Diagnostic agents for measuring immune levels

Abstract

The invention discloses a diagnostic preparation for measuring immune levels, belonging to the field of biomedicine. The present invention first modifies magnetic mesoporous silica by aminosilane coupling agent, γ-mercaptopropyltrimethoxysilane, N-isopropylacrylamide, N-hydroxypropylacrylamide, concentrated sulfuric acid and hydrogen peroxide to obtain modified mesoporous silica, then mixes the modified mesoporous silica with polyallylamine hydrochloride, adds chloroauric acid, stirs and mixes, then adds trisodium citrate for reduction, obtains a product blank, and finally mixes the product blank with an antigen to obtain a diagnostic preparation for measuring immune levels. The diagnostic preparation for measuring immune levels prepared by the present invention has sensitive immunodiagnostic performance and has a good judgment effect on immune levels.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, in particular to a diagnostic preparation for determining immune levels. Background Art

[0002] As a new immunological method, the immunocolloidal gold technique has developed rapidly and has been increasingly widely used in various biomedical research fields, especially in medical testing. Colloidal gold has become a non-radioactive labeling agent commonly used in immunolabeling technology after fluorescein, radioisotopes and enzymes.

[0003] Colloidal gold is formed by the polymerization of chloroauric acid under the action of reducing agents such as tannic acid and trisodium citrate to form gold particles of a specific size, and due to electrostatic action, it becomes a stable colloidal state. It has the characteristics of high electron density and can bind to a variety of biological macromolecules. Immunogold labeling technology mainly uses this feature of gold particles. When gold-labeled proteins are bound, dark brown particles can be seen under a microscope. When these markers are gathered in large quantities at the corresponding ligands, red or purple spots can be seen with the naked eye. Therefore, it is used in qualitative or semi-quantitative rapid immunoassay methods.

[0004] Immunomagnetic particle technology is a new technology developed in recent years, and is mostly used in separation and concentration tests. Magnetic particles can covalently bind to proteins without affecting their activity, so they are often used to capture specific proteins or separate proteins. The principle is as follows: a magnetic particle labeled with an antibody is placed in a solution containing the target antigen. After sufficient mixing and reaction, an antigen-antibody-magnetic particle mixture is formed. The solution is passed through a magnetic column, and the target antigen is retained due to the adsorption of the magnetic particles. At the same time, it can also be used for quantitative detection, and the amount of captured antibody or antigen can be determined according to the strength of the magnetism.

[0005] At present, the application of immunocolloidal gold technology in gold-labeled detection reagents has the following disadvantages:

[0006] 1. Due to methodological limitations, diagnostic preparations established using only immunocolloidal gold technology have low sensitivity.

[0007] 2. For some samples with extremely low antigen or antibody content, the rapid diagnostic preparations established using this technology cannot make direct and effective judgments on the immune level due to sensitivity limitations. Summary of the invention

[0008] The object of the present invention is to provide a diagnostic preparation for measuring immune level and a preparation method thereof, so as to solve the problems in the prior art.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A diagnostic preparation for measuring immune levels, characterized in that the diagnostic preparation for measuring immune levels mainly comprises the following raw material components in parts by weight: 20 to 30 parts of modified mesoporous silica, 10 to 12 parts of antigens, 2 to 4 parts of trisodium citrate and 5 to 8 parts of chloroauric acid; the addition of modified mesoporous silica can adsorb antigens, thereby achieving effective contact between antibodies and antigens during the immune diagnosis process.

[0011] A diagnostic preparation for measuring immune level, characterized in that the diagnostic preparation for measuring immune level also includes the following raw material components in parts by weight: 5 to 10 parts of polyallylamine hydrochloride; the addition of polyallylamine hydrochloride can make the gold chloride ions in chloroauric acid adsorbed inside the modified mesoporous silica, and under the treatment of trisodium citrate, gold single substance can be formed inside the modified mesoporous silica and blocked by antigens; when the antigen is engulfed by antibodies, the macromolecular nature of the antigen disappears, thereby allowing the modified mesoporous gold single substance and magnetic particles to combine with antibodies to form red or purple spots visible to the naked eye, thereby realizing the immune diagnosis function.

[0012] As an optimization, the modified mesoporous silica is prepared by jointly modifying magnetic mesoporous silica with an aminosilane coupling agent, γ-mercaptopropyltrimethoxysilane, N-isopropylacrylamide, N-hydroxypropylacrylamide, concentrated sulfuric acid and hydrogen peroxide; the magnetic mesoporous silica is prepared by mixing cellulose, mesoporous silica and magnetic particles; the addition of cellulose can improve the adsorption performance of mesoporous silica on antigens.

[0013] As an optimization, the diagnostic preparation for determining the immune level mainly includes the following raw material components in parts by weight: 28 parts of modified mesoporous silica, 10 parts of antigen, 4 parts of trisodium citrate, 6 parts of chloroauric acid and 8 parts of polyallylamine hydrochloride.

[0014] As an optimization, a method for preparing a diagnostic preparation for measuring immune levels mainly includes the following preparation steps:

[0015] (1) The magnetic mesoporous silica is first treated with concentrated sulfuric acid and hydrogen peroxide, and then modified with aminosilane coupling agent, γ-mercaptopropyltrimethoxysilane, N-isopropylacrylamide and N-hydroxypropylacrylamide, filtered and dried;

[0016] (2) mixing the substance obtained in step (1) with a polyallylamine hydrochloride solution, adding chloroauric acid and trisodium citrate in sequence, stirring for reaction, filtering, and drying;

[0017] (3) mixing the substance obtained in step (2) with the antigen dispersion under alkaline conditions, stirring for reaction, filtering and drying;

[0018] (4) Performing index analysis on the substance obtained in step (3).

[0019] As an optimization, a method for preparing a diagnostic preparation for measuring immune levels mainly includes the following preparation steps:

[0020] (1) 98% concentrated sulfuric acid and 30% hydrogen peroxide are mixed in a volume ratio of 7:3 to obtain a mixed treatment liquid, and the mixed treatment liquid is mixed with magnetic mesoporous silica in a mass ratio of 10:1-12:1, stirred for reaction, filtered, washed, and dried to obtain a modified mesoporous silica blank; the modified mesoporous silica blank is mixed with anhydrous ethanol in a mass ratio of 1:50, and 0.1-0.3 times the mass of the modified mesoporous silica blank is added with aminopropyltrimethoxysilane and 0.1-0.2 times the mass of the modified mesoporous silica blank is added, stirred for reaction, and centrifuged to obtain a pretreated modified mesoporous silica blank, and the pretreated modified mesoporous silica blank is mixed with anhydrous ethanol in a mass ratio of 1:50. The blank is mixed with N,N-dimethylformamide at a mass ratio of 1:10, and succinic anhydride is added in an amount twice the mass of the pre-treated modified mesoporous silica blank, stirred for reaction, filtered, and dried to obtain pre-treated modified mesoporous silica; the pre-treated modified mesoporous silica is mixed with tetrahydrofuran at a mass ratio of 1:20-1:30, and N-isopropylacrylamide in an amount of 10-12 times the mass of the pre-treated modified mesoporous silica, N-hydroxymethylacrylamide in an amount of 0.8-1.2 times the mass of the pre-treated modified mesoporous silica, and azobisisobutyronitrile in an amount of 0.01-0.03 times the mass of the pre-treated modified mesoporous silica are added, stirred for reaction under a nitrogen atmosphere, filtered, washed, and dried;

[0021] (2) polyallylamine hydrochloride and water are mixed at a mass ratio of 1:10, and the substance obtained in step (1) is added in an amount of 4 to 5 times the mass of polyallylamine hydrochloride, and the mixture is stirred to obtain a mixed dispersion, the mixed dispersion is mixed with chloroauric acid at a mass ratio of 50:1 to 80:1, and trisodium citrate is added in an amount of 0.02 to 0.05 times the mass of the mixed dispersion, and the mixture is stirred for reaction, filtered, and dried;

[0022] (3) Mix the antigen and water in a mass ratio of 1:10, disperse them ultrasonically at pH 8, add the substance obtained in step (2) with a mass of 4 to 6 times that of the antigen, stir for reaction, filter in a magnetic filter, remove the filtrate, and dry;

[0023] (4) Performing index analysis on the substance obtained in step (3).

[0024] As an optimization, the magnetic mesoporous silica in step (1) is prepared by mixing an aqueous solution of 2% hexadecyltrimethylammonium bromide with nano-ferrosilicate in a mass ratio of 200:1 to 300:1, and adding 1,3,5-trimethylbenzene in an amount of 0.1 to 0.2 times the mass of the aqueous solution of hexadecyltrimethylammonium bromide and ammonia in an amount of 0.1 to 0.3 times the mass of the aqueous solution of hexadecyltrimethylammonium bromide, and then ultrasonically dispersing the mixture to obtain a mixed aqueous solution of hexadecyltrimethylammonium bromide. The mixed aqueous solution of hexadecyltrimethylammonium bromide is mixed with tetraethyl orthosilicate in a mass ratio of 40:1, and then adding microfibrillated cellulose fibers in an amount of 4 to 8 times the mass of tetraethyl orthosilicate. After stirring for reaction, the mixture is filtered and dried to obtain a magnetic mesoporous silica blank. The magnetic mesoporous silica blank is mixed with ammonium nitrate in a mass ratio of 1:2, and then adding anhydrous ethanol in an amount of 10 times the mass of the magnetic mesoporous silica blank. After condensation and reflux, the mixture is filtered and dried to obtain a magnetic mesoporous silica blank.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention uses modified mesoporous silica when preparing a diagnostic preparation for measuring immune levels, and simultaneously adds polyallylamine hydrochloride; first, after the modified mesoporous silica is modified, acrylamide is grafted onto the pores inside the modified mesoporous silica, thereby making the modified mesoporous silica pH-sensitive, that is, under acidic conditions, the pores of the modified mesoporous silica will expand, and when the pH is alkaline, the pores of the modified mesoporous silica will shrink. Therefore, after the modified mesoporous silica is mixed with chloroauric acid, when the chloroauric acid is reduced under the action of trisodium citrate, the pores inside the modified mesoporous silica will shrink at the same time, so that the gold element formed by the reduction of chloroauric acid by trisodium citrate can be fixed in the voids inside the modified mesoporous silica. Secondly, after the modified mesoporous silica is mixed with the antigen, the shrinkage of the mesopores in the modified mesoporous silica can prevent the gold particles from binding to the antigen, resulting in a color reaction and improving the sensitivity of the product during use; furthermore, polyallylamine hydrochloride is added during the product preparation process. Since the internal surface of the modified mesoporous silica carries a negative charge, when the modified mesoporous silica is mixed with polyallylamine hydrochloride, the polyallylamine hydrochloride can be adsorbed on the inner wall of the modified mesoporous silica under the action of electrostatic force. When the modified mesoporous silica treated with polyallylamine hydrochloride is mixed with chloroauric acid, gold chloride ions can be adsorbed inside the modified mesoporous silica, thereby providing conditions for the subsequent adsorption and fixation of gold by the modified mesoporous silica.

[0027] (2) The present invention modifies silica when preparing a diagnostic preparation for measuring immune levels, so that the mesoporous silica has pH sensitivity. After the mesoporous silica adsorbs the antigen, the mesopores can be closed to effectively prevent the loss of gold particles inside the modified mesoporous silica. After the antigen is adsorbed, due to the large molecular weight of the antigen, the pH around the mesopores inside the modified mesoporous silica can be kept stable when the antigen is not eliminated, thereby improving the sensitivity of the product. When the antigen is eliminated, no matter how much of the antigen is eliminated, the pH around the pores inside the modified mesoporous silica will change, thereby causing the gold particles inside the modified mesoporous silica to leak out. Moreover, due to the connectivity of the pores inside the modified mesoporous silica, an obvious color reaction can be formed, thereby further improving the sensitivity of the product. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] In order to more clearly illustrate the method provided by the present invention, the following examples are used to describe in detail the test methods for each index of the diagnostic preparation for determining the immune level prepared in the following examples are as follows:

[0030] Color development: Place the diagnostic preparations for determining the immune level obtained in each example and the comparative example product in the antibody solution corresponding to the antigen (concentration is 2g / L) and observe whether there is any color change.

[0031] Immune level determination: The diagnostic preparations for determining the immune level obtained in each example and the comparative example products were placed in antibody solutions corresponding to antigens of different concentrations, and the time for the color reaction to occur was observed; the shorter the color reaction time, the higher the immune level.

[0032] Example 1

[0033] A diagnostic preparation for measuring immune level mainly comprises the following raw material components by weight: 28 parts of modified mesoporous silica, 10 parts of antigen, 4 parts of trisodium citrate, 6 parts of chloroauric acid and 8 parts of polyallylamine hydrochloride.

[0034] A method for preparing a diagnostic preparation for measuring immune levels, the method for preparing a diagnostic preparation for measuring immune levels mainly comprises the following preparation steps:

[0035] (1) 98% concentrated sulfuric acid and 30% hydrogen peroxide were mixed in a volume ratio of 7:3 to obtain a mixed treatment liquid, and the mixed treatment liquid was mixed with magnetic mesoporous silica in a mass ratio of 12:1. The mixture was stirred at a temperature of 45°C and a rotation speed of 300 r / min for 2 h, and then filtered to obtain a filter cake. The filter cake was washed with water for 5 times, and then dried at a temperature of 80°C for 6 h to obtain a modified mesoporous silica blank. The modified mesoporous silica blank was mixed with anhydrous ethanol in a mass ratio of 1:1. :50 were mixed in a beaker, and 0.3 times the mass of the modified mesoporous silica blank aminopropyltrimethoxysilane and 0.2 times the mass of the modified mesoporous silica blank γ-mercaptopropyltrimethoxysilane were added to the beaker, and the reaction was stirred at a temperature of 65 ° C and a speed of 320 r / min for 8 hours, and then centrifuged to obtain a pretreated modified mesoporous silica blank. The pretreated modified mesoporous silica blank was mixed with N,N-dimethylformamide at a mass ratio of 1:10 In a flask, add succinic anhydride twice the mass of the pretreated modified mesoporous silica blank into the flask, stir and react for 6 hours at a temperature of 60°C and a rotation speed of 300r / min, filter to obtain a filter residue, and dry the filter residue at a temperature of 80°C for 3 hours to obtain pretreated modified mesoporous silica; mix the pretreated modified mesoporous silica and tetrahydrofuran in a four-necked flask at a mass ratio of 1:30, and add the pretreated modified mesoporous silica into the four-necked flask. 12 times the mass of N-isopropylacrylamide, 1.2 times the mass of N-hydroxymethylacrylamide and 0.03 times the mass of azobisisobutyronitrile were introduced into a four-necked flask at a rate of 50 mL / min. The mixture was stirred at 80°C and 280 r / min for 24 h, filtered, the filtrate was removed, washed with deionized water for 3 times, and dried at 60°C for 2 h.

[0036] (2) polyallylamine hydrochloride and water are mixed at a mass ratio of 1:10, and the substance obtained in step (1) is added to the mixture of polyallylamine hydrochloride and water in an amount of 5 times the mass of polyallylamine hydrochloride, and the mixture is stirred for 30 minutes at a temperature of 40° C. and a rotation speed of 300 r / min to obtain a mixed dispersion, the mixed dispersion is mixed with chloroauric acid at a mass ratio of 80:1, and trisodium citrate is added to the mixture of the mixed dispersion and chloroauric acid in an amount of 0.05 times the mass of the mixed dispersion, and the mixture is stirred for reaction for 3 hours at a temperature of 40° C. and a rotation speed of 280 r / min, and then filtered and dried in vacuo;

[0037] (3) The antigen and water were mixed at a mass ratio of 1:10, and the pH of the mixture of the antigen and water was adjusted to 8 with a 2% sodium hydroxide solution. After ultrasonic dispersion at a frequency of 45 kHz for 10 minutes, the substance obtained in step (2) was added to the mixture of the antigen and water in an amount of 6 times the mass of the antigen. After stirring for reaction at a temperature of 30°C and a rotation speed of 300 r / min for 5 hours, the filtrate was removed by magnetic filtration to obtain a blank, and the blank was dried at a temperature of 40°C for 12 hours.

[0038] (4) Performing index analysis on the substance obtained in step (3).

[0039] As an optimization, the magnetic mesoporous silica in step (1) is prepared by mixing an aqueous solution of 2% hexadecyltrimethylammonium bromide with nano-ferrosilicate in a mass ratio of 300:1, and adding 1,3,5-trimethylbenzene in an amount of 0.2 times the mass of the hexadecyltrimethylammonium bromide aqueous solution and ammonia water in an amount of 0.3 times the mass of the hexadecyltrimethylammonium bromide aqueous solution, and then ultrasonically dispersing to obtain a mixed aqueous solution of hexadecyltrimethylammonium bromide; the mixed aqueous solution of hexadecyltrimethylammonium bromide is mixed with tetraethyl orthosilicate in a mass ratio of 40:1, and then adding microfibrillated cellulose fibers in an amount of 8 times the mass of tetraethyl orthosilicate, stirring for reaction, filtering, and drying to obtain a magnetic mesoporous silica blank; the magnetic mesoporous silica blank is mixed with ammonium nitrate in a mass ratio of 1:2, and then adding anhydrous ethanol in an amount of 10 times the mass of the magnetic mesoporous silica blank, and then condensing and refluxing, filtering, and drying to obtain a magnetic mesoporous silica blank.

[0040] Example 2

[0041] A diagnostic preparation for measuring immune level mainly comprises the following raw material components by weight: 28 parts of modified mesoporous silica, 10 parts of antigen, 4 parts of trisodium citrate, 6 parts of chloroauric acid and 8 parts of polyallylamine hydrochloride.

[0042] A method for preparing a diagnostic preparation for measuring immune levels, the method for preparing a diagnostic preparation for measuring immune levels mainly comprises the following preparation steps:

[0043] (1) 98% concentrated sulfuric acid and 30% hydrogen peroxide were mixed in a volume ratio of 7:3 to obtain a mixed treatment liquid, and the mixed treatment liquid was mixed with magnetic mesoporous silica in a mass ratio of 12:1. The mixture was stirred at a temperature of 45°C and a rotation speed of 300 r / min for 2 h, and then filtered to obtain a filter cake. The filter cake was washed with water for 5 times, and then dried at a temperature of 80°C for 6 h to obtain a modified mesoporous silica blank. The modified mesoporous silica blank was mixed with anhydrous ethanol in a mass ratio of 1:1. :50 were mixed in a beaker, and 0.3 times the mass of the modified mesoporous silica blank aminopropyltrimethoxysilane and 0.2 times the mass of the modified mesoporous silica blank γ-mercaptopropyltrimethoxysilane were added to the beaker, and the reaction was stirred at a temperature of 65 ° C and a speed of 320 r / min for 8 hours, and then centrifuged to obtain a pretreated modified mesoporous silica blank. The pretreated modified mesoporous silica blank was mixed with N,N-dimethylformamide at a mass ratio of 1:10 In a flask, add succinic anhydride twice the mass of the pretreated modified mesoporous silica blank into the flask, stir and react for 6 hours at a temperature of 60°C and a rotation speed of 300r / min, filter to obtain a filter residue, and dry the filter residue at a temperature of 80°C for 3 hours to obtain pretreated modified mesoporous silica; mix the pretreated modified mesoporous silica and tetrahydrofuran in a four-necked flask at a mass ratio of 1:30, and add the pretreated modified mesoporous silica into the four-necked flask. 12 times the mass of N-isopropylacrylamide, 1.2 times the mass of N-hydroxymethylacrylamide and 0.03 times the mass of azobisisobutyronitrile were introduced into a four-necked flask at a rate of 50 mL / min. The mixture was stirred at 80°C and 280 r / min for 24 h, filtered, the filtrate was removed, washed with deionized water for 3 times, and dried at 60°C for 2 h.

[0044] (2) polyallylamine hydrochloride and water are mixed at a mass ratio of 1:10, and the substance obtained in step (1) is added to the mixture of polyallylamine hydrochloride and water in an amount of 5 times the mass of polyallylamine hydrochloride, and the mixture is stirred for 30 minutes at a temperature of 40° C. and a rotation speed of 300 r / min to obtain a mixed dispersion, the mixed dispersion is mixed with chloroauric acid at a mass ratio of 80:1, and trisodium citrate is added to the mixture of the mixed dispersion and chloroauric acid in an amount of 0.05 times the mass of the mixed dispersion, and the mixture is stirred for reaction for 3 hours at a temperature of 40° C. and a rotation speed of 280 r / min, and then filtered and dried in vacuo;

[0045] (3) The antigen and water were mixed at a mass ratio of 1:10, and the pH of the mixture of the antigen and water was adjusted to 8 with a 2% sodium hydroxide solution. After ultrasonic dispersion at a frequency of 45 kHz for 10 minutes, the substance obtained in step (2) was added to the mixture of the antigen and water in an amount of 6 times the mass of the antigen. After stirring for reaction at a temperature of 30°C and a rotation speed of 300 r / min for 5 hours, the filtrate was removed by magnetic filtration to obtain a blank, and the blank was dried at a temperature of 40°C for 12 hours.

[0046] (4) Performing index analysis on the substance obtained in step (3).

[0047] As an optimization, the magnetic mesoporous silica in step (1) is prepared by mixing an aqueous solution of 2% hexadecyltrimethylammonium bromide with nano-ferrosilicate in a mass ratio of 300:1, and adding 1,3,5-trimethylbenzene in an amount of 0.2 times the mass of the aqueous solution of hexadecyltrimethylammonium bromide and ammonia in an amount of 0.3 times the mass of the aqueous solution of hexadecyltrimethylammonium bromide, and then ultrasonically dispersing the mixture to obtain a mixed aqueous solution of hexadecyltrimethylammonium bromide. The mixed aqueous solution of hexadecyltrimethylammonium bromide is mixed with ethyl orthosilicate in a mass ratio of 40:1, and after stirring for reaction, the mixture is filtered and dried to obtain a magnetic mesoporous silica blank. The magnetic mesoporous silica blank is mixed with ammonium nitrate in a mass ratio of 1:2, and anhydrous ethanol in an amount of 10 times the mass of the magnetic mesoporous silica blank is added. After condensation and reflux, the mixture is filtered and dried to obtain a magnetic mesoporous silica blank.

[0048] Example 3

[0049] A diagnostic preparation for measuring immune level mainly comprises the following raw material components in parts by weight: 28 parts of modified mesoporous silica, 10 parts of antigen, 4 parts of trisodium citrate and 6 parts of chloroauric acid.

[0050] A method for preparing a diagnostic preparation for measuring immune levels, the method for preparing a diagnostic preparation for measuring immune levels mainly comprises the following preparation steps:

[0051] (1) 98% concentrated sulfuric acid and 30% hydrogen peroxide were mixed in a volume ratio of 7:3 to obtain a mixed treatment liquid, and the mixed treatment liquid was mixed with magnetic mesoporous silica in a mass ratio of 12:1. The mixture was stirred at a temperature of 45°C and a rotation speed of 300 r / min for 2 h, and then filtered to obtain a filter cake. The filter cake was washed with water for 5 times, and then dried at a temperature of 80°C for 6 h to obtain a modified mesoporous silica blank. The modified mesoporous silica blank was mixed with anhydrous ethanol in a mass ratio of 1:1. :50 were mixed in a beaker, and 0.3 times the mass of the modified mesoporous silica blank aminopropyltrimethoxysilane and 0.2 times the mass of the modified mesoporous silica blank γ-mercaptopropyltrimethoxysilane were added to the beaker, and the reaction was stirred at a temperature of 65 ° C and a speed of 320 r / min for 8 hours, and then centrifuged to obtain a pretreated modified mesoporous silica blank. The pretreated modified mesoporous silica blank was mixed with N,N-dimethylformamide at a mass ratio of 1:10 In a flask, add succinic anhydride twice the mass of the pretreated modified mesoporous silica blank into the flask, stir and react for 6 hours at a temperature of 60°C and a rotation speed of 300r / min, filter to obtain a filter residue, and dry the filter residue at a temperature of 80°C for 3 hours to obtain pretreated modified mesoporous silica; mix the pretreated modified mesoporous silica and tetrahydrofuran in a four-necked flask at a mass ratio of 1:30, and add the pretreated modified mesoporous silica into the four-necked flask. 12 times the mass of N-isopropylacrylamide, 1.2 times the mass of N-hydroxymethylacrylamide and 0.03 times the mass of azobisisobutyronitrile were introduced into a four-necked flask at a rate of 50 mL / min. The mixture was stirred at 80°C and 280 r / min for 24 h, filtered, the filtrate was removed, washed with deionized water for 3 times, and dried at 60°C for 2 h.

[0052] (2) mixing the substance obtained in step (1) with water in a mass ratio of 1:10, and adding 0.3 times the mass of chloroauric acid and 0.05 times the mass of trisodium citrate of the substance obtained in step (1) to the mixture of the substance obtained in step (1) and water, stirring the mixture at a temperature of 40° C. and a rotation speed of 280 r / min for 3 h, filtering, and vacuum drying;

[0053] (3) The antigen and water were mixed at a mass ratio of 1:10, and the pH of the mixture of the antigen and water was adjusted to 8 with a 2% sodium hydroxide solution. After ultrasonic dispersion at a frequency of 45 kHz for 10 minutes, the substance obtained in step (2) was added to the mixture of the antigen and water in an amount of 6 times the mass of the antigen. After stirring for reaction at a temperature of 30°C and a rotation speed of 300 r / min for 5 hours, the filtrate was removed by magnetic filtration to obtain a blank, and the blank was dried at a temperature of 40°C for 12 hours.

[0054] (4) Performing index analysis on the substance obtained in step (3).

[0055] As an optimization, the magnetic mesoporous silica in step (1) is prepared by mixing an aqueous solution of 2% hexadecyltrimethylammonium bromide with nano-ferrosilicate in a mass ratio of 300:1, and adding 1,3,5-trimethylbenzene in an amount of 0.2 times the mass of the hexadecyltrimethylammonium bromide aqueous solution and ammonia water in an amount of 0.3 times the mass of the hexadecyltrimethylammonium bromide aqueous solution, and then ultrasonically dispersing to obtain a mixed aqueous solution of hexadecyltrimethylammonium bromide; the mixed aqueous solution of hexadecyltrimethylammonium bromide is mixed with tetraethyl orthosilicate in a mass ratio of 40:1, and then adding microfibrillated cellulose fibers in an amount of 8 times the mass of tetraethyl orthosilicate, stirring for reaction, filtering, and drying to obtain a magnetic mesoporous silica blank; the magnetic mesoporous silica blank is mixed with ammonium nitrate in a mass ratio of 1:2, and then adding anhydrous ethanol in an amount of 10 times the mass of the magnetic mesoporous silica blank, and then condensing and refluxing, filtering, and drying to obtain a magnetic mesoporous silica blank.

[0056] Example 4

[0057] A diagnostic preparation for measuring immune level mainly comprises the following raw material components by weight: 28 parts of modified mesoporous silica, 10 parts of antigen, 4 parts of trisodium citrate, 6 parts of chloroauric acid and 8 parts of polyallylamine hydrochloride.

[0058] A method for preparing a diagnostic preparation for measuring immune levels, the method for preparing a diagnostic preparation for measuring immune levels mainly comprises the following preparation steps:

[0059] (1) 98% concentrated sulfuric acid and 30% hydrogen peroxide were mixed in a volume ratio of 7:3 to obtain a mixed treatment liquid, and the mixed treatment liquid was mixed with magnetic mesoporous silica in a mass ratio of 12:1. The mixture was stirred at a temperature of 45°C and a rotation speed of 300 r / min for 2 hours, and then filtered to obtain a filter cake. The filter cake was washed with water for 5 times and then dried at a temperature of 80°C for 6 hours.

[0060] (2) polyallylamine hydrochloride and water are mixed at a mass ratio of 1:10, and the substance obtained in step (1) is added to the mixture of polyallylamine hydrochloride and water in an amount of 5 times the mass of polyallylamine hydrochloride, and the mixture is stirred for 30 minutes at a temperature of 40° C. and a rotation speed of 300 r / min to obtain a mixed dispersion, the mixed dispersion is mixed with chloroauric acid at a mass ratio of 80:1, and trisodium citrate is added to the mixture of the mixed dispersion and chloroauric acid in an amount of 0.05 times the mass of the mixed dispersion, and the mixture is stirred for reaction for 3 hours at a temperature of 40° C. and a rotation speed of 280 r / min, and then filtered and dried in vacuo;

[0061] (3) The antigen and water were mixed at a mass ratio of 1:10, and the pH of the mixture of the antigen and water was adjusted to 8 with a 2% sodium hydroxide solution. After ultrasonic dispersion at a frequency of 45 kHz for 10 minutes, the substance obtained in step (2) was added to the mixture of the antigen and water in an amount of 6 times the mass of the antigen. After stirring for reaction at a temperature of 30°C and a rotation speed of 300 r / min for 5 hours, the filtrate was removed by magnetic filtration to obtain a blank, and the blank was dried at a temperature of 40°C for 12 hours.

[0062] (4) Performing index analysis on the substance obtained in step (3).

[0063] As an optimization, the magnetic mesoporous silica in step (1) is prepared by mixing an aqueous solution of 2% hexadecyltrimethylammonium bromide with nano-ferrosilicate in a mass ratio of 300:1, and adding 1,3,5-trimethylbenzene in an amount of 0.2 times the mass of the hexadecyltrimethylammonium bromide aqueous solution and ammonia water in an amount of 0.3 times the mass of the hexadecyltrimethylammonium bromide aqueous solution, and then ultrasonically dispersing to obtain a mixed aqueous solution of hexadecyltrimethylammonium bromide; the mixed aqueous solution of hexadecyltrimethylammonium bromide is mixed with tetraethyl orthosilicate in a mass ratio of 40:1, and then adding microfibrillated cellulose fibers in an amount of 8 times the mass of tetraethyl orthosilicate, stirring for reaction, filtering, and drying to obtain a magnetic mesoporous silica blank; the magnetic mesoporous silica blank is mixed with ammonium nitrate in a mass ratio of 1:2, and then adding anhydrous ethanol in an amount of 10 times the mass of the magnetic mesoporous silica blank, and then condensing and refluxing, filtering, and drying to obtain a magnetic mesoporous silica blank.

[0064] Comparative Example

[0065] A diagnostic preparation for measuring immune level mainly comprises the following raw material components in parts by weight: 28 parts of modified mesoporous silica, 10 parts of antigen, 4 parts of trisodium citrate and 6 parts of chloroauric acid.

[0066] A method for preparing a diagnostic preparation for measuring immune levels, the method for preparing a diagnostic preparation for measuring immune levels mainly comprises the following preparation steps:

[0067] (1) 98% concentrated sulfuric acid and 30% hydrogen peroxide were mixed in a volume ratio of 7:3 to obtain a mixed treatment liquid, and the mixed treatment liquid was mixed with magnetic mesoporous silica in a mass ratio of 12:1. The mixture was stirred at a temperature of 45°C and a rotation speed of 300 r / min for 2 hours, and then filtered to obtain a filter cake. The filter cake was washed with water for 5 times and then dried at a temperature of 80°C for 6 hours.

[0068] (2) mixing the substance obtained in step (1) with water in a mass ratio of 1:10, and adding 0.3 times the mass of chloroauric acid and 0.05 times the mass of trisodium citrate of the substance obtained in step (1) to the mixture of the substance obtained in step (1) and water, stirring the mixture at a temperature of 40° C. and a rotation speed of 280 r / min for 3 h, filtering, and vacuum drying;

[0069] (3) The antigen and water were mixed at a mass ratio of 1:10, and the pH of the mixture of the antigen and water was adjusted to 8 with a 2% sodium hydroxide solution. After ultrasonic dispersion at a frequency of 45 kHz for 10 minutes, the substance obtained in step (2) was added to the mixture of the antigen and water in an amount of 6 times the mass of the antigen. After stirring for reaction at a temperature of 30°C and a rotation speed of 300 r / min for 5 hours, the filtrate was removed by magnetic filtration to obtain a blank, and the blank was dried at a temperature of 40°C for 12 hours.

[0070] (4) Performing index analysis on the substance obtained in step (3).

[0071] As an optimization, the magnetic mesoporous silica in step (1) is prepared by mixing an aqueous solution of 2% hexadecyltrimethylammonium bromide with nano-ferrosilicate in a mass ratio of 300:1, and adding 1,3,5-trimethylbenzene in an amount of 0.2 times the mass of the aqueous solution of hexadecyltrimethylammonium bromide and ammonia in an amount of 0.3 times the mass of the aqueous solution of hexadecyltrimethylammonium bromide, and then ultrasonically dispersing the mixture to obtain a mixed aqueous solution of hexadecyltrimethylammonium bromide. The mixed aqueous solution of hexadecyltrimethylammonium bromide is mixed with ethyl orthosilicate in a mass ratio of 40:1, and after stirring for reaction, the mixture is filtered and dried to obtain a magnetic mesoporous silica blank. The magnetic mesoporous silica blank is mixed with ammonium nitrate in a mass ratio of 1:2, and anhydrous ethanol in an amount of 10 times the mass of the magnetic mesoporous silica blank is added. After condensation and reflux, the mixture is filtered and dried to obtain a magnetic mesoporous silica blank.

[0072] Effect example

[0073] Table 1 below shows the analysis results of the colorimetric properties and immune level determination of the diagnostic preparations and preparation methods for determining immune levels of Examples 1 to 4 of the present invention and the comparative example.

[0074]

[0075] From the comparison of the implementation data of Example 1 and the comparative example in Table 1, it can be found that when polyallylamine hydrochloride is added to the product and the mesoporous silica is modified, the product can have effective immunodiagnostic ability, and from the comparison of the color development time under different antibody concentration conditions, it can be found that the product prepared by the present invention has the ability to efficiently measure the immune level. From the comparison between Example 1 and Example 2 in Table 1, it can be found that when cellulose is not added to the product, the antigen adsorption of the modified mesoporous silica becomes poor, so that the gold particles inside the modified mesoporous silica are easily lost, resulting in a decrease in the color development time of the product. From the comparison between Example 1 and Example 3 in Table 1, it can be found that when polyallylamine hydrochloride is not added to the product, the adsorption of the modified mesoporous silica to the gold particles is reduced, so that the gold particles cannot be fixed in the modified mesoporous silica, making the product unable to diagnose the immune level; from the comparison between Example 1 and Example 4, it can be found that when the modified mesoporous silica added to the product does not have pH sensitivity, the modified mesoporous silica cannot fix the gold particles during the chloroauric acid reduction process, resulting in the gold particles binding to the antigen, making it impossible for the subsequent product to form a color development reaction.

Claims

1. A diagnostic preparation for measuring immune levels, which mainly comprises the following raw material components in parts by weight: 28 parts of modified mesoporous silica, 10 parts of antigen, 4 parts of trisodium citrate, 6 parts of chloroauric acid and 8 parts of polyallylamine hydrochloride; The method for preparing the diagnostic preparation for determining the immune level comprises the following preparation steps: (1) 98% concentrated sulfuric acid and 30% hydrogen peroxide were mixed in a volume ratio of 7:3 to obtain a mixed treatment liquid, and the mixed treatment liquid was mixed with magnetic mesoporous silica in a mass ratio of 12:1, and stirred for reaction at a temperature of 45°C and a rotation speed of 300 r / min for 2 hours, and then filtered to obtain a filter cake, and the filter cake was washed with water for 5 times, and then dried at a temperature of 80°C for 6 hours to obtain a modified mesoporous silica blank; The modified mesoporous silica blank and anhydrous ethanol are mixed in a beaker at a mass ratio of 1:50, and aminopropyltrimethoxysilane in an amount of 0.3 times the mass of the modified mesoporous silica blank and γ-mercaptopropyltrimethoxysilane in an amount of 0.2 times the mass of the modified mesoporous silica blank are added to the beaker, and the mixture is stirred for reaction for 8 hours at a temperature of 65°C and a rotation speed of 320 r / min, and then centrifuged to obtain a pretreated modified mesoporous silica blank. The pretreated modified mesoporous silica blank and N,N-dimethylformamide are mixed in a flask at a mass ratio of 1:10, and succinic anhydride in an amount of 2 times the mass of the pretreated modified mesoporous silica blank is added to the flask, and the mixture is stirred for reaction for 6 hours at a temperature of 60°C and a rotation speed of 300 r / min, and then filtered to obtain a filter residue, and the filter residue is dried at a temperature of 80°C for 3 hours to obtain a pretreated modified mesoporous silica. The pretreated modified mesoporous silica and tetrahydrofuran were mixed in a four-necked flask at a mass ratio of 1:30, and N-isopropylacrylamide (12 times the mass of the pretreated modified mesoporous silica), N-hydroxymethylacrylamide (1.2 times the mass of the pretreated modified mesoporous silica) and azobisisobutyronitrile (0.03 times the mass of the pretreated modified mesoporous silica) were added to the four-necked flask, nitrogen was introduced into the four-necked flask at a rate of 50 mL / min, and the mixture was stirred at 80°C and 280 r / min for 24 h, filtered, the filtrate was removed, washed with deionized water 3 times, and dried at 60°C for 2 h; (2) polyallylamine hydrochloride and water are mixed at a mass ratio of 1:10, and the substance obtained in step (1) is added to the mixture of polyallylamine hydrochloride and water in an amount of 5 times the mass of polyallylamine hydrochloride, and the mixture is stirred for 30 minutes at a temperature of 40° C. and a rotation speed of 300 r / min to obtain a mixed dispersion, the mixed dispersion is mixed with chloroauric acid at a mass ratio of 80:1, and trisodium citrate is added to the mixture of the mixed dispersion and chloroauric acid in an amount of 0.05 times the mass of the mixed dispersion, and the mixture is stirred for reaction for 3 hours at a temperature of 40° C. and a rotation speed of 280 r / min, and then filtered and dried in vacuo; (3) The antigen and water were mixed at a mass ratio of 1:10, and the pH of the mixture of the antigen and water was adjusted to 8 with a 2% sodium hydroxide solution. After ultrasonic dispersion at a frequency of 45 kHz for 10 minutes, the substance obtained in step (2) was added to the mixture of the antigen and water in an amount of 6 times the mass of the antigen. After stirring for reaction at a temperature of 30°C and a rotation speed of 300 r / min for 5 hours, the filtrate was removed by magnetic filtration to obtain a blank, and the blank was dried at a temperature of 40°C for 12 hours. (4) performing index analysis on the substance obtained in step (3); The magnetic mesoporous silica of step (1) is prepared by mixing an aqueous solution of 2% hexadecyltrimethylammonium bromide with nano-ferrosilicate in a mass ratio of 300:1, adding 1,3,5-trimethylbenzene in an amount of 0.2 times the mass of the aqueous solution of hexadecyltrimethylammonium bromide and ammonia water in an amount of 0.3 times the mass of the aqueous solution of hexadecyltrimethylammonium bromide, and dispersing by ultrasonication to obtain a mixed aqueous solution of hexadecyltrimethylammonium bromide; the mixed aqueous solution of hexadecyltrimethylammonium bromide is mixed with ethyl orthosilicate in a mass ratio of 40:1, stirring for reaction, filtering, and drying to obtain a magnetic mesoporous silica blank; the magnetic mesoporous silica blank is mixed with ammonium nitrate in a mass ratio of 1:2, and anhydrous ethanol in an amount of 10 times the mass of the magnetic mesoporous silica blank is added, and after condensation and reflux, filtering, and drying to obtain a magnetic mesoporous silica blank.

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