Patent Application
20240182698
Immunoassays are useful for discovering, detecting, and monitoring biological processes which express a distinctive proteomic “biomarker” signature by utilizing specific chemical interactions between antibodies and antigens. Immunoassays are becoming more important as both medical and research tools, and also in various scientific fields such as analytical chemistry, pharmacology, molecular cell biology, and clinical biochemistry. Immunoassays are now used in many laboratories worldwide, and many companies are marketing diagnostic or detection tools that employ immunoassays to determine the levels of specific analytes of interest, such as pollutants, hormones, disease markers, and pathogens.
However, immunoassays often suffer from low signal-to-noise (S/N) ratios at the early-stage of detection where the biomarker's concentration level is relatively low. A high sensitivity test is needed because a low S/N ratio gives rise to poor assay confidence, resulting in false positive or false negative results. This could arise from the high background noise caused by the nonspecific adsorption of both target and non-target molecules onto the assay platform. As a result, background noise increases and obstructs improvement of sensitivity. Among the different immunoassay techniques, chemiluminescence provides the highest detection limit as compared to colour change or fluorescence. However, the S/N of chemiluminescence immunoassays still needs to be further improved.
In the chemiluminescence immunoassay (CLIA), a potential cause of high noise level is the nonspecific adsorption of secondary antibodies which conjugate with the signal generators, such as antibodies labelled with acridinium esters or isoluminol derivates, alkaline phosphates and horseradish peroxidase. In the case of diagnosis in clinical samples, a specific substance such as an antigen is detected in the coexisting living body molecules. Substances such as serums and the like, which coexist in the living body molecules, tend to adsorb on the surface of particles. They can then render the surface to be more prone to non-specific adsorption of the secondary antibody labelled with signal generators. As a result, the noise increases and obstructs improvement of S/N ratios.
A general approach to improving S/N ratios of CLIA is to prevent nonspecific adsorption by using organism-derived substances such as albumin, casein, gelatin and the like as additive agents. However, even after using these additive agents, nonspecific adsorption remains. Furthermore, these biological additives often have huge batch to batch variation. For example, one batch of albumin may provide good results, but a subsequent batch may not work at all. There will be plenty of trial-and-error to pick out the workable batches. In addition, when using a living body-derived additive agent, organism pollution can be a problem. Furthermore, the prevention of nonspecific adsorption by these additive agents is hindered by washing with acid or alkali, surfactant aqueous solution and the like, and shows inferior durability. Efforts were made by using synthetic materials to passivate the surface. Examples of synthetic polymers prepared with the aim of preventing nonspecific adsorption include polyoxy ethylene (JP-A-5-103831, JP-A-2006 177914 and JP-A-2006-299045), and copolymers having a phosphorylcholine group (JP-A-2006-176720 and JP-A-2006-258585). However, their nonspecific adsorption preventive effect was insufficient. The possible reasons are (1) the structure of the copolymer is usually linear, so it may be difficult to achieve high coverage by using linear polymer; (2) the molecular weight of such block copolymer is usually low, so it may be difficult to achieve a thick coating layer on the target surface. Other polymers include aminodextrans and modified dextrans of various molecular weights (EP2230515A1). These polymers may be able to prevent non-specific adsorption against proteins and bio-substances having negative charge, but are not as effective against those showing having positive charge. Since biological samples are usually a mixture of antigens and antibodies, they may have biomolecules with various different charges in the same sample, and so these polymers may not be useful against many biological samples. There is therefore an unmet need for a polymers that are able to prevent non-specific adsorption to a complex mixture.
The inventors have surprisingly found that by conjugating a polymer as defined herein onto the surface of a substrate, non-specific adsorption of biological molecules/material can be minimised. This may advantageously minimise background binding, improving the signal to noise ratio in an assay and leading to more accurate and reliable detection.
Aspects and embodiments of the invention are described in the following numbered clauses.
wherein:
wherein
wherein
It has been surprisingly found that the problems above can be solved in whole or in part by the use of a water-soluble crosslinked copolymer as defined herein.
Biological samples are usually a complex mixture of lipids, antigens and antibodies. Since they may have totally opposite charge in the same environment, it is important that a surface inhibits non-specific adsorption of a complex mixture. For example, the isoelectric point of Beta-HCG is 6.2-6.6 while the isoelectric point of Thyrotropin receptor (TSHR) is 8-10. This means under neutral pH, Beta-HCG is positively charged and Thyrotropin receptor (TSHR) is negatively charged. An ideal surface preventing non-specific adsorption of bio-substances should not adsorb any of them under the same buffer pH. It has surprisingly been found that by conjugating the polymer of the invention on the surface of a substrate, the non-specific adsorption can be minimised. It is believed that chemiluminescence immunoassays utilising this coating will improve the signal to noise ratio and minimise background binding, leading to accurate and reliable detection. As will be appreciated by a person skilled in the art, the polymer of the invention may be useful in coatings for other materials, where non-specific adsorption (e.g. of biomolecules such as proteins) is desired.
In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g., the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa.
In embodiments herein, various features may be described in the singular or the plural. It is herein explicitly contemplated that references to the singular are to be understood as including the plural, and references to the plural are to be understood as including the singular, unless such an interpretation would be technically illogical.
The water-soluble crosslinked copolymer of the invention may be referred to herein as the polymer (of the invention), the copolymer (of the invention), the water-soluble copolymer (of the invention), the water-soluble polymer (of the invention), or the like.
Some of the advantages of the water-soluble crosslinked copolymer and the relevant methods to realise the surface preventing non-specific adsorption of bio-substances on substrates include the following.
The water-soluble copolymers are useful for preventing non-specific adsorption of a substance including lipids, proteins, saccharides or nucleic acids, and cells. According to the present invention, the water-soluble copolymer may be formed by crosslinking a first polymer chain to a second polymer chain comprising a polycarboxylic acid, wherein
The water-soluble crosslinked polymer of the invention may be a water-soluble crosslinked copolymer comprising:
wherein:
The weight average molecular weight of the water-soluble crosslinked copolymer of the invention is from 2,000 to 1,000,000 Daltons, preferably 5,000 to 500,000 Daltons, more preferably from 10,000 to 250,000 Daltons. When the weight average molecular weight of the water-soluble copolymer is lower than 2,000 Daltons, the effect of preventing non-specific adsorption of bio-substances becomes negligible. On the other hand, when the weight average molecular weight of the water-soluble copolymer is higher than 1,000,000 Daltons, it will become infeasible to prepare a solution due to the increased viscosity of the polymer solution.
The first chain of the water-soluble crosslinked polymer may be a random copolymer comprising constitutional unit (A′) and/or (A″), constitutional unit (B) and constitutional unit (C). For example, in some embodiments the first chain of the water-soluble crosslinked polymer may be a random copolymer comprising constitutional unit (A′), constitutional unit (B) and constitutional unit (C). In some embodiments, the first chain of the water-soluble crosslinked polymer may be a random copolymer comprising constitutional unit (A″), constitutional unit (B) and constitutional unit (C).
The proportion of carboxylic acid groups in the second chain that are crosslinked to amine groups in the first chain may be from 5% to 75%, for example from 10% to 50%. In some embodiments, the first chain comprises a constitutional unit (A′) where X is O, R1 is methylene and E is a covalent bond. In some embodiments the first chain comprises a constitutional unit (A″).
In some embodiments, constitutional unit (B) represents a constitutional unit formed from the polymerisation of one of the group consisting of allylamine hydrochloride, 4-vinylaniline, 2-aminoethyl methacrylate hydrochloride, 2-aminoethyl acrylate, amino acrylate, N-(2-aminoethyl) methacrylamide hydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, and 2-aminoethyl methacrylate phenothiazine.
In some embodiments, constitutional unit (B) represents a constitutional unit formed from the polymerisation of one of the group consisting of allylamine hydrochloride, 2-aminoethyl methacrylate hydrochloride, 2-aminoethyl acrylate, amino acrylate, N-(2-aminoethyl) methacrylamide hydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, and 2-aminoethyl methacrylate phenothiazine (e.g. N-(2-aminoethyl) methacrylamide hydrochloride).
In some embodiments, constitutional unit (B) represents a constitutional unit formed from the polymerisation of one of the group consisting of allylamine hydrochloride, 2-aminoethyl methacrylate hydrochloride, 2-aminoethyl acrylate, N-(2-aminoethyl) methacrylamide hydrochloride, and N-(3-aminopropyl)methacrylamide hydrochloride.
In some embodiments, constitutional unit (B) represents a constitutional unit formed from the polymerisation of allylamine hydrochloride.
In some embodiments, constitutional unit (B) is a constitutional unit as defined above that comprises a primary amine group.
In some embodiments, constitutional unit (B) has the formula
wherein
In embodiments, R6a represents —Y—CH2—NH2, such that R6a comprises a primary amine. In some embodiments Y represents a C1-3 alkylene group.
In some embodiments constitutional unit (C) has the formula
wherein
In some embodiments R2 represents an ethylene glycol or a propylene glycol having from 1 to 20 repeating units. In some embodiments R2 represents a linear or branched alkylene group having from 1 to 5 carbon atoms.
In some embodiments, R2 represents a polyoxyalkylene group (e.g. ethylene glycol or propylene glycol) with a unit number of from 1 to 20, such as a unit number of from 3 to 10.
In some embodiments m is 1. In some embodiments n is 1, 2 or 3, such as 1 or 2.
In some embodiments constitutional unit (C) is a constitutional unit formed from the polymerisation of one of the group consisting of 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, hydroxypolyethoxy (10) Allyl Ether, N-(2-hydroxypropyl)methacrylamide, glycerol monomethacrylate, 3-phenoxy 2 hydroxy propyl methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, 2-hydroxypropyl acrylate, N-hydroxyethyl acrylamide, poly(ethylene glycol) methacrylate, poly(propylene glycol) methacrylate, and N-[tris(hydroxymethyl)methyl]acrylamide (e.g. 2-hydroxyethyl methacrylate).
In some embodiments, constitutional unit (C) is a constitutional unit formed from the polymerisation of one of the group consisting of hydroxypolyethoxy (10) Allyl Ether and poly(ethylene glycol) methacrylate, poly(propylene glycol) methacrylate.
In some embodiments the weight average molecular weight of the first chain is from 500 to 100,000 Daltons, preferably from 2,000 Daltons to 50,000 Daltons.
In some embodiments constitutional unit (D) is a constitutional unit formed from the polymerisation of one of the group consisting of poly(acrylic acid), polymethacrylic acid, polystyrene-block-poly(acrylic acid), polymaleic acid, poly(acrylic acid-co-maleic acid), poly(D,L-lactide-block-acrylic acid), poly(acrylamide-co-acrylic acid), poly(N-isopropylacrylamide-co-acrylic acid), and poly(ethylene-co-acrylic acid), such as a constitutional unit formed from the polymerisation of one of the group consisting of acrylic acid, methacrylic acid and 2-carboxyethyl acrylate.
In some embodiments the weight average molecular weight of the second chain is from 2,000 to 700,000 Daltons, preferably 5,000 to 350,000 Daltons, more preferably from 10,000 to 250,000 Daltons.
The invention also provides a method for the preparation of the water-soluble crosslinked copolymer of the invention, comprising the steps:
The method may further comprise a step (d):
Alternatively, immobilisation on a substrate may be performed before the water-soluble crosslinked copolymer is fully synthesised. For example, the method may further comprise a step (b2) or (c0):
More specific methods for preparing the copolymers of the invention are provided below.
Optionally, the terpolymer can be used to conjugate to the polyacid backbone which has been coated on a substrate, then step (f) is not required.
The substrate may be selected from particles, hollow filters, plastic tubes, glass fibers, glass slides, microplates and microfluidic devices, (e.g. wherein the substrate is selected from particles, hollow filters, microplates and microfluidic devices, such as particles, hollow filters, and microfluidic devices).
The material of the substrate may be selected from at least one of the following polymeric, organic or composite materials, such as polyurethanes, polyacrylonitriles, polymethacrylate, carbon fibers, cellulosic materials, polyacrylamide, polyacrylate, polyolefins, poly(4-methylbutene, polystyrene, poly(ethylene terephthalate), polysiloxanes, nylon, poly(vinyl butyrate), ferromagnetic materials, silica, or mixtures or composites of any of the above.
The abovementioned substrate may be further functionalised with anchoring groups, which can further react with the water-soluble copolymer in the present embodiment, selected from primary amine, epoxide group, tosyl group and aldehyde group.
The water-soluble copolymer in the present invention may be conjugated on a substrate by (1) dissolving water-soluble branched copolymer in the buffer with pH 4.5-8.5, (2) activating the water-soluble branched copolymer, (3) mixing the water-soluble branched copolymer with the substrate after activation; (4) wash off the unbonded water-soluble branched copolymer.
Optionally, the water-soluble copolymer in the present embodiment may be conjugated on a substrate by (1) dissolving the terpolymer in the buffer with pH 4.5-8.5; (2) activating the substrate surface and (3) mixing the substrate with the terpolymer solution; (4) wash off the unbonded water-soluble branched copolymer.
Optionally, the water-soluble copolymer in the present embodiment may be conjugated on a substrate by (1) dissolving water-soluble branched copolymer in the buffer with pH 4.5-8.5, (2) mixing with the substrate with functional groups selected from epoxide group, tosyl group and aldehyde group.
The materials were purchased from the sources as provided below.
Mechanical stirring (200 rpm) was used for all the polymerization processes. Prior to use, DI water and ethanol were bubbled with nitrogen for 20 min to remove oxygen. All the chemicals were used as received without purification. All the polymerizations and reactions were carried out under the protection of nitrogen using standard Schlenk line techniques.
To a 100 mL three-necked round bottom glass reactor equipped with a mechanical stirrer, 50 g of water, 0.5 g of 2-Methacryloyloxyethyl-phosphorylcholine as the monomer (1), 0.01 g of N-(2-aminoethyl) methacrylamide hydrochloride as the monomer (2), 1 g of 2-hydroxyethyl methacrylate as the monomer (3) were added and nitrogen was introduced from one side-neck to remove oxygen. After 10 minutes of nitrogen flow, the glass reactor was heated up to 75° C. 5 mg of sodium persulfate as initiator was added after the temperature of the glass reactor was stable at 75° C. The mixture was mixed for 24 hours to allow polymerization complete. The obtained solution was purified by dialysis.
To a 100 mL three-necked round bottom glass reactor equipped with a mechanical stirrer, 50 g of water and 1 g of 2-carboxyethyl acrylate as the monomer (4) were added into the glass reactor and nitrogen was introduced from one side-neck to remove oxygen. After 10 minutes of nitrogen flow, the glass reactor was heated up to 75° C. 5 mg of sodium persulfate as initiator was added after the temperature of glass reactor was stable at 75° C. The mixture was mixed for 24 h to allow polymerization complete. The obtained polycarboxylic acid solution was purified by dialysis with MES buffer.
EDC and NHS were dissolved separately in 25 mM MES buffer to achieve concentration 50 mg/mL. 20 μl of EDC solution was added per 1 mg of polycarboxylic acid backbone and followed by 20 μl of NHS solution. The solution was mixed for 30 min before adding into terpolymer solution with the ratio of 25 mg of terpolymer per 1 mg of polycarboxylic acid. The mixture was mixed for another 2 h. The obtained water-soluble copolymer solution was purified by dialysis.
To a 250 mL three-necked round bottom glass reactor equipped with a mechanical stirrer, AIBN solution (2 g, 12 wt % in acetone) was added and nitrogen was introduced from one side-neck to remove acetone solvent. After 10 minutes of nitrogen flow, dried AIBN powder was observed at the bottom of the reactor. Then PVP (0.2 g), ethanol (80 mL), and DI water (20 mL) were added. The mixture was stirred at room temperature for 5 minutes to obtain a clear solution, which was then heated to 60° C.with an oil bath, followed by addition of styrene (7.5 mL). A white colloidal solution was generated after 4 hours, indicating polymerization of styrene. A solution of DVB (3 mL DVB in 7 mL ethanol) was then slowly added with a constant pressure dropping funnel over 30 minutes. After addition of DVB solution was completed, the reaction (cross-linking polymerization) was continued for 3 hours. Then a monomer solution of 2-hydroxyethyl methacrylate (0.75 g in 5 mL ethanol) and acrylamide monomers (0.75 g in 5 mL ethanol, neutralized with ammonia solution, 25% W/W) was added with a syringe. The reaction (polymerization) was continued for 6 hours to give a milk-like colloidal solution. The obtained beads were named as “A-1”. The beads were washed with ethanol by using centrifuge with 7000G force for 9 min. The supernatants were discarded and microspheres were resuspended in 25 mM MES buffer.
EDC and NHS were dissolved separately in 25 mM MES buffer to concentration 50 mg/mL. 20 μl of EDC solution was added per 1 mg of water-soluble copolymer and followed by 20 μl of NHS solution. The suspension was mixed with 3 mg of beads A-1 with to achieve concentration of 10 mg/ml. The mixture was mixed for another 2 h. The resultant beads A-2 were washed with Tris buffer by using centrifuge with 9000 rpm for 11 min. The supernatants were discarded and microspheres were resuspended in 1×PBS buffer.
To a 250 mL three-necked round bottom glass reactor equipped with a mechanical stirrer, AIBN solution (2 g, 12 wt % in acetone) was added and nitrogen was introduced from one side-neck to remove acetone solvent. After 10 minutes of nitrogen flow, dried AIBN powder was observed at the bottom of the reactor. Then PVP (0.2 g), ethanol (80 mL), and DI water (20 mL) were added. The mixture was stirred at room temperature for 5 minutes to obtain a clear solution, which was then heated to 60° C. with an oil bath, followed by addition of styrene (7.5 mL). A white colloidal solution was generated after 4 hours, indicating polymerization of styrene. A solution of DVB (3 mL DVB in 7 mL ethanol) was then slowly added with a constant pressure dropping funnel over 30 minutes. After addition of DVB solution was completed, the reaction (cross-linking polymerization) was continued for 3 hours. Then a monomer solution of 2-hydroxyethyl methacrylate (0.75 g in 5 mL ethanol) and carboxyethyl acrylate oligomers (0.75 g in 5 mL ethanol, neutralized with ammonia solution, 25% W/W) was added with a syringe. The reaction (polymerization) was continued for 6 hours to give a milk-like colloidal solution. The obtained beads were named as “B-1”. The beads were washed with ethanol by using centrifuge with 7000G force for 9 min. The supernatants were discarded and microspheres were resuspended in 25 mM MES buffer.
EDC and NHS were dissolved separately in 25 mM MES buffer to concentration 50 mg/mL. 20 μl of EDC solution was added per 3 mg of beads B-1 and followed by 20 μl of NHS solution. The mixture was mixed for 30 min. The resulted activated beads were centrifuged with 7000G force for 9 min to remove supernatant. The terpolymer solution (20 mg/ml) was then added to the active beads and mixed for 2 h. The resultant beads B-2 were washed with Tris buffer by using centrifuge with 9000 rpm for 11 min. The supernatants were discarded and microspheres were resuspended in 1×PBS buffer.
The water-soluble copolymer (II) was synthesized following the exact steps in example 1, 2, 3. Except monomer (1) was changed to 2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide. The microsphere sample C-1 was prepared following steps in example 4.
The water-soluble copolymer (III) was synthesized following the exact steps. Except monomer (3) was not added during terpolymer synthesis in step 1. The microsphere sample C-2 was prepared following steps in Example 4.
The microsphere sample B-1 was conjugated with Amine-PEG-Carboxylic acid hydrochloride (Mn 2000 Dalton) by using similar protocol in Example 5. EDC and NHS were dissolved separately in 25 mM MES buffer to concentration 50 mg/mL. 20 μl of EDC solution was added per 3 mg of beads B-1 and followed by 20 μl of NHS solution. The mixture was mixed for 30 min. The resulted activated beads were centrifuged with 9000 rpm for 11 min to remove supernatant. The terpolymer solution (20 mg/ml) was then added to the active beads and mixed for 2 h. The resultant beads C-3 were washed with Tris buffer by using centrifuge with 9000G force for 11 min. The supernatants were discarded and microspheres were resuspended in 1×PBS buffer.
BSA and lysozyme have different isoelectric points. BSA (isoelectric point 5.5) and lysozyme (isoelectric point 11) are commonly used model proteins for studies. In this evaluation, both were used to evaluate nonspecific adsorption effect of different type of beads surface.
BSA and lysozyme were dissolved in 1×PBS buffer solution to prepare stock solution with concentration of 0.2 mg/ml, respectively. A total dry mass of 0.1 mg of protein was added per 1 mg dry mass of beads sample (10 mg/ml, 0.1 ml). A series of beads samples prepared in Examples 4, 5 and 6 were studied.
The beads-protein suspensions were mixed at 25° C. for 2 h to achieve adsorption equilibrium. The samples were centrifuged with 9000 rpm for 11 min to precipitate the particles. The protein content in supernatants were determined by using UV-Vis spectroscopy under 280 nm. The amount of adsorbed protein on the beads surface was calculated by equation (1).
As shown in
It is therefore clear that the invention provides an improved coating for preventing non-specific adsorption of biomolecules onto surfaces. The invention is able to prevent non-specific adsorption on both positively-charged (amine, Example 4) and negatively-charged (carboxylic acid, Example 5) surfaces.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202103164U | Mar 2021 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2022/050158 | 3/23/2022 | WO |
