A surface sealer based on a bola-form surfactant

Abstract

The application provides a Bola type surfactant-based blocking agent, which can be used for modifying various object surface materials, especially the surface of a carrier material for immune diagnosis, so that the non-specific adsorption of the carrier material is reduced, and the sensitivity and signal-to-noise ratio of a reagent are improved. Compared with traditional blocking agents, the Bola type surfactant-based blocking agent provided by the application not only has the excellent characteristics of traditional surfactant blocking agents, but also has increased repulsion to non-specific molecules and more compact blocking due to the rigid hairpin structure after combination.

Description

Technical Field

[0001] This invention relates to the field of surface materials, and more specifically to a surface sealing agent based on a Bola surfactant formulation, which is used in the field of in vitro diagnostic reagents. Background Technology

[0002] In today's in vitro diagnostics field, immunodiagnostics is one of the most diverse, widely applied, and largest-scale technologies. Specifically, it involves methods based on the specific binding between antigens, haptens, and antibodies to capture, enrich, and detect them. Mainstream immunodiagnostic methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, immunochromatography, immunoturbidimetry, and flow cytometry. Immunodiagnostic technology has long focused on improving sensitivity, specificity, and detection efficiency. Reducing background noise interference is one of the most effective methods, significantly improving reagent sensitivity. Therefore, after coating the carrier with antibodies, blocking agents are needed to block the unbound antibody sites on the carrier to prevent non-specific adsorption and background noise during subsequent detection.

[0003] Currently, there are three types of blocking agents commonly used in immunodiagnostic reagents: 1) Biomacromolecules, which mainly rely on physical adsorption or covalent bonding to bind to unreacted antibody sites on the carrier, forming a blockage. Common blocking agents include BSA, casein, and fish serum albumin. However, these protein blocking agents are prone to detachment during storage, causing poor reagent storage stability. 2) Small molecules, which mainly rely on the interaction of small molecule active groups such as amino, thiol, and hydroxyl groups with unreacted carrier sites to form covalent bonds and achieve a blocking effect. Examples include PEG, ethanolamine, and Tris. These small molecule blocking agents are not easily detached after forming covalent bonds. However, small molecule blocking agents are prone to detachment during the blocking process. It takes a long time and requires incubation at a certain temperature to achieve good results, which may affect the already bound antibody protein; 3) Surfactants: Common surfactants contain a hydrophilic head and a hydrophobic tail. When used as blocking agents, depending on the hydrophilicity and hydrophobicity of the carrier, one end will bind to the carrier surface, while the other end will be flexibly bound and exposed to the outside, forming a block, such as the MPC blocking agent of the Biolipidure series in Japan. However, these surfactants may also have a small amount of detachment, and the exposed flexible end may bring other non-specific adsorption. On the other hand, as the amount of blocking agent added increases, a multilayer structure will be formed, which brings potential risks. Summary of the Invention

[0004] This invention provides a blocking agent based on a Bola-type surfactant. This blocking agent can be used to modify the surface of carrier materials used in in vitro diagnostic reagents, achieving a blocking effect, reducing the proportion of non-specific adsorption during detection, increasing the signal-to-noise ratio of the reagent itself, and thus improving product performance. Specifically, the blocking agent described in this invention can be used in common immunodiagnostic fields such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, immunochromatography, and immunoturbidimetry.

[0005] The present invention provides a blocking agent (or blocking agent composition) comprising a Bola-type surfactant.

[0006] Bola-type surfactants have a molecular structure similar to the shape of the South American projectile weapon "bola". They consist of two hydrophilic polar groups (hydrophobic nonpolar groups) connected by one or more hydrophobic chains (hydrophilic chains). Common Bola-type surfactants have hydrophilic heads.

[0007] According to an embodiment of the present invention, the Bola-type surfactant is composed of the heads of two hydrophilic polar groups connected by one or more hydrophobic chains, or is composed of the heads of two hydrophobic nonpolar groups connected by one or more hydrophilic chains.

[0008] According to an embodiment of the present invention, the Bola-type surfactant is selected from any one, two, or more of the following: (i) Alkylene chains based on 1-20 carbons (-C 1-20 -), with hydrophilic groups attached to both ends; preferably, the hydrophilic groups may be the same or different, and are independently selected from quaternary ammonium salts, polyethylene glycol groups, polyol groups, carboxylates, water-soluble sugar groups, hydroxyl groups, and carboxyl groups; preferably, its structural formula is R 1 -(CH2) n -R 2 Where n is an integer selected from 1 to 20; R 1 R 2 They may be the same or different, and are independently selected from -COOH, -COONa, -COOK, and -NMe3. + Cl - , -C(CH2OH)3, -CH(CH2OH)2, -(CH2CH2O) m (m is selected from integers from 1 to 20), glucosyl, sucrose-syl, dextran-syl, sophoryl, etc.; (ii) Based on a 6-20 carbon arylene or biarylene chain, with hydrophilic groups attached to both ends; preferably, the hydrophilic groups are the same or different, and are independently selected from quaternary ammonium salts, polyethylene glycol groups, polyol groups, carboxylates, water-soluble sugar groups, hydroxyl groups, and carboxyl groups; preferably, the hydrophilic groups are the same or different, and are independently selected from -COOH, -COONa, -COOK, and -NMe3. + Cl - , -C(CH2OH)3, -CH(CH2OH)2, -(CH2CH2O) m (m is selected from integers from 1 to 20), glucosyl, sucrose-syl, dextran-syl, sophoryl, etc.; (iii) Polyethylene glycol chains (-(PEG)) based on degree of polymerization 1-20000 1-2000 -), with a hydrophobic group attached to each end; preferably, the hydrophobic groups are the same or different, and are independently selected from alkyl, alkenyl, alkynyl, aryl, heterocyclic aryl, etc.; preferably, the degree of polymerization of the polyethylene glycol chain is 1-20, 1-200, or 1-500; preferably, the alkyl group is C 1-20 Alkyl group; preferably, alkenyl group is C10. 2-20 Alkenyl group; preferably, the ynyl group is C. 2-20 Alkyne group; preferably, the aryl group is C. 6-20 Aryl group; preferably, the heterocyclic aryl group is a 5-20 membered heterocyclic aryl group; (iv) The 008-Fluorosurfactant series developed by Ran Biotechnologies (USA) has the structural formula F-[CF(CF3)CF2O]. n -CF(CF3)-CONH-(CH2CH2O) m -CH2CH2-NHCO-CF(CF3)-[OCF2(CF3)CF] n -F, where m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; (v) Bola-type cationic surfactants developed by BASF (Germany), with the structural formula [RN + -(CH2CH2O) n -(CH2) m -N + R]·2Br - Where R is C 1-20 Alkyl group, m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; (vi) The Bola-type surfactant developed by Gansu Zhilun New Materials (China) has the structural formula [Me3N + -(CH2) 12 -N + Me3]·2Br- ; (vii) The Bola series surfactants developed by Kenyou (Suzhou) have the structural formula Sophorose-O-(CH2). n -COOH, where n is an integer selected from 1 to 20; (viii) The structural formula is HO-(CH2). n -OH, where n is an integer selected from 2 to 30 (e.g., 5-25, 10-20, 15-18, 16); for example, Bola A-16 (purchased from Sigma-Alderich) has the structural formula HO-(CH2). 16 -OH.

[0009] Quaternary ammonium salts: Compounds formed by replacing all four hydrogen atoms in an ammonium ion with hydrocarbon groups, with the general formula R4NX; where R may be the same or different and are independently selected from C. 1-20 Alkyl group; X is selected from halide ions (halogens such as F, Cl, Br, I), and acid radicals (such as HSO4-, R0COO-, etc.; R0 is selected from C). 1-20 alkyl).

[0010] Polyethylene glycol: Chemical formula HO(CH2CH2O) n H, HO(CH2CH2O) n -; where n is selected from integers of 1-200, 1-2000, 1-500, 1-20, and 3-20.

[0011] Polyols: Alcohols containing two or more hydroxyl groups in their molecules; their general formula is C2. n H 2n+2-x (OH) x , where x≥2, and n is an integer selected from 1 to 20.

[0012] Carboxylates: Carboxylates are salt compounds formed by the neutralization reaction of carboxylic acids with bases.

[0013] Water-soluble sugars: Sugars that can dissolve in water, including monosaccharides and disaccharides. Examples include glucose, sucrose, fructose, and sophorose.

[0014] The basic information and chemical structural formulas of the above-mentioned Bola-type surfactants are shown in Table 1. When using them, one or more of them can be selected and mixed together.

[0015] Table 1: Basic information and chemical structure of Bola-type surfactants

[0016] According to an embodiment of the present invention, the Bola-type surfactant is selected from any one, two or more of the following (1)-(3): (1) The structural formula is HO-(CH2). n -OH, where n is an integer selected from 2 to 30 (e.g., 5-25, 10-20, 15-18, 16); for example, the structural formula is HO-(CH2). 16 -OH; (2) The structural formula is Sophorose-O-(CH2). n -COOH, where n is an integer selected from 1 to 20; (3) The structural formula is [RN + -(CH2CH2O) n -(CH2) m -N + R]·2Br - Where R is selected from C 1-20 Alkyl group, m is selected from integers 1-20, n is selected from integers 1-20; for example, di(trimethylammonium)tetraethylene glycol, with the structural formula: [(Me3N + )-(CH2CH2O)3-CH2CH2-N + Me3]·2Br - .

[0017] According to an embodiment of the present invention, the content (mass percentage) of Bola-type surfactant in the blocking agent is 0.01%-10%, preferably 0.1-8%, and more preferably 1%-5%; for example, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%.

[0018] According to an embodiment of the present invention, the sealing agent further comprises: a pH adjuster.

[0019] According to an embodiment of the present invention, the pH adjuster is selected from any one, two, or more of the following: disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate, sodium citrate, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 2-morpholine ethanesulfonic acid (MES), 3-morpholine propanesulfonic acid (MOPS), 2-[[tris(hydroxymethyl)methyl]amino]ethanesulfonic acid (TES), piperazine-N,N'-di(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane (Tris), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), and tris(hydroxymethyl)aminopropanesulfonic acid (TAPS). Preferably, the pH adjuster is disodium hydrogen phosphate, sodium dihydrogen phosphate, 2-morpholine ethanesulfonic acid (MES), or 3-morpholine propanesulfonic acid (MOPS), and more preferably, disodium hydrogen phosphate or sodium dihydrogen phosphate. According to an embodiment of the present invention, the pH adjuster is selected from a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate, or 3-morpholinopropanesulfonic acid (MOPS); preferably, the content ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 1:5-5:1, for example 1:5, 1:3, 1:1, 3:1, 5:1, for example a combination of 25 mM disodium hydrogen phosphate and 25 mM sodium dihydrogen phosphate, or a combination of 100 mM disodium hydrogen phosphate and 100 mM sodium dihydrogen phosphate.

[0020] According to an embodiment of the present invention, the content of the pH adjuster is 10 μM-1000 mM, preferably 1 mM-500 mM, and more preferably 10 mM-300 mM; for example, 10 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 150 mM, 180 mM, 200 mM, 220 mM, 250 mM, 280 mM, and 300 mM.

[0021] According to an embodiment of the present invention, the pH value of the sealing agent is 5.0-8.0, preferably 5.5-7.8, for example 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.4, 7.5, 7.8, 8.0.

[0022] According to an embodiment of the present invention, the sealing agent further comprises: an inorganic salt.

[0023] According to embodiments of the present invention, the inorganic salt is selected from any one, two, or more of the following: sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, sodium citrate, sodium ascorbate, potassium phosphate, sodium phosphate, ammonium sulfate, ammonium chloride, and sodium gluconate. Preferably, it is sodium chloride, potassium chloride, or magnesium chloride; more preferably, it is sodium chloride or magnesium chloride. According to embodiments of the present invention, the inorganic salt is selected from sodium chloride, magnesium chloride, or a combination of sodium chloride and magnesium chloride; preferably, the content ratio of sodium chloride to magnesium chloride is 1:5-5:1, for example, 1:5, 1:3, 1:1.5, 1:1, 1.5:1, 3:1, 5:1, for example, 3:2, for example, a combination of 30 mM sodium chloride and 20 mM magnesium chloride.

[0024] According to an embodiment of the present invention, the content of the inorganic salt is 10 μM-1000 mM, preferably 1 mM-200 mM, more preferably 10 mM-100 mM, for example 10 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM.

[0025] According to an embodiment of the present invention, the sealing agent further comprises: an antibacterial agent.

[0026] According to embodiments of the present invention, the active ingredient of the antibacterial agent is an inorganic or organic compound with antibacterial activity; preferably, the active ingredient of the antibacterial agent is selected from any one, two, or more of the following: sodium azide, potassium sorbate, potassium sorbitol, sodium phenylpropionate, isothiazolinone, methylchloroisothiazolinone, methylisothiazolinone; more preferably, sodium azide, methylchloroisothiazolinone, methylisothiazolinone, Proclin-300; more preferably, sodium azide, or methylchloroisothiazolinone and methylisothiazolinone in a mass ratio of 3:1. In some embodiments, the antibacterial agent is Proclin-300.

[0027] According to an embodiment of the present invention, the content (mass percentage) of the antibacterial agent is 0.001-1%, preferably 0.005-0.5%, and more preferably 0.01%-0.1%; for example, 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.05%, 0.08%, 0.1%.

[0028] According to an embodiment of the present invention, the sealing agent comprises the following components: a Bola-type surfactant, a pH adjuster, an inorganic salt, and an antibacterial agent.

[0029] According to an embodiment of the present invention, the sealing agent further comprises water; for example, purified water, distilled water, filtered water, etc.

[0030] According to an embodiment of the present invention, the sealing agent comprises: 0.1%-10% Bola-type surfactant, 10 mM-300 mM pH adjuster, 10 mM-100 mM inorganic salt, balance water; The pH adjuster is disodium hydrogen phosphate, sodium dihydrogen phosphate, 2-morpholine ethanesulfonic acid (MES), and 3-morpholine propanesulfonic acid (MOPS). Preferably, the Bola-type surfactant is selected from any one, two, or more of the following (i)-(viii): (i) Based on an alkylene chain of 1-20 carbons, with hydrophilic groups attached to both ends; preferably, the hydrophilic groups are the same or different, and are independently selected from quaternary ammonium salts, polyethylene glycol groups, polyol groups, carboxylates, water-soluble sugar groups, hydroxyl groups, and carboxyl groups; (ii) Based on a 6-20 carbon arylene or biarylene chain, with hydrophilic groups attached to both ends; preferably, the hydrophilic groups are the same or different, and are independently selected from quaternary ammonium salts, polyethylene glycol groups, polyol groups, carboxylates, water-soluble sugar groups, hydroxyl groups, and carboxyl groups; (iii) Based on polyethylene glycol chains with a degree of polymerization of 1-200, each end is connected to a hydrophobic group; preferably, the hydrophobic groups are the same or different, and are independently selected from C 2-20 alkenyl, C 2-20 alkynyl group, C 6-20 Aryl, 5-20 membered heterocyclic aryl; (iv) The structural formula is F-[CF(CF3)CF2O] n -CF(CF3)-CONH-(CH2CH2O) m -CH2CH2-NHCO-CF(CF3)-[OCF2(CF3)CF] n -F, where m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; (v) The structural formula is [RN + -(CH2CH2O) n -(CH2) m -N + R]·2Br - Where R is C 1-20 Alkyl group, m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; (vi) The structural formula is [Me3N + -(CH2) 12 -N + Me3]·2Br - ; (vii) The structural formula is Sophorose-O-(CH2). n-COOH, where n is an integer selected from 1 to 20; (viii) The structural formula is HO-(CH2). n -OH, where n is an integer selected from 2 to 30 (e.g., 5-25, 10-20, 15-18, 16); for example, the structural formula is HO-(CH2). 16 -OH; Preferably, the Bola-type surfactant is selected from any one, two, or more of the following (iv)-(viii): (iv) The structural formula is F-[CF(CF3)CF2O] n -CF(CF3)-CONH-(CH2CH2O) m -CH2CH2-NHCO-CF(CF3)-[OCF2(CF3)CF] n -F, where m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; (v) The structural formula is [RN + -(CH2CH2O) n -(CH2) m -N + R]·2Br - Where R is C 1-20 Alkyl group, m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; (vi) The structural formula is [Me3N + -(CH2) 12 -N + Me3]·2Br - ; (vii) The structural formula is Sophorose-O-(CH2). n -COOH, where n is an integer selected from 1 to 20; (viii) The structural formula is HO-(CH2). n -OH, where n is an integer selected from 2 to 30 (e.g., 5-25, 10-20, 16); for example, the structural formula is HO-(CH2). 16 -OH; Preferably, the Bola-type surfactant is selected from any one, two or more of the following (1)-(3): (1) The structural formula is HO-(CH2). n -OH, where n is an integer selected from 2 to 30 (e.g., 5-25, 10-20, 15-18, 16); for example, the structural formula is HO-(CH2). 16 -OH; (2) The structural formula is Sophorose-O-(CH2). n-COOH, where n is an integer selected from 1 to 20; (3) The structural formula is [RN + -(CH2CH2O) n -(CH2) m -N + R]·2Br - Where R is selected from C 1-20 Alkyl group, m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; Preferably, the content of the Bola-type surfactant is 1%-5%; Preferably, the pH adjuster is selected from: a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate, and 3-morpholinopropanesulfonic acid (MOPS). Preferably, the pH adjuster content is 20 mM-200 mM; Preferably, the inorganic salt is selected from sodium chloride, magnesium chloride, or a combination of sodium chloride and magnesium chloride; Preferably, the content of the inorganic salt is 20 mM-50 mM; Preferably, the sealing agent further comprises an antibacterial agent; preferably, the antibacterial agent is selected from Proclin-300; preferably, the content of the antibacterial agent is 0.005-0.5%; Preferably, the pH of the sealing agent is 6.0-7.5.

[0031] According to an embodiment of the present invention, the blocking agent comprises: 1% Bola-type surfactant, 25 mM disodium hydrogen phosphate, 25 mM sodium dihydrogen phosphate, 50 mM sodium chloride, 0.01% Proclin-300, with the balance being water, pH = 7.4; the structural formula of the Bola-type surfactant is HO-(CH2). 16 -OH.

[0032] According to an embodiment of the present invention, the blocking agent comprises: 3% Bola-type surfactant, 100 mM disodium hydrogen phosphate, 100 mM sodium dihydrogen phosphate, 25 mM magnesium chloride, 0.01% Proclin-300, and the balance being water, with a pH of 7.4; the Bola-type surfactant has the structural formula Sophorose-O-(CH2). n -COOH, where n is an integer selected from 1 to 20.

[0033] According to an embodiment of the present invention, the blocking agent comprises: 5% Bola-type surfactant, 20 mM MOPS, 30 mM sodium chloride, 20 mM magnesium chloride, 0.01% Proclin-300, with the balance being water, pH = 6.0; the Bola-type surfactant is di(trimethylammonium)tetraethylene glycol, with the structural formula: [(Me3N + )-(CH2CH2O)3-CH2CH2-N + Me3]·2Br - .

[0034] The present invention also provides the application of the above-mentioned sealing agent in the modification of material surface or in the preparation of material surface modification reagents.

[0035] The present invention also provides the application of the above-mentioned blocking agent in immunodiagnostics or in the preparation of immunodiagnostic reagents; preferably, the immunodiagnostics include: enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, immunochromatography, and immunoturbidimetry.

[0036] Beneficial effects This invention provides a blocking agent based on a Bola-type surfactant. This blocking agent can modify various surface materials, particularly for modifying and blocking carrier materials used in immunodiagnostics. It can reduce non-specific adsorption, thereby improving reagent sensitivity and signal-to-noise ratio. Compared to traditional blocking agents, the Bola-type surfactant-based blocking agent provided by this invention not only possesses the excellent characteristics of traditional surfactant blocking agents, but also exhibits a rigid hairpin structure after binding, which increases the repulsion of non-specific molecules and results in a more dense and effective blockage. Attached Figure Description

[0037] Figure 1 The bonding methods of different types of sealants on the surface of carrier materials. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0039] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0040] Bola A-16: Purchased from Sigma-Alderich, structural formula is shown in Table 1.

[0041] Proclin-300: Preservative, purchased from Sigma-Alderich.

[0042] KU-Bola-SL: The structural formula is shown in Table 1.

[0043] bis(trimethylammonium)tetraethylene glycol, with the structural formula: [(Me3N + )-(CH2CH2O)3-CH2CH2-N + Me3]·2Br - .

[0044] Example 1: Use of Bola-type surfactant blocking agents in enzyme-linked immunosorbent assay (ELISA) This embodiment primarily aims to investigate the use of the Bola-type surfactant involved in this invention in enzyme-linked immunosorbent assay (ELISA) reagents. The specific implementation process is as follows: 1) Preparation of Bola-type surfactant blocking agent: 1% Bola A-16, 25 mM disodium hydrogen phosphate, 25 mM sodium dihydrogen phosphate, 50 mM sodium chloride, 0.01% Proclin-300, pH = 7.4.

[0045] 2) Myeloperoxidase (MPO) enzyme-linked immunosorbent assay (ELISA) reagent was selected as the model reagent for this example. Following the general antibody coating protocol for ELISA, anti-MPO antibody was added to a 96-well microplate and incubated for 30 min. After removing the supernatant, the blocking agent prepared in step 1) was added and incubated for 10 min. Then, the supernatant was removed, and the plate was washed with PBS (phosphate buffer) and air-dried for later use.

[0046] 3) The reference blocking agent is 1% BSA, and the coating is carried out according to the above scheme.

[0047] 4) The reagents used in the testing process, such as labeled antibodies, sample diluents, washing solutions, and TMB colorimetric solutions, are all commercially available products.

[0048] 5) MPO antigen diluent was selected as the test sample, with concentrations of 0, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 400 ng / mL, numbered S0-S4.

[0049] 6) The detection was performed according to the general detection method of enzyme-linked immunosorbent assay (ELISA) reagents, and the results are summarized as follows.

[0050] Table 2. Use of Bola-type surfactant blocking agents in enzyme-linked immunosorbent assay (ELISA) (Unit: ng / mL)

[0051] Example 2: Use of Bola-type surfactant blocking agents in chemiluminescence This embodiment is mainly intended to demonstrate the performance of Bola-type surfactant blocking agents in chemiluminescence. The specific implementation scheme is shown below: 1) Formulation of Bola-type surfactant blocking agent: 3% KU-Bola-SL Bola series surfactant developed by Kenyou (Suzhou), 100 mM disodium hydrogen phosphate, 100 mM sodium dihydrogen phosphate, 25 mM magnesium chloride, 0.01% Proclin-300, pH= 7.4.

[0052] 2) The anti-Müllerian hormone (AMH) chemiluminescent assay reagent was selected as the model reagent for this example. Carboxyl magnetic beads were used as the carrier in this example. The coating was performed according to the general method for coating magnetic beads with chemiluminescent antibodies. The specific procedure was as follows: Carboxyl magnetic beads were activated with EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide), incubated in MES (50 mM, pH = 5.6) buffer for 1 h, then the supernatant was removed, anti-AMH antibody was added, and incubation was carried out at room temperature for 2 h. After removing the supernatant, the blocking agent prepared in step 1) above was added and blocked at room temperature for 2 h. After removing the supernatant, the beads were washed and diluted to obtain the working solution of the magnetic beads.

[0053] 3) Select 3% BSA as the reference sealant and coat the magnetic beads in the same way.

[0054] 4) The reagents used in the detection process, such as acridinium ester labeled antibody, sample diluent, washing solution and substrate solution, are all commercially available products. The Shenzhen Yingkai Shine i2910 fully automated chemiluminescence analyzer was selected as the detection instrument for this example.

[0055] 5) AMH antigen diluent was selected as the test sample, with concentrations of 0, 0.01 ng / mL, 0.05 ng / mL, 0.20 ng / mL, and 1.00 ng / mL, numbered S0-S4.

[0056] 6) The results obtained after the test are summarized as follows.

[0057] Table 3. Use of Bola-type surfactant blocking agents in chemiluminescence (unit: ng / mL)

[0058] Example 3: Use of Bola-type surfactant blocking agents in immunochromatography This embodiment demonstrates the performance of Bola-type surfactant blocking agents in immunochromatography, and the specific implementation scheme is shown below: 1) Preparation of Bola-type surfactant: 5% di(trimethylammonium)tetraethylene glycol, 20 mM MOPS, 30 mM sodium chloride, 20 mM magnesium chloride, 0.01% Proclin-300, pH = 6.0.

[0059] 2) Procalcitonin (PCT) immunochromatographic assay reagent was selected as the model reagent for this example. Carboxyl time-resolved fluorescent microspheres were chosen as the carrier for this example. Coating was performed according to the general method for chemiluminescent antibody-coated microspheres. The specific procedure was as follows: the microspheres were activated using EDC and NHS, incubated for 1 h in MES (50 mM, pH = 5.6) buffer, the supernatant was removed, anti-PCT antibody was added, and incubation was carried out at room temperature for 2 h. After removing the supernatant, the blocking agent prepared in step 1) was added and blocked at room temperature for 2 h. After removing the supernatant, the microspheres were washed and diluted to obtain the working solution.

[0060] 3) Select 3% BSA blocking agent as the reference blocking agent and coat the microspheres in the same way.

[0061] 4) Using a standard immunochromatographic method, the fluorescent microspheres were loaded onto a labeling pad using a gold-spraying membrane scribing device. Paired anti-PCT antibodies were then scribed onto an NC membrane, assembled, and cut into 3 mm wide test strips for later use. A Hangzhou Hemai dry fluorescence spectrometer was selected as the detection instrument for this example.

[0062] 5) PCT antigen diluent was selected as the test sample, with concentrations of 0, 0.05 ng / mL, 0.10 ng / mL, 1.00 ng / mL, and 10.00 ng / mL, numbered S0-S4.

[0063] 6) The results obtained after the test are summarized as follows.

[0064] Table 4. Use of Bola-type surfactant blocking agents in immunochromatography (unit: ng / mL)

[0065] Example 4: Test of non-specific protein adsorption by Bola-type surfactant-based blocking agents In this embodiment, different types of blocking agents were selected to activate and block the magnetic beads. Fluorescently labeled BSA (Flu-BSA) was then used as a model protein to test the non-specific adsorption of the magnetic beads under different blocking conditions. The specific procedures are as follows: 1) Six types of blocking agents were used, namely the Bola-type surfactant blocking agents (blocking agents 1-3) from Examples 1-3, BSA (1%), ethanolamine (100 mM), and MPC blocking agent (Biolipidure, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer). The blocking process was as follows: Carboxylated magnetic beads were activated with EDC and NHS, incubated in MES (50 mM, pH = 5.6) buffer for 1 h, then the supernatant was removed, and the aforementioned blocking agents were added (the amount of blocking agent used was related to the amount of magnetic beads, maintaining a reaction concentration of 10 mg / mL) for 2 h at room temperature. After removing the supernatant, the beads were washed and diluted to obtain the working solution.

[0066] 2) Flu-BSA protein was prepared into two concentrations: 10 ng / mL and 100 ng / mL.

[0067] 3) Add 100 μL of Flu-BSA to the blocked magnetic beads obtained in step 1), mix well and incubate for 10 min. After removing the supernatant, wash the magnetic beads with PBS and finally prepare the magnetic bead dispersion to be tested.

[0068] 4) Measure the fluorescence signal of the magnetic bead dispersion using a fluorescence spectrometer and calculate the content of Flu-BSA in the magnetic beads.

[0069] Table 5. Tests on the adsorption of nonspecific proteins by Bola-type surfactant blocking agents (unit: ng / mL)

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A blocking agent comprising a Bola-type surfactant; The Bola-type surfactant is composed of two hydrophilic polar groups whose heads are connected by one or more hydrophobic chains, or of two hydrophobic nonpolar groups whose heads are connected by one or more hydrophilic chains. The content of Bola-type surfactant in the blocking agent is 0.01%-10%.

2. The sealant according to claim 1, wherein The Bola-type surfactant is selected from any one, two, or more of the following: (i) Based on an alkylene chain of 1-20 carbons, with hydrophilic groups attached to both ends; the hydrophilic groups may be the same or different, and are independently selected from quaternary ammonium salts, polyethylene glycol groups, polyol groups, carboxylates, water-soluble sugar groups, hydroxyl groups, and carboxyl groups; (ii) Based on a 6-20 carbon arylene or biarylene chain, with hydrophilic groups attached to both ends; the hydrophilic groups may be the same or different, and are independently selected from quaternary ammonium salts, polyethylene glycol groups, polyol groups, carboxyl salts, water-soluble sugar groups, hydroxyl groups, and carboxyl groups; (iii) Based on a polyethylene glycol chain with a degree of polymerization of 1-20000, with a hydrophobic group attached to each end; the hydrophobic groups may be the same or different, and are independently selected from alkyl, alkenyl, alkynyl, aryl, and heterocyclic aryl groups; (iv) The structural formula is F-[CF(CF3)CF2O] n -CF(CF3)-CONH-(CH2CH2O) m -CH2CH2-NHCO-CF(CF3)-[OCF2(CF3)CF] n -F, where m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; (v) of the formula [RN + -(CH2CH2O) n -(CH2) m -N + R]·2Br - wherein R is C 1-20 alkyl, m is selected from an integer from 1 to 20, and n is selected from an integer from 1 to 20; (vi) a compound having the structural formula [Me3N + -(CH2) 12 -N + Me3]·2Br - ; (vii) Sophorose-O-(CH2) n -COOH, wherein n is selected from an integer from 1 to 20; (viii) of the formula HO-(CH2) n -OH, n being an integer selected from 2 to 30.

3. The sealant of claim 2, wherein, The Bola-type surfactant is selected from any one of the following: (1) a structure of HO-(CH2) n -OH, n is an integer selected from 2-30; (2) Sophorose-O-(CH2) n -COOH, wherein n is selected from an integer from 1 to 20; (3) The structural formula is [RN + -(CH2CH2O) n -(CH2) m -N + R]·2Br - Where R is selected from C 1-20 Alkyl group, m is selected from integers 1-20, and n is selected from integers 1-20.

4. The sealant according to any one of claims 1 to 3, characterized in that, The sealing agent further includes: a pH adjuster; And / or, the sealing agent further comprises: an inorganic salt; And / or, the sealing agent further comprises: an antibacterial agent.

5. The sealant of claim 4, wherein The pH adjuster is selected from any one, two or more of the following: disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate, sodium citrate, 4-hydroxyethylpiperazine ethanesulfonic acid, 2-morpholine ethanesulfonic acid, 3-morpholine propanesulfonic acid, 2-[[tris(hydroxymethyl)methyl]amino]ethanesulfonic acid, piperazine-N,N'-di(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride, and tris(hydroxymethyl)methylaminopropanesulfonic acid; And / or, the pH adjuster content is 10 μM-1000 mM; And / or, the pH value of the sealing agent is 5.0-8.0; And / or, the inorganic salt is selected from any one, two or more of the following: sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, sodium citrate, sodium ascorbate, potassium phosphate, sodium phosphate, ammonium sulfate, ammonium chloride, sodium gluconate; And / or, the content of the inorganic salt is 10 μM-1000 mM; And / or, the active ingredient of the antibacterial agent is selected from any one, two or more of the following: sodium azide, potassium sorbate, potassium sorbitol, sodium phenylpropionate, isothiazolinone, methylchloroisothiazolinone, methylisothiazolinone; And / or, the content of the antibacterial agent is 0.001-1%.

6. The sealant according to any one of claims 1 to 3, wherein The sealing agent comprises: 0.1%-10% Bola-type surfactant, 10 mM-300 mM pH adjuster, 10 mM-100 mM inorganic salt, and the balance being water.

7. The sealant of claim 6, wherein The Bola-type surfactant is selected from any one of the following: (1) a structure of HO-(CH2) n -OH, n is an integer selected from 2-30; (2) Sophorose-O-(CH2) n -COOH, wherein n is selected from an integer from 1 to 20; (3) The structural formula is [RN + -(CH2CH2O) n -(CH2) m -N + R]·2Br - Where R is selected from C 1-20 Alkyl group, m is selected from integers from 1 to 20, and n is selected from integers from 1 to 20; And / or, the content of the Bola-type surfactant is 1%-5%; And / or, the pH adjuster is selected from: a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate, 3-morpholine propanesulfonic acid; And / or, the pH adjuster content is 20 mM-200 mM; And / or, the inorganic salt is selected from: sodium chloride, magnesium chloride, or a combination of sodium chloride and magnesium chloride; And / or, the content of the inorganic salt is 20 mM-50 mM; And / or, the pH of the sealing agent is 6.0-7.

5.

8. The sealant of claim 6, wherein, The sealing agent further includes an antibacterial agent; the antibacterial agent is selected from Proclin-300; the content of the antibacterial agent is 0.005-0.5%.

9. The sealant of claim 8, wherein, The sealing agent comprises: 1% Bola surfactant, 25 mM disodium hydrogen phosphate, 25 mM sodium dihydrogen phosphate, 50 mM sodium chloride, 0.01% Proclin-300, the balance being water, pH = 7.4; the structural formula of the Bola surfactant is HO-(CH2) 16 -OH; Alternatively, the blocking agent comprises: 3% Bola-type surfactant, 100 mM disodium hydrogen phosphate, 100 mM sodium dihydrogen phosphate, 25 mM magnesium chloride, 0.01% Proclin-300, with the balance being water, pH = 7.4; the Bola-type surfactant has the structural formula Sophorose-O-(CH2). n -COOH, where n is an integer selected from 1 to 20; Alternatively, the blocking agent comprises: 5% Bola-type surfactant, 20 mM MOPS, 30 mM sodium chloride, 20 mM magnesium chloride, 0.01% Proclin-300, with the balance being water, pH = 6.0; the Bola-type surfactant is di(trimethylammonium)tetraethylene glycol, with the structural formula: [(Me3N + )-(CH2CH2O)3-CH2CH2-N + Me3]·2Br - .

10. The use of the blocking agent according to any one of claims 1-9 in the preparation of reagents for material surface modification or immunodiagnosis.

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