, and the leaching of bioactive agents can also influence the regulatory status of the device. Coating of the silicone surface with biocompatible polymers and proteins can also be used to improve biocompatibility and reduce implant-related issues like scar tissue formation.
Coating silicone: Silicone is generally considered chemically inert, as it is lacking reactive groups, and therefore, for coating silicone the surface is typically first activated to introduce groups that can be targeted for chemical modification. The commonly used approach is plasma (ozone) treatment, to oxidize the surface and introduce —OH groups, but treatment with other oxidizing agents such as H2O2 has also been used for the same purpose (Bračič et al. Bioactive Functionalisation of Silicones with Polysaccharides, SpringerBriefs in Molecular Science Biobased Polymers ISBN 978-3-030-02275-4 (eBook) 2018 https://doi.org/10.1007/978-3-030-02275-4). This is often followed by a second step where the surface is treated with silanization reagent (R—Si—(OR′)3) such as APTMS (3-Aminopropyl) trimethoxysilane) to introduce a more nucleophilic amino group on the surface. The silicone surface can then be coated by using the nucleophilic groups on the surface to initiate the polymerization of monomeric building blocks or by reacting with electrophilic moieties on a polymer or other compound used for the coating or by reacting them with a crosslinker and then reacting with a electrophilic moieties on a polymer or other compound used for the coating. Thus, a stable coating is obtained, where a polymer coating is covalently attached to the surface. Alternatively, the oxidized surface can be treated with silanization reagent such as (3-Glycidyloxypropyl)trimethoxysilane to introduce electrophilic epoxy groups on the surface. This can be further reacted with a nucleophilic polymer or other compound to covalently coat the surface. US 2016/0130403 (Brook et al.) discloses that a hydrophilic or reactive polymer linked to a linear or branched hydrophobic silicone polymer chain can be physically absorbed onto the surface silicone elastomer to form a stable hydrophilic coating. U.S. Pat. No. 10,465,055 discloses treating a silicone substrate with a polymer containing at least three reactive sites to promote “covalent grafting” of said polymer to the substrate but presupposes that the substrate has accessible Si—H groups, which can be obtained according to the disclosure by dipping a silicone surface into hydrogen fluoride aqueous solution.
The invention provides a new process for surface modifying silicone material and for coating silicone material. The invention provides a simple process for activation of the surface such that it can readily be further modified. This process is based on the surprising finding that amino groups can be introduced on the silicone surface without prior surface oxidation, with processes described herein.
Accordingly, the invention sets forth new methods for modifying a silicone surface and thereby make said surface amenable towards further modification. Specifically, the methods presented herein comprise treating a silicone surface with a substance selected from a specified group further described herein below, which substance contains an amino group. A variety of substances have been tested by the inventor, and different conditions and settings, these can vary depending on the specific surface type and/or object being modified and what type of subsequent modification, chemically and functionally, is desired and intended.
Advantageously, the method can be performed with readily available and economical reagents, mild conditions and environmentally benign solvents.
Accordingly, in a first aspect, the invention relates to a method for modifying a solid silicone surface, in particular silicone elastomer surface, to introduce amino groups to said surface that thus can be further modified, comprising treating the silicone elastomer surface with a substance that is selected from
wherein, at least in the case where said substance is a silanization reagent, the surface is not pre-treated with oxidizing agent including ozone, hydrogen peroxide or plasma treatment prior to the above treating step.
The term “silicone surface” encompasses any type of surface on solid silicone objects which encompasses objects that may be from essentially only silicone as well as silicone objects that comprise common additives and further ingredients that are known in the art as minor ingredients in silicone, e.g. fillers, reinforcement agents, e.g. mineral fillers such as silica, alumina, calcium carbonate, carbon black, zinc oxide, titanium dioxide, glass, graphene crosslinkers, catalysts, boric acid, polyhydroxyl alcohols and such. Thus, silicone objects as used herein include objects from silicone elastomer or silicone rubber.
The reagent substance can be optionally dissolved in a solvent selected from water-miscible solvents, such as but not limited to methanol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, t-butyl alcohol, glycerol, water or an aqueous solution, and any miscible mixture of one or more of these. Then the silicone surface is placed in said solvent with the dissolved reagent.
Various amines have been found useful as a reagent in the method of the invention. Non-limiting examples of specific reagents are listed in Table 1.
Thus, in some embodiments, the reagent is an amine that comprises a straight or branched alkane or alkene chain, at least one primary or secondary amine and/or amide group, and at least one further nucleophilic group selected from OH, SH COOH, and NH2, amide, and NHR where R is alkyl. In some embodiments, the reagent is an ester, ether or alcohol with at least one primary or secondary amine group. The reagent can also be an amine with two or more primary or secondary amino groups, some examples of which are shown in Table 4.
Useful reagents also include amino acids and amino acid derivatives, including the naturally occurring amino acids as well as derivatives thereof such as salts and esters thereof. Preferred amino acids are those with nucleophilic groups on sidechains such as the basic amino acids (histidine, lysine) and those with —OH or —SH groups on sidechain (tyrosine, serine, threonine, cysteine). Also useful are small peptides, such as but not limited to dipeptides, tripeptides, and other soluble oligopeptides, preferably including at least one amino acid of the preferred type with a basic side chain —OH or —SH or polyethyleneimine oligomers or polymers that contain large number of secondary amino groups and also primary amino groups.
In the embodiment of the invention using a silanization reagent Si—(OR1)(OR2)(OR3)R5NHR4, R4 may be selected from H, straight or branched alkyl that may be further substituted with amide, benzyl, carbamate, and amine. Such reagent may be in particular embodiments comprise a trimethoxysilane or triethoxysilane. Specific non-limiting examples include the compounds depicted in Table 2.
In some embodiments the reagent substance is a molecule represented by the formula W(X)—NHR4 where W is a molecule comprising an aromatic structure and X represents OH, SH, COOH, amide, NH2 or NHR where R is alkyl or alkenyl, and R4 are as defined above. Each of the groups X and NHR4 may independently be linked directly on carbon atoms of the aromatic structure or on alkyl or alkylene sidechains therefrom. Non-limiting include the compounds depicted in Table 3.
Table 4 shows a non-limiting list of other useful reagents.
As discussed above, the method of the present invention has the distinct advantage that the silicone surface to be treated according to the invention does not need to be pre-treated with an oxidizing agent such as ozone, hydrogen peroxide, or plasma treatment prior to the treatment with the reagent with the method of the invention. This makes the method of the invention much simpler than prior art methods for modifying silicone surfaces discussed above in the Background section. This applies to any reagent that may be selected, but in particular, when a silanization reagent as defined above is selected no pre-oxidation step is applied. When other reagents are used, a pre-oxidation step may or may not be applied but is, as mentioned, not necessary.
Also, pre-treatment with strong alkali, such as NaOH, KOH, LiOH, BaOH, NH4OH, and CaOH2 can be omitted and is omitted in preferred embodiments.
The method can advantageously be performed by use of any of a variety of common solvents or a mixture of solvents. It is an advantage of the invention that non-hazardous environmentally benign solvents can readily be used, such as common alcohol solvents (solvents that are not highly toxic or serious carcinogens and not hazardous to the environment when discarded). In some embodiments, an organic solvent is used, which may be selected from but is not limited to diethylene glycol, diethyl ether, diethylene glycol, dimethyl ether, methyl t-butyl ether (MTBE), tetrahydrofuran (THF), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), ethylene glycol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, t-butyl alcohol ethyl acetate, and acetone. Alternatively, water or an aqueous solution can be used or a miscible solution of water and one or more organic solvents. In these embodiments, the silicone surface to be treated is placed in the solvent and the reagent is mixed in the solvent. Tests have shown that the method can be successfully operated at ambient room temperature but higher temperatures can be advantageous in some embodiments for faster reaction. After a period of time the treated silicon surface is preferably rinsed, preferably with the same solvent used for the reaction or another solvent and preferably with water or an aqueous solution or non-toxic alcohol other environmentally benign solvents. In embodiments where a gas phase reagent is used the silicon surface is placed in suitable contained atmosphere which in some embodiments is air but in other embodiments an inert atmosphere such as nitrogen, ad the reagent is injected or leaked into the contained atmosphere.
Applicable reaction conditions vary according to the specific reagent and solvent selected and can be suitably optimized. Thus in some embodiments the reaction can be performed at ambient room temperature (about 20-25° C.), in other embodiments the reaction is performed at a temperature in the range of about 10°−100° C., such as a range from about 10°, or from about 15°, or from about 20°, or from about 25° or from about 30°, to about 100°, or to about 90° or to about 70° or more preferably to about 50° or to about 40° C. In some embodiments, using the applicable solvent(s), is useful to apply lower temperature and thus the reaction may take place at a temperature in the range of about −30° to about 10° C.
The reaction may also be aided by application of ultrasound and/or microwave heating.
The obtained surface-modified silicone with reactive amino groups can advantageously be further modified to obtain silicone with desired modified properties. Thus, in some embodiments the method of the invention includes further steps of modifying further the silicone surfaces. In one embodiment the method comprises a step of treating the silicone surface with diazo transfer reagent (sometimes referred to as azide-transfer reagent) to convert at least some of said introduced amino groups to azide groups. These azide groups can then be selectively reacted with alkynes in a 1,3-dipolar cycloaddition (i.e. “click reaction”) to give 1,2,3-triazole derivatives (see e.g. U.S. Pat. No. 9,302,997B2). Such 1,3-dipolar cycloadditions have been used to covalently link alkyne derivatives of various bioactive molecules, such as proteins, peptides, biotin and polymers to surfaces (Escorihuela et al. Adv. Mater. Interfaces 2015, 2, 1500135-https://doi.org/10.1002/admi.201500135). Accordingly, in some embodiments of the method, the silicone surface, after having been reacted with a selected azide-transfer reagent, is reacted with a selected desired molecule that binds via 1,3-dipolar cycloaddition to the introduced azide groups. The diazo transfer reagent may be selected from but is not limited to imidazole-1-sulfonyl azide, triflyl azide, azidotrimethylsilane and sodium azide. The molecule which is bound in this way may be selected from but is not limited to a peptide, protein, biotin, a polymer, e.g. PEG, but other reagents are as well applicable.
In another embodiment, the method comprises a step of treating the surface with introduced amino groups with a crosslinker which is preferably selected from but is not limited to glutaraldehyde, ethylene glycol dimethacrylate, genepin, tannic acid, and polyethylene glycol diglycidyl ether. After the selected crosslinker has been reacted with the modified surface, with methods that are such well known in the art, then a desired a biopolymer can be linked thereto, or other polymer, peptide, protein or another bioactive molecule were nucleophilic reactive groups are present in the structure. Thus such surface of the invention with introduced crosslinker can be reacted to, for example, synthetic polymer (e.g. PEG), protein, peptide, carbohydrate, a small bioactive molecule, such as but not limited to a bioactive drug molecule, a cytokine or hormone, to name just a few.
The methods of the invention introduce amino groups to the accessible surface on the treated silicone. The exact structure and nature of the association of the amino groups to the silicone has yet to be elucidated and is being investigated by the inventors; the tests included in the below examples demonstrate that significant activity remains after extensive washing, indicating covalent association, though non-covalent association may also play a role.
An aspect of the invention provides silicone objects with modified surface obtained with the above described methods. Such objects of the invention can be produced and provided commercially for subsequent use as useful intermediate products, or silicone objects can be treated with the methods of the invention and further modified by subsequent steps to bind thereto selected useful moieties, as described above. Thus, the present invention as well encompasses silicone objects obtainable with the methods of the invention, where one or more molecule has been bound (preferably covalently) to at least some of the amino groups introduced on the silicone surface. Such molecule can comprise but is not limited to any of the above mentioned, e.g. synthetic polymer, protein, peptide, carbohydrate, and small bioactive molecule, such as but not limited to a bioactive drug molecule, a cytokine or hormone, or any combination of one or more of these.
As used herein, including in the claims, singular forms of terms are to be construed as also including the plural form and vice versa, unless the context indicates otherwise. Thus, it should be noted that as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
Throughout the description and claims, the terms “comprise”, “including”, “having”, and “contain” and their variations should be understood as meaning “including but not limited to”, and are not intended to exclude other components.
The term “at least one” should be understood as meaning “one or more”, and therefore includes both embodiments that include one or multiple components. Furthermore, dependent claims that refer to independent claims that describe features with “at least one” have the same meaning, both when the feature is referred to as “the” and “the at least one”.
It will be appreciated that variations to the foregoing embodiments of the invention can be made while still falling within the scope of the invention can be made while still falling within the scope of the invention. Features disclosed in the specification, unless stated otherwise, can be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed represents one example of a generic series of equivalent or similar features.
Use of exemplary language, such as “for instance”, “such as”, “for example” and the like, is merely intended to better illustrate the invention and does not indicate a limitation on the scope of the invention unless so claimed. Any steps described in the specification may be performed in any order or simultaneously, unless the context clearly indicates otherwise.
All of the features and/or steps disclosed in the specification can be combined in any combination, except for combinations where at least some of the features and/or steps are mutually exclusive. In particular, preferred features of the invention are applicable to all aspects of the invention and may be used in any combination.
Medical silicone elastomer sheets from NuSil Technology (MED82-5010-80 and MED82-5010-40) were used in the experiment. Circular discs of silicone were cut from the sheets with a hollow puncher to give samples with a 1.2 cm diameter and 0.2 cm (thin discs) or 0.4 cm (thick discs) thickness and a 3.0 cm2 or 3.8 cm2 total surface area, respectively. The weight of the thin disc samples was 0.14 g, and the weight of the thick disc samples was 0.26 g. The thin discs were used, and they were immersed in acetone and ultrasonicated for 30 minutes to remove all surface contamination, rinsed with deionized water, and dried in an 80° C. oven before use unless otherwise stated.
In a second set of experiments we made 2 mm thick silicone sheet by pouring a silicone blend made from two types of liquid silicone, one containing the catalyst and the other the cross-linker and cure inhibitor, into a mold and heating the mold at 100° C. to cure the silicone into an elastomer. Three types of silicone membranes were made with different stiffness (shore OO) referred to as A (CF1350), B (CF15) and C (CF13).
Ninhydrin reagent used to quantify free amino groups (—NH2) in the sample: Lithium acetate dihydrate was dissolved in deionized water, pH adjusted to 5.2 with glacial acetic acid to obtain 4M lithium acetate buffer solution. Ninhydrin (1.0 g) and hydrindantin (0.15 g) were weighed directly into a round bottom flask and dissolved in DMSO (37.5 ml). This flask was then stoppered with a septum. A syringe attached to a nitrogen balloon was inserted through the septum into the solution, and the oxygen was flushed out by inserting another syringe into the septum. 12.5 ml of the 4M lithium acetate buffer solution was then added, and the solution was flushed again with nitrogen.
1-3 Calibration of the Ninhydrin Assay with Glucosamine Hydrochloride.
25 mL of CH3COOH/H2O (1:250 v/v) solution was prepared by adding 0.1 ml of CH3COOH to 25 mL of H2O. Then 0.25 gr of chitosan was added to the solution and stir the solution with a magnetic stirrer until the chitosan is completely dissolved to make a stock solution with a concentration of 1 mg/ml. Then, the stock solution was used to prepare six standard solutions with the specified concentrations: 15.62 μg/ml, 31.25 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml, and 500 μg/ml.
The similar process was also performed to prepare glucosamine standard solution.
A blank was made by mixing 1 ml of deionized water with 1 ml of ninhydrin reagent. The test tubes were then heated in a water bath (90° C.) for 30 minutes to complete the reaction. After cooling in another water bath, the solution was diluted with 5 ml of 50% (v/v) EtOH/H2O mixture to give a 7 ml final volume. These solutions were vortexed again for 15 sec each in order to oxidize the excess of hydrindantin. The absorbance at 570 nm was measured with a UV-vis spectrophotometer (GENESYS 150). The results used to make a calibration curve (
Treated discs or untreated discs were inserted into test tubes containing 1 ml of the ninhydrin reagent and 1 ml of water. The test tubes were heated in a water bath (90° C.) for 30 minutes to complete the ninhydrin reaction. The discs were then removed, the solutions cooled and then diluted with 5 ml of 50% (v/v) ethanol/water mixture to give a 7 ml final volume. The absorbance of the diluted solutions was measured in a 1 cm pathlength cuvette with a UV spectrophotometer. In the case of a strong response (absorbance >, 1-2), the solutions were further diluted with 50% (v/v) ethanol/water to allow measurements in the optimal 0-2 absorbance range.
The surface functionalization and coatings were investigated with Nicolet iZ10 FT-IR spectro-photometer (Thermo scientific) in reflection mode, and the spectra were analyzed with OMNIC software.
In this experiment, 1%, 2%, and 4% solutions of 3-Aminopropyl) trimethoxysilane (APTMS) were prepared by adding 0.5, 1, and 2 mL of APTMS to 49.5, 49, and 48 mL of toluene, respectively.
1 ml of (3-Aminopropyl) trimethoxysilane (APTMS) was diluted in 50 ml toluene to obtain 2% APTMS solution. Thin silicone discs were threaded with 19G BD microlance syringe needles to weigh them down to allow full immersion into the APTMS solution. The discs were then immersed in the APTMS solution in an Erlenmeyer flask, and the solution was stirred with a magnetic stirrer. The discs were removed after set treatment time (20 min., 40 min., 1 h, 1,5 h, 2 h, or 24 h), thoroughly rinsed with water, and then washed 6 times on each side using a transfer pipette to remove any excess reagent. This was followed by drying in an oven (80° C.) for 2 hours. The response to ninhydrin increased with up to 24 hours (
2-2 ATPMS Treated Silicone with Glutaraldehyde Crosslinker.
Silicone discs treated with 2% APTMS solution in toluene for 2 hours and rinsed were immersed in a 25% glutaraldehyde solution in H2O, which was stirred occasionally. The discs were removed from the solution after 20 min, 40 min, 1 hour, 1.5 hours, 2 hours, or 3 hours and washed 4 times on each side with methanol using a transfer pipette and air-dried overnight. The sheets were analyzed by FT-IR and ninhydrin tests. All tests were carried out in triplicate. Prior to the coating of amino-functionalized silicone with chitosan, the samples must be treated with a suitable crosslinker that can react with the amino groups on the silicone surface and the polysaccharide. Glutaraldehyde is a crosslinker that reacts readily with primary amino groups to form an imine. The imines can then be reduced to form stable dialkyl amines. The reagent blocks amino groups on the surface of the silicone, so the response to the ninhydrin test should decrease as the reaction processes. The ninhydrin response of the samples decreased with increasing reaction time in the glutaraldehyde solution due to the conversion of the amino groups to an imine. After 3 hours of reaction, there was almost no response to the ninhydrin test. The crosslinking reaction was therefore almost total after 3 hours of crosslinking (
2-3 Treatment of ATPMS and Glutaraldehyde Treated Silicone with Chitosan.
Silicone discs that had been treated with 2% APTMS solution in toluene for 2 hours and rinsed, were immersed in a 25% glutaraldehyde solution as previously described, were immersed a 2% (v/v) acetic acid/H2O solution containing 0.04 g/ml chitosan and 0.001 g/ml NaBH4, for 2 min, 40 min, 1 hour, 1.5 hour or 2 hours. This was done at room temperature with occasional stirring with the needle. Then removed from the solution and rinsed thoroughly by immersing them in deionized water and washed 10 times on each side using a plastic pipette containing the same solvent and dried in an oven (80° C.) for 1 hour. The sheets were analyzed by FT-IR and ninhydrin tests. All tests were carried out in triplicate.
The chitosan polymer chain contains many amino groups (—NH2), and the response in the ninhydrin test should increase again as glutaraldehyde treated silicone samples have been immersed in chitosan solutions. An increase in the ninhydrin response was observed (
Untreated silicone discs were measured to identify characteristic peaks that could be compared with spectra of treated silicone. The characteristic silicone peak at 2961 cm-1 (CH3 stretch) was observed in the untreated silicone sample (
Digital images showed the appearance of the silicone discs samples after different treatments. Untreated silicone has an opaque grayish color and becomes dark after treatment with the ninhydrin reagent (
Results are shown in
Silicone disc samples that had been treated 2% APTMS for 2 h were washed thoroughly with different solvents to try to extract the reagent that was not firmly (covalently) attached to the silicone (
Procedure for toluene solutions. The silicone discs were immersed in the 2% (v/v) ATPMS in toluene standard solutions for 20 min at room temperature. Subsequently, the discs were thoroughly rinsed with anhydrous toluene to remove excess reagent residues. The silicone samples were then heated in an oven (80° C.) to dry, and a ninhydrin assay was performed as described in example 1.
Procedure for deionized water solutions: The samples were immersed in 5% APTMS (heated at the temperature of 85° C.) solution for 5 or 30 minutes and dried with nitrogen gas. Then the samples are kept at 65° C. for 10 minutes.
Procedure for pure APTMS liquid. The silicone samples are immersed for 5 min into 97% APTMS, then washed in deionized water and dried in an oven at 80° C.
Procedures for ethanol and ethyl acetate solutions: An anhydrous solution is prepared by mixing APTMS and anhydrous ethanol or ethyl acetate to obtain a 1% solution. The solution was stirred for 10 min under nitrogen at room temperature. The silicone discs were then placed in the solution under nitrogen at room temperature. After 20 min, the substrates are washed with ethanol and blown dry with nitrogen. Results are shown in Table 6.
Protocol for the azide transfer reaction: Silicone disc samples that had been treated for 2 h with 2% APTMS in toluene were threaded with a syringe needle and immersed in 50 mL of MeOH in a 100 ml round bottom flask. Imidazole sulfonyl azide (50 mg) was then added to the reaction medium during agitation. Following this, 50 mg of K2CO3 dissolved in 1 mL deionized water were added and 5.75 mg CuSO4·5H2O dissolved in 1 mL deionized water. The flask was stoppered, and the reaction medium stirred for 16 h to 20 h. Once this reaction time had elapsed, the samples were cleaned with acetone, then with water and again with acetone, and finally dried at room temperature for one hour. FTIR was used to analyze the treated discs. The spectrum of the treated disc was consistent with the introduction of the azide group on the surface as a new peak was observed at 2097 cm−1.
Protocol for the Cycloaddition: 15 mL of DMSO was added to a 25 mL round bottom flask that was fitted with a reflux condenser. Heating was applied until solvent reached is at 50° C. Then the azide functionalized discs (threaded on a needle) were added to the reaction medium which was stirred with a magnetic stirrer. Then 1.5 mg CuSO4 and 4.7 mg sodium ascorbate are then dissolved in 1 mL deionized water were separately and added to the reaction medium. Finally, 23.3 mg N,N,N-trimethylprop-2-yn-1-aminium was added under a nitrogen atmosphere. The solution was then stirred for 48 h at 50° C. After this time, the samples are removed from the medium and washed with deionized water at least 3 times. The samples were then dried in the open air. The treated discs were analysed by FTIR. Following the cycloaddition reaction the azide peak had disappeared but a new peak in the C—H stretch region could be observed at 2922 cm-1 in the spectrum, consistent with the triazole functionalization.
Silicone discs were cut from a 0.4 cm thick silicone sheets. The silicone disks were soaked in deionized water in an Erlenmeyer flask and shaken. The water washing solution was replaced, and the discs were allowed to soak at room temperature for one hour. This was repeated twice. The discs were finally dried in an 80° C. oven for 2 hours.
Ethanolamine (EA) was mixed with water, isopropanol, ethanol, or ethyl acetate to give treatment solutions with the desired concentration. The discs were soaked in the treatment solutions, which were gently stirred at 1-hour intervals (The thick discs did not float in the solution and thus, it was not necessary to thread them with a needle).
After the reaction, the discs were removed and washed thoroughly 4 times in water (15 minutes total washing time), and then ninhydrin assay was performed as previously described (in example 1).
Results are shown in Table 7.
In this example silicon discs from fabricated sheets A, B, C were modified with APTMS. Silicon discs were made by using a 12 mm hollow steel hole punch, washed with deionized water and finally dried in an oven at 80° C. The samples (silicon discs) were added to 50 ml of the APTMS solutions or just solvent (control) and stirred at room temperature for 1 hour. Then the supernatant solution was discarded, and the samples were washed six times with toluene (for this, the appropriate amount of toluene was added to each disc to cover the disc completely (20 mL) and shaken for about 30 seconds, then the solution was replaced with 20 ml of fresh toluene and this was repeated six times). Then the samples were washed with methanol and deionized water respectively 6 times as mentioned above to remove any remaining traces. Finally, the sample was dried in an oven at 80° C. and used for ninhydrin assays.
The ninhydrin assay with silicone discs was performed the same way as with the standards for the calibration curve. The discs were immersed in 1 ml water and 1 ml Ninhydrin reagent in a test tube. The test tubes were then heated in a water bath (90° C.) for 30 minutes to complete the reaction. The solutions were then diluted with 26 milliliters of 50% (v/v) EtOH/H2O mixture before the UV measurement. All tests were carried out in triplicate. Results are shown in Table 8.
In this experiment, five different reagents were prepared by adding 2.2 mL of DAP, EA, A12P, 3AP, and EDA, respectively, to 20 mL of isopropanol.
Discs were made from the silicon sheets of type A by using 12 mm hollow steel hole puncher as before, and washed 2× with 20 mL of isopropanol and water (30 min) respectively as previously. The samples (silicon discs) were added to DAP, EA, A12P, 3AP, and EDA solutions separately (3 discs for each treatment) and stirred for 24 hours. Then the supernatant solution was discarded, and the samples were washed two times with Isopropanol, and water, as mentioned previously. Finally, the samples were dried in an oven at 80° C. and used for ninhydrin assays.
The ninhydrin assay was done as in example 8 except that the solutions were diluted with 40 mL of 50% (v/v) EtOH/H2O mixture after the heating. All tests were carried out in triplicate. Results are shown in Table 9.
| Number | Date | Country | Kind |
|---|---|---|---|
| 050359 | Mar 2022 | IS | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IS2023/050003 | 3/21/2023 | WO |
