Surface hydroxyl and carboxylation modified microspheres and preparation method thereof

Abstract

The invention provides surface hydroxyl and carboxylation modified microspheres and a preparation method thereof. The preparation method comprises the following steps: preparing polymer microspheres, carrying out modification treatment on the surfaces of the polymer microspheres to enable the surfaces of the microspheres to carry hydroxyl groups, finally treating the microspheres by using an oxidizing agent, and regulating and controlling the ratio of the oxidizing agent to the microspheres to enable 30-70% of the hydroxyl groups carried on the surfaces of the microspheres to be oxidized into carboxyl groups. The surface hydroxyl and carboxylation modified microspheres are obtained. The surface of the prepared microsphere is modified with hydroxyl and carboxyl at the same time, non-specific adsorption of the microsphere caused by hydrophobic interaction and electrostatic interaction can be remarkably reduced, most importantly, the preparation method is simple in process, easy and convenient to operate and short in time consumption, meanwhile, the surface structure of the microsphere is simple, the proportion of hydroxyl to carboxyl on the surface of the microsphere is adjustable, and the microsphere is wide in applicability.

Description

Technical Field

[0001] The present invention relates to the field of microspheres for biological detection, and in particular to microspheres with surface hydroxyl and carboxyl modification and a preparation method thereof. Background Art

[0002] Photoinduced chemiluminescence technology has shown significant advantages in the field of biological detection, especially in the detection of small molecules such as hormones, due to its high detection accuracy, high sensitivity, high specificity, low cost and high efficiency of homogeneous detection. Photoinduced chemiluminescence technology uses nanoscale microspheres as reaction carriers. When using microspheres as adsorption carriers, there is often a problem of nonspecific adsorption. The main causes of nonspecific adsorption are hydrophobic and electrostatic effects. The electrostatic effect is mainly due to the fact that proteins have positively charged amino acid residues and negatively charged amino acid residues randomly distributed on their surfaces. Any net positively charged material or negatively charged material will easily adsorb proteins (charge adsorption).

[0003] At present, the means of reducing non-specific adsorption include modifying the surface of the microparticles. The prior art involves modifying carboxyl groups on the surface of microspheres. Modifying carboxyl groups can make microspheres have improved hydrophilicity. However, carboxyl groups are negatively charged, and non-specific adsorption is still widely present due to electrostatic and hydrophobic effects, thereby affecting the accuracy of detection. However, carboxyl groups are easy to couple antibodies, and the surface of biological microspheres needs to be carboxyl groups. A method for reducing non-specific adsorption is recorded in Chinese patent CN116718782A. The method is to combine polyethylene glycol derivatives containing functional groups (such as carboxyl, amino, sulfhydryl, aldehyde, etc.) on existing carboxyl receptor microspheres, so that in the detection process, since the surface of the receptor microspheres is hydrophilic, the non-specific adsorption of the hydrophobic region of the protein and the hydrophobic region of the microspheres is reduced, thereby achieving the purpose of reducing interference. However, the process described is complicated, time-consuming, and inconvenient to operate.

[0004] Therefore, how to prepare biological microspheres with low time consumption, low cost and low non-specific adsorption has been a major problem that has troubled technicians in the field of biological microspheres for many years. Summary of the invention

[0005] The purpose of the present invention is to provide a surface hydroxyl and carboxyl modified microsphere and a preparation method thereof, which can significantly reduce the nonspecific adsorption of the microspheres. The most important thing is that the preparation method is simple in process, easy to operate, and short in time. At the same time, the surface structure of the microspheres is simple, the ratio of hydroxyl and carboxyl groups on the surface of the microspheres is adjustable, and the microspheres have a wide range of applicability.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In one aspect of the present invention, a method for preparing surface hydroxyl and carboxyl modified microspheres is provided, comprising the following steps:

[0008] (1) Preparation of polymer microspheres;

[0009] (2) modifying the surface of the polymer microspheres so that the microspheres carry hydroxyl groups;

[0010] (3) treating the microspheres of step (2) with an oxidant, and adjusting the ratio of the oxidant to the microspheres so that 30%-70% of the hydroxyl groups on the surface of the microspheres are oxidized to carboxyl groups, thereby obtaining surface hydroxyl and carboxyl modified microspheres.

[0011] Furthermore, in step (3), the mass ratio of the oxidant to the microspheres of step (2) is 1:0.05-1:2, and the oxidant is selected from sodium periodate and sodium borohydride.

[0012] In some embodiments, in step (3), the mass ratio of the oxidant to the microspheres of step (2) is 1:0.2 to 1:1, and the oxidant is preferably sodium periodate.

[0013] Furthermore, in step (2), a compound containing both hydroxyl groups and double bonds is used to modify the surface of the microspheres so that the surface of the microspheres carries hydroxyl groups.

[0014] Furthermore, the compound containing both a hydroxyl group and a double bond is selected from hydroxyethyl methacrylate and 3-butene-1-ol.

[0015] In some embodiments, the compound containing both a hydroxyl group and a double bond is preferably hydroxyethyl methacrylate.

[0016] In some embodiments, the polymer microspheres are polystyrene microspheres.

[0017] Another aspect of the present invention provides a method for preparing surface hydroxyl and carboxyl modified polystyrene microspheres, comprising the following steps:

[0018] (1) After styrene is washed with a 0.05% NaOH aqueous solution, water and SDS are added in sequence, and then stirred until the SDS is completely dissolved, and then an initiator-potassium persulfate is added, and the mixture is heated to 80° C. in a water bath, and stirred continuously for 2 h to obtain a solution containing polystyrene microspheres;

[0019] (2) adding hydroxyethyl methacrylate and potassium persulfate to the polystyrene microsphere solution, continuing the reaction for 4 hours, and after the reaction is completed, washing away the unreacted substances with ethanol, and then drying to obtain polystyrene microspheres with hydroxyl groups on the surface;

[0020] (3) resuspending the polystyrene microspheres carrying hydroxyl groups on the surface in water, adding sodium periodate, stirring at room temperature for 2 hours, removing unreacted substances, and then drying to obtain polystyrene microspheres modified with surface hydroxyl groups and carboxyl groups.

[0021] The present invention also provides a hydroxyl and carboxyl modified microsphere, which is prepared by the above method.

[0022] The present invention also provides the use of the hydroxyl and carboxyl modified microspheres in chemiluminescence detection.

[0023] The preparation method of the present invention and the obtained product have the following advantages and beneficial effects:

[0024] (1) The present invention prepares a polymer microsphere or polystyrene microsphere whose surface is modified with both carboxyl and hydroxyl groups, which has good hydrophilicity. At the same time, the surface structure of the microsphere is simple, and only has hydroxyl and carboxyl groups. In addition, compared with microspheres whose surfaces are all carboxyl-modified, the surface of the microsphere has a certain hydroxyl ratio, so the charge is less, which can reduce the nonspecific adsorption caused by hydrophobic effect and charge electrostatic adsorption.

[0025] (2) The preparation method of the present invention comprises first modifying the hydroxyl groups on the surface of the polymer microspheres, and then utilizing the property of the hydroxyl groups being easily oxidized to oxidize part of the hydroxyl groups to carboxyl groups, thereby forming sites that can bind to proteins. The method of the present invention is simple in process, easy to operate, and short in time. In addition, the ratio of hydroxyl groups to carboxyl groups carried on the surface of the microspheres is adjustable, that is, it can be achieved by regulating the ratio of the oxidant to the microspheres. The microspheres prepared by the present invention have a wide range of applicability. DETAILED DESCRIPTION

[0026] In order to make the technical problems solved by the present invention, the technical solutions adopted and the technical effects achieved clearer, the technical solutions of the embodiments of the present invention are further described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. The present invention includes but is not limited to the following embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] A surface hydroxyl and carboxyl modified microsphere, wherein the carboxyl modification accounts for 35%, and the preparation method thereof comprises the following steps:

[0029] (1) After styrene is washed with a 0.05% NaOH aqueous solution, 10 ml of the solution is taken, and 56 ml of water and 0.1 g of SDS are added in sequence, followed by stirring until the SDS is completely dissolved, and then 0.03 g of potassium persulfate is added. The solution is heated to 80° C. in a water bath, and the stirring is continued. The reaction is continued for 2 h to obtain a solution containing polystyrene microspheres;

[0030] (2) adding 3 ml of hydroxyethyl methacrylate and potassium persulfate to the polystyrene microsphere solution, and continuing the reaction for 4 hours. After the reaction is completed, washing away the unreacted substances with ethanol, and then drying to obtain polystyrene microspheres with hydroxyl groups on the surface;

[0031] (3) The polystyrene microspheres carrying hydroxyl groups on the surface are resuspended in water, 500 mg of sodium periodate is added, stirred at room temperature for 2 hours, unreacted substances are removed, and then dried to obtain polystyrene microspheres modified with surface hydroxyl groups and carboxyl groups, wherein the carboxyl groups account for 35%.

[0032] Example 2

[0033] A surface hydroxyl and carboxyl modified microsphere, wherein the carboxyl modification accounts for 65%, and the preparation method thereof comprises the following steps:

[0034] The amount of sodium periodate in Example 1 was changed to 50 mg, and the rest was the same as Example 1. Increasing the amount of sodium periodate can increase the proportion of surface carboxylation modification. The hydroxyl and carboxylation modified polystyrene microspheres obtained in Example 2 had a carboxylation ratio of 65%.

[0035] Comparative Example 1

[0036] A microsphere with only carboxyl modification on its surface, the preparation method of which comprises the following steps:

[0037] The amount of sodium periodate in Example 1 was changed to 2 g, and the rest was the same as Example 1. Excessive addition of sodium periodate can oxidize all the hydroxyl groups on the surface of the microspheres to carboxyl groups.

[0038] Comparative Example 2

[0039] A microsphere with surface only hydroxylation modification, the preparation method of which comprises the following steps:

[0040] The steps (1) and (2) in Example 1 were used to prepare microspheres whose surfaces were modified only by hydroxylation.

[0041] Antibody conjugation validation:

[0042] The modified microspheres obtained in the above-mentioned Examples 1-2 and Comparative Examples 1-2 are subjected to antibody coupling verification by the double antibody sandwich method. The specific method steps are as follows: (1) the modified microspheres obtained in Examples 1-2 and Comparative Examples 1-2 are further processed to form luminescent microspheres; (2) a pair of specific antibodies R1 and R2 are used to coat the luminescent microspheres (FG-Ab) and the labeled biotin (Bio-Ab) of the examples or comparative examples, respectively, and added to the microwells, and then the sample Ag or the calibration substance to be tested is added, and after starting the first stage of warm bath, a double antibody sandwich complex (FG-Ab-Ag-Ab-Bio) is formed on the surface of the luminescent microspheres; (3) a photosensitive microsphere solution (universal solution) is prepared to coat streptavidin (GG-SA), and it is added to the microwell solution of the above step, and the second stage of warm bath is started, and the luminescent microspheres are bound to the streptavidin on the surface of the photosensitive microspheres through the surface biotin, and the two microspheres are close to each other; (4) a Cosma SMART 6500 device is used to induce light-induced chemiluminescence, and the intensity of the light signal is detected. The optical signal intensity is related to the antigen-antibody binding strength. The optical signal intensity detection results of Example 1-2 and Comparative Example 1-2 are shown in Table 1.

[0043] Table 1: Antibody coupling verification results of microspheres of Example 1-2 and Comparative Example 1-2:

[0044]

[0045] In Example 1-2 and Comparative Example 1-2, antibody coupling verification was performed. When the antigen concentration of the added test sample was zero, that is, there was no antigen, and in the absence of nonspecific adsorption, the reading should be zero; and at this time, if the reading of the value 0 is higher, it means that the nonspecific adsorption is more serious. As can be seen from Table 1, the hydroxyl and carboxyl modified microspheres of Examples 1 and 2 were tested for antibody coupling verification. Compared with Comparative Example 1 or 2, the nonspecific adsorption was significantly reduced, and the readings of other concentrations were also significantly improved.

Claims

1. A method for preparing surface hydroxyl and carboxyl modified microspheres, characterized in that: The following steps are involved: (1) Preparation of polymer microspheres; (2) modifying the surface of the polymer microspheres so that the microspheres carry hydroxyl groups; (3) treating the microspheres of step (2) with an oxidant, and adjusting the ratio of the oxidant to the microspheres so that 30%-70% of the hydroxyl groups on the surface of the microspheres are oxidized to carboxyl groups, thereby obtaining surface hydroxyl and carboxyl modified microspheres.

2. The method for preparing surface hydroxyl and carboxyl modified microspheres according to claim 1, characterized in that: In step (3), the mass ratio of the oxidant to the microspheres of step (2) is 1:0.05-1:2, and the oxidant is selected from sodium periodate and sodium borohydride.

3. The method for preparing surface hydroxyl and carboxyl modified microspheres according to claim 2, characterized in that: In step (3), the mass ratio of the oxidant to the microspheres of step (2) is 1:0.2-1:1, and the oxidant is preferably sodium periodate.

4. The method for preparing surface hydroxyl and carboxyl modified microspheres according to claim 3, characterized in that: In step (2), the surface of the microspheres is modified using a compound containing both hydroxyl groups and double bonds, so that the surface of the microspheres carries hydroxyl groups.

5. The method for preparing surface hydroxyl and carboxyl modified microspheres according to claim 4, characterized in that: The compound containing both hydroxyl groups and double bonds is selected from hydroxyethyl methacrylate and 3-butene-1-ol, preferably hydroxyethyl methacrylate.

6. The method for preparing surface hydroxyl and carboxyl modified microspheres according to claim 5, characterized in that: The polymer microspheres are polystyrene microspheres.

7. The method for preparing surface hydroxyl and carboxyl modified microspheres according to claim 6, characterized in that: The polystyrene microspheres include the following specific preparation steps: (1) After styrene is washed with a 0.05% NaOH aqueous solution, water and SDS are added in sequence, and then stirred until the SDS is completely dissolved, and then an initiator-potassium persulfate is added, and the mixture is heated to 80° C. in a water bath, and stirred continuously for 2 h to obtain a solution containing polystyrene microspheres; (2) adding hydroxyethyl methacrylate and potassium persulfate to the polystyrene microsphere solution, continuing the reaction for 4 hours, and after the reaction is completed, washing away the unreacted substances with ethanol, and then drying to obtain polystyrene microspheres with hydroxyl groups on the surface; (3) resuspending the polystyrene microspheres carrying hydroxyl groups on the surface in water, adding sodium periodate, stirring at room temperature for 2 hours, removing unreacted substances, and then drying to obtain polystyrene microspheres modified with surface hydroxyl groups and carboxyl groups.

8. A microsphere modified with surface hydroxyl and carboxyl groups, characterized in that: It is prepared by the method according to any one of claims 1 to 7.

9. Use of the surface hydroxyl and carboxyl modified microspheres according to claim 8 in chemiluminescence detection.

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