Composite stabilizer of colloidal gold in-vitro diagnostic reagent and preparation method thereof

Abstract

The invention relates to the technical field of biological stabilizers, in particular to a compound stabilizer of a colloidal gold in-vitro diagnostic reagent and a preparation method of the compound stabilizer, and the compound stabilizer comprises the following components: polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate, an anticorrosive biological protective agent microcapsule and water. According to the invention, trehalose and a volatile oil extract are processed to obtain an antiseptic biological protective agent microcapsule, the antiseptic biological protective agent microcapsule is compounded with polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate and other components, and through the synergistic effect of the components, more kinds of substances capable of improving the stability are provided; the method enriches the means for improving the stability, so that the in-vitro diagnostic reagent can be stored for a long time, can be widely applied to various in-vitro diagnostic reagents, has a better stabilizing effect on the reagents, and effectively prolongs the validity period of the reagents.

Description

Technical Field

[0001] The invention relates to the technical field of biological stabilizers, in particular to a composite stabilizer for a colloidal gold in vitro diagnostic reagent and a preparation method thereof. Background Art

[0002] In vitro diagnostic reagents are chemical, biological or immunological substances that can be used alone or in combination with instruments, appliances, equipment or systems to examine human samples in vitro and to detect or measure certain components in samples. In vitro diagnostic reagent systems include many components such as test reagents, reagent supplies, calibrators, quality control materials, etc. In vitro diagnostic reagents have been widely used in medical research and clinical testing, and are an indispensable and important part of routine medical treatment. In vitro diagnostic reagents with stable and reliable quality can provide accurate clinical test data and play an important role in the prevention and control of diseases and the timely diagnosis and treatment of diseases.

[0003] There are many varieties and types of in vitro diagnostic reagents. From the perspective of clinical specialty, they can be divided into hematology, biochemistry, immunology, microbiology, cell histology and molecular biology; from the perspective of properties, they can be divided into liquid, dry powder, freeze-dried culture medium, test strips, etc.; from the perspective of methodology, they can be divided into enzyme immunoassay, radioimmunoassay, immunohistochemistry, immunocytochemistry, fluorescence and chemiluminescence, etc.; from the perspective of detection performance, they can be divided into qualitative, semi-quantitative and quantitative; from the perspective of detection methods, they can be divided into chemical colorimetric reagents, immunoturbidimetric reagents, enzyme reagents, ELISA reagents, colloidal gold reagents, culture medium reagents, flow cytometry reagents, chemiluminescent immunoassay reagents and polymerase chain reaction (PCR) reagents, etc. For many varieties or types of in vitro diagnostic reagents, their production processes vary greatly, and the quality requirements for products also have different emphases; at the same time, due to the different production modes of reagents and the technical capabilities of R&D and production personnel, it is easy to cause uneven quality of reagent products from various manufacturers.

[0004] Therefore, how to improve the stability of in vitro diagnostic reagents, improve the accuracy of clinical testing, and extend the shelf life of composite stabilizers used for in vitro diagnostic reagents is an important issue. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a composite stabilizer for colloidal gold in vitro diagnostic reagents and a preparation method thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A composite stabilizer for colloidal gold in vitro diagnostic reagent, the composite stabilizer comprising polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate, antiseptic bioprotectant microcapsules and water;

[0008] The molecular weight of the polyethyleneimine is 2000, and the content is 0.1-1% W / V;

[0009] The content of bovine serum albumin is 20-35g / L;

[0010] The content of Tween 20 is 10-13 g / L;

[0011] The content of dithiothreitol is 0.02-0.05% W / V;

[0012] The content of the xanthan gum is 1-10g / L;

[0013] The content of sodium borate is 0.01-1g / L;

[0014] The content of sodium tripolyphosphate is 0.05-0.5% W / V;

[0015] The content of the antiseptic bioprotectant microcapsule is 1-3% W / V; the balance is distilled water.

[0016] As a further preferred embodiment of the present invention, the preparation method of the antiseptic bioprotectant microcapsule is as follows:

[0017] 1) placing turnip seed powder in a container, adding distilled water, potassium hydrogen phthalate-sodium hydroxide buffer with a pH value of 7.0 and vitamin C, hydrolyzing in an ice bath at 40-43° C. for 2-3 hours, and then continuing distillation for 5-8 hours to obtain a volatile oil extract;

[0018] 2) Immersing the porous nanomaterial into the volatile oil extract, stirring thoroughly, evacuating to 10-50 Pa, and maintaining under ultrasonic action for 20-30 minutes, after the treatment is completed, taking out the product to obtain the anticorrosive nanomaterial,

[0019] 3) Add 56-85 mL of trehalose solution, 20-28 mL of distilled water and 3-7 g of antiseptic nanomaterial to 30-50 mL of gum arabic solution, stir thoroughly to obtain a mixed emulsion, then pour 60-90 mL of gelatin solution into the mixed emulsion while stirring, add acid solution dropwise to adjust the pH value to 4.0-4.5, maintain for 20-30 minutes, add 100-150 mL of distilled water at a temperature of 30-32° C. to dilute, place in an ice water bath, cool to 10-12° C., add 3-5 mL of 37 wt % formaldehyde solution, add alkali solution dropwise to adjust the pH value to 8.0-8.5, discard the supernatant after centrifugation, wash repeatedly with distilled water until neutral, and obtain antiseptic bioprotectant microcapsules after drying.

[0020] As a further preferred embodiment of the present invention, the ratio of the turnip seed powder, distilled water, potassium hydrogen phthalate-sodium hydroxide buffer solution and vitamin C is (200-300) g: (100-200) mL: (400-700) mL: (0.4-0.8) g.

[0021] As a further preferred embodiment of the present invention, the solid-liquid ratio of the porous nanomaterial to the volatile oil extract is 1 g: (50-80) mL;

[0022] The power of the ultrasonic action is 150-250W.

[0023] As a further preferred embodiment of the present invention, the concentration of the trehalose solution is 3-5wt%;

[0024] The concentration of the gelatin solution is 2-3wt%;

[0025] The concentration of the gum arabic solution is 2-3 wt %.

[0026] As a further preferred embodiment of the present invention, the acid solution is a hydrochloric acid solution with a concentration of 10-13wt%;

[0027] The alkali solution is a sodium hydroxide solution with a concentration of 10-13wt%;

[0028] The centrifugal speed is 10000-15000 r / min, and the processing time is 20-30 min.

[0029] As a further preferred embodiment of the present invention, the preparation method of the porous nanomaterial is as follows:

[0030] 1) adding polyvinyl alcohol to deionized water, stirring in a water bath at 80-83° C. for 4-6 hours, cooling to room temperature, adding boric acid solution, continuing stirring for 4-6 hours, then adding polytetrafluoroethylene dispersion, continuing stirring for 12-15 hours to obtain spinning solution, and then performing electrospinning, drying the obtained product, placing it in a muffle furnace, heating it from room temperature to 280-300° C. at 3-5° C. / min, and keeping it warm for 2-3 hours, cooling it naturally to room temperature, heating it to 500-520° C. at 5-8° C. / min under argon protection, and then heating it to 900-920° C. at 3-5° C. / min and keeping it warm for 2-3 hours to obtain porous nanofibers;

[0031] 2) Sodium molybdate, thiourea and 1-butyl-3-methylimidazolium tetrafluoroborate are dissolved in deionized water, and then the porous nanofibers are added, stirred at 800-1200 r / min for 30-50 min, and then transferred to a reactor, sealed, and reacted at 200-210° C. for 24-28 h under 100-160 W ultrasound. After the reaction is completed, the product is repeatedly washed with deionized water and ethanol at a low temperature, and the porous nanomaterial is obtained after drying.

[0032] As a further preferred embodiment of the present invention, the usage ratio of the polyvinyl alcohol, deionized water, boric acid, and polytetrafluoroethylene dispersion is (2-5) g: (13-30) mL: (30-50) μL: (10-18) g;

[0033] The concentration of the boric acid solution is 2.5-2.8wt%;

[0034] The concentration of the polytetrafluoroethylene dispersion is 60-65wt%;

[0035] The conditions for the electrospinning are a flow rate of 0.2-0.3 mL / h, a voltage of 18-20 kV, and a receiving distance of 15-20 cm.

[0036] As a further preferred embodiment of the present invention, the usage ratio of sodium molybdate, thiourea, 1-butyl-3-methylimidazolium tetrafluoroborate, deionized water and porous nanofibers is (1.5-2.0) mmol: (4.5-5.0) mmol: (7.5-8.2) mmol: (50-80) mL: (3-5) g.

[0037] A method for preparing a composite stabilizer for a colloidal gold in vitro diagnostic reagent comprises the following steps:

[0038] According to the proportion, polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate and preservative bioprotectant microcapsules are added to water in sequence, and shaken and stirred evenly.

[0039] The beneficial effects of the present invention are:

[0040] In the present invention, a porous nanofiber with a porous structure is obtained by electrospinning and heat treatment, and then the porous nanofiber is used as a deposition matrix, and an ionic liquid-assisted hydrothermal method is adopted to synthesize a large number of nanosheets on the deposition matrix to obtain a porous nanomaterial. In addition, ultrasonic action is applied during the synthesis process to promote the embedding of the nanosheets into the pores of the porous nanofiber, thereby reducing the porosity of the porous nanofiber and playing a certain solid limiting role on the material. At the same time, a large number of nanosheets embedded in the pores play the role of a barrier plate, increasing the flow distance of the material, thereby delaying the outflow time of the material and playing a sustained release role. Then, the volatile oil extract of turnip seeds is infiltrated into the porous nanomaterial by vacuum impregnation to obtain an anticorrosive nanomaterial, and the anticorrosive nanomaterial can be slowly released through the sustained release effect, thereby improving the action time and efficiency of the volatile oil extract and avoiding the low utilization rate caused by the volatile oil extract being directly added to the composite stabilizer. at the same time, since the volatile oil extract has a good effect of inhibiting the growth of microorganisms and has an antiseptic effect, it can inhibit the reproduction of corrupt microorganisms in the composite stabilizer, thereby extending the shelf life of the composite stabilizer; trehalose is used as the capsule core, gelatin and gum arabic are used as wall materials, and antiseptic nanomaterials are added to the wall materials, and a composite coagulation method is adopted to obtain an antiseptic bioprotectant microcapsule; through microencapsulation treatment, the trehalose in the capsule core and the volatile oil extract loaded with the antiseptic nanomaterials in the wall material can be sustained-released, thereby greatly improving the utilization rate of trehalose and volatile oil extracts and achieving the shelf life and effect of the composite stabilizer; moreover, the antiseptic nanomaterials are introduced into the wall material, on the one hand, they can be protected by the wall material, can be isolated from the outside world, and avoid the deterioration of the volatile oil extract, and at the same time, the strength of the wall material can be increased, the protection of the capsule core can be improved, and the wall material can be prevented from rupturing and losing the sustained-release effect.

[0041] In the present invention, trehalose and volatile oil extracts are processed to obtain antiseptic bioprotectant microcapsules, which are then compounded with polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate and other components. Polyethyleneimine has polar groups and hydrophobic group structures, and can be combined with protein substances such as enzymes to provide active centers, thereby improving enzyme activity and its mechanical properties and thermal stability. Xanthan gum has a macromolecular network structure, which can produce a sieve effect, spatially limit enzyme protein molecules, reduce collisions between enzyme molecules, and improve the thermal stability of enzyme molecules to a certain extent. In addition, the hydrophobic groups of the xanthan gum macromolecules enable the xanthan gum macromolecules to adsorb the enzymes, increase the hydrophilicity of the enzyme molecule surfaces, and improve the stability of the enzymes. Through the synergistic effect of the components, more types of substances that can improve stability are provided, and the means for improving stability are enriched, so that the xanthan gum can be stored for a long time, and can be widely used in various in vitro diagnostic reagents, and the xanthan gum has a better stabilizing effect on the reagents, effectively extending the shelf life of the reagents. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] A composite stabilizer for colloidal gold in vitro diagnostic reagent, the composite stabilizer comprising polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate, antiseptic bioprotectant microcapsules and water;

[0045] The molecular weight of polyethyleneimine is 2000 and the content is 0.1% W / V;

[0046] The content of bovine serum albumin is 20g / L;

[0047] The content of Tween 20 is 10g / L;

[0048] The content of dithiothreitol is 0.02% W / V;

[0049] The content of xanthan gum is 1g / L;

[0050] The content of sodium borate is 0.01g / L;

[0051] The content of sodium tripolyphosphate is 0.05% W / V;

[0052] The content of the antiseptic bioprotectant microcapsule is 1% W / V; the balance is distilled water;

[0053] The preparation method of the composite stabilizer specifically comprises the following steps:

[0054] According to the proportion, polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate and preservative bioprotectant microcapsules are added to water in sequence, and shaken and stirred evenly.

[0055] The preparation method of the antiseptic bioprotectant microcapsule is as follows:

[0056] 1) 2 g of polyvinyl alcohol was added to 13 mL of deionized water, stirred in a water bath at 80° C. for 4 h, cooled to room temperature, and then 30 μL of a 2.5 wt% boric acid solution was added, and stirring was continued for 4 h, followed by adding 10 g of a 60 wt% polytetrafluoroethylene dispersion, and stirring was continued for 12 h to obtain a spinning solution, and then electrospinning was performed at a flow rate of 0.2 mL / h, a voltage of 18 kV, and a receiving distance of 15 cm. After drying the obtained product, it was placed in a muffle furnace, heated from room temperature to 280° C. at 3° C. / min, and kept warm for 2 h. After naturally cooling to room temperature, it was heated to 500° C. at 5° C. / min under argon protection, and then heated to 900° C. at 3° C. / min and kept warm for 2 h to obtain porous nanofibers;

[0057] 2) 1.5mmol sodium molybdate, 4.5mmol thiourea and 7.5mmol 1-butyl-3-methylimidazolium tetrafluoroborate were dissolved in 50mL deionized water, and then 3g porous nanofibers were added, stirred at 800r / min for 30min, and then transferred to a reactor, sealed, and reacted at 200°C for 24h under 100W ultrasound. After the reaction was completed, the product was repeatedly washed with deionized water and ethanol at a low temperature, and then dried to obtain a porous nanomaterial;

[0058] 3) placing 200 g of turnip seed powder in a container, adding 100 mL of distilled water, 400 mL of potassium hydrogen phthalate-sodium hydroxide buffer with a pH value of 7.0 and 0.4 g of vitamin C, hydrolyzing in an ice bath at 40° C. for 2 h, and then continuing distillation for 5 h to obtain a volatile oil extract;

[0059] 4) Immerse the porous nanomaterial into the volatile oil extract at a solid-liquid ratio of 1 g:50 mL, stir thoroughly, evacuate to 10 Pa, and maintain under 150 W ultrasound for 20 min. After the treatment is completed, take out the product to obtain the anticorrosive nanomaterial.

[0060] 5) Dissolve trehalose in water to obtain a trehalose solution with a concentration of 3wt%, dissolve gelatin and gum arabic in water respectively to prepare a gelatin solution with a concentration of 2wt% and a gum arabic solution with a concentration of 2wt%, add 56mL of trehalose solution, 20mL of distilled water and 3g of antiseptic nanomaterial to 30mL of gum arabic solution, stir well to obtain a mixed emulsion, then pour 60mL of gelatin solution into the mixed emulsion under stirring, and add dropwise a 10wt% hydrochloric acid solution to adjust the pH value to 4.0, maintain for 20min, add 100mL of distilled water with a temperature of 30°C to dilute, place in an ice water bath, cool to 10°C, add 3mL of 37wt% formaldehyde solution, and add dropwise a 10wt% sodium hydroxide solution to adjust the pH value to 8.0, centrifuge at 10000r / min for 20min, discard the supernatant, wash repeatedly with distilled water until neutral, and dry to obtain antiseptic bioprotectant microcapsules.

[0061] Example 2

[0062] A composite stabilizer for colloidal gold in vitro diagnostic reagent, the composite stabilizer comprising polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate, antiseptic bioprotectant microcapsules and water;

[0063] The molecular weight of polyethyleneimine is 2000 and the content is 0.5% W / V;

[0064] The content of bovine serum albumin is 26g / L;

[0065] The content of Tween 20 is 12g / L;

[0066] The content of dithiothreitol is 0.03% W / V;

[0067] The content of xanthan gum is 6g / L;

[0068] The content of sodium borate is 0.5g / L;

[0069] The content of sodium tripolyphosphate is 0.3% W / V;

[0070] The content of the antiseptic bioprotectant microcapsule is 2% W / V; the balance is distilled water;

[0071] The preparation method of the composite stabilizer specifically comprises the following steps:

[0072] According to the proportion, polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate and preservative bioprotectant microcapsules are added to water in sequence, and shaken and stirred evenly.

[0073] The preparation method of the antiseptic bioprotectant microcapsule is as follows:

[0074] 1) 3 g of polyvinyl alcohol was added to 21 mL of deionized water, stirred in a water bath at 82° C. for 5 h, cooled to room temperature, and then 40 μL of a 2.7 wt% boric acid solution was added, and stirring was continued for 5 h, followed by adding 15 g of a 63 wt% polytetrafluoroethylene dispersion, and stirring was continued for 13 h to obtain a spinning solution, and then electrospinning was performed at a flow rate of 0.25 mL / h, a voltage of 19 kV, and a receiving distance of 18 cm. After drying the obtained product, it was placed in a muffle furnace, heated from room temperature to 290° C. at 4° C. / min, and kept warm for 2.5 h. After naturally cooling to room temperature, it was heated to 510° C. at 7° C. / min under argon protection, and then heated to 940° C. at 4° C. / min and kept warm for 2.5 h to obtain porous nanofibers;

[0075] 2) 1.7 mmol sodium molybdate, 4.6 mmol thiourea and 7.8 mmol 1-butyl-3-methylimidazolium tetrafluoroborate were dissolved in 70 mL deionized water, and then 4 g of porous nanofibers were added, stirred at 1000 r / min for 40 min, and then transferred to a reactor, sealed, and reacted at 205° C. for 26 h under 150 W ultrasound. After the reaction was completed, the product was repeatedly washed with deionized water and ethanol at a low temperature, and then dried to obtain a porous nanomaterial;

[0076] 3) placing 260 g of turnip seed powder in a container, adding 170 mL of distilled water, 600 mL of potassium hydrogen phthalate-sodium hydroxide buffer with a pH value of 7.0 and 0.7 g of vitamin C, hydrolyzing in an ice bath at 42° C. for 2.5 h, and then continuing distillation for 7 h to obtain a volatile oil extract;

[0077] 4) Immerse the porous nanomaterial into the volatile oil extract at a solid-liquid ratio of 1 g: 70 mL, stir thoroughly, evacuate to 30 Pa, and maintain under 200 W ultrasound for 25 min. After the treatment is completed, take out the product to obtain the anticorrosive nanomaterial.

[0078] 5) dissolving trehalose in water to obtain a trehalose solution with a concentration of 4 wt %, dissolving gelatin and gum arabic in water to prepare a gelatin solution with a concentration of 2.5 wt % and a gum arabic solution with a concentration of 2.5 wt %, respectively, adding 72 mL of the trehalose solution, 24 mL of distilled water and 5 g of the antiseptic nanomaterial to 40 mL of the gum arabic solution, stirring thoroughly to obtain a mixed emulsion, then pouring 80 mL of the gelatin solution into the mixed emulsion while stirring, and dropping a 2.5 wt % gelatin solution. The pH value was adjusted to 4.5 with a 12wt% hydrochloric acid solution, maintained for 25 minutes, and then 130mL of distilled water at a temperature of 31°C was added for dilution. The mixture was placed in an ice water bath, cooled to 11°C, and then 4mL of a 37wt% formaldehyde solution was added. A 12wt% sodium hydroxide solution was added dropwise to adjust the pH value to 8.5. The mixture was centrifuged at 12000r / min for 25 minutes, and the supernatant was discarded. The mixture was repeatedly washed with distilled water until neutral, and dried to obtain antiseptic bioprotectant microcapsules.

[0079] Example 3

[0080] A composite stabilizer for colloidal gold in vitro diagnostic reagent, the composite stabilizer comprising polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate, antiseptic bioprotectant microcapsules and water;

[0081] The molecular weight of polyethyleneimine is 2000 and the content is 1% W / V;

[0082] The content of bovine serum albumin is 35g / L;

[0083] The content of Tween 20 is 13g / L;

[0084] The content of dithiothreitol is 0.05% W / V;

[0085] The content of xanthan gum is 10g / L;

[0086] The content of sodium borate is 1g / L;

[0087] The content of sodium tripolyphosphate is 0.5% W / V;

[0088] The content of the antiseptic bioprotectant microcapsule is 3% W / V; the balance is distilled water;

[0089] The preparation method of the composite stabilizer specifically comprises the following steps:

[0090] According to the proportion, polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate and preservative bioprotectant microcapsules are added to water in sequence, and shaken and stirred evenly.

[0091] The preparation method of the antiseptic bioprotectant microcapsule is as follows:

[0092] 1) 5 g of polyvinyl alcohol was added to 30 mL of deionized water, stirred in a water bath at 83° C. for 6 h, cooled to room temperature, and then 50 μL of a 2.8 wt % boric acid solution was added, and stirring was continued for 6 h, followed by adding 18 g of a 65 wt % polytetrafluoroethylene dispersion, and stirring was continued for 15 h to obtain a spinning solution, and then electrospinning was performed at a flow rate of 0.3 mL / h, a voltage of 20 kV, and a receiving distance of 20 cm. After drying the obtained product, it was placed in a muffle furnace, heated from room temperature to 300° C. at 5° C. / min, and kept warm for 3 h. After naturally cooling to room temperature, it was heated to 520° C. at 8° C. / min under argon protection, and then heated to 920° C. at 5° C. / min and kept warm for 3 h to obtain porous nanofibers;

[0093] 2) 2.0mmol sodium molybdate, 5.0mmol thiourea and 8.2mmol 1-butyl-3-methylimidazolium tetrafluoroborate were dissolved in 80mL deionized water, and then 5g porous nanofibers were added, stirred at 1200r / min for 50min, and then transferred to a reactor, sealed, and reacted at 210°C for 28h under 160W ultrasound. After the reaction was completed, the product was repeatedly washed with deionized water and ethanol at a low temperature, and then dried to obtain a porous nanomaterial;

[0094] 3) placing 300 g of turnip seed powder in a container, adding 200 mL of distilled water, 700 mL of potassium hydrogen phthalate-sodium hydroxide buffer with a pH value of 7.0 and 0.8 g of vitamin C, hydrolyzing in an ice bath at 43° C. for 3 h, and then continuing distillation for 8 h to obtain a volatile oil extract;

[0095] 4) Immerse the porous nanomaterial into the volatile oil extract at a solid-liquid ratio of 1 g: 80 mL, stir thoroughly, evacuate to 50 Pa, and maintain under 250 W ultrasound for 30 min. After the treatment is completed, take out the product to obtain the anticorrosive nanomaterial.

[0096] 5) Dissolve trehalose in water to obtain a 5wt% trehalose solution, dissolve gelatin and gum arabic in water to prepare a 3wt% gelatin solution and a 3wt% gum arabic solution, respectively, add 85mL of trehalose solution, 28mL of distilled water and 7g of antiseptic nanomaterial to 50mL of gum arabic solution, stir thoroughly to obtain a mixed emulsion, then pour 90mL of gelatin solution into the mixed emulsion under stirring, and add dropwise a 13wt% hydrochloric acid solution to adjust the pH value to 4.5, maintain for 30min, add 150mL of 32°C distilled water to dilute, place in an ice water bath, cool to 12°C, add 5mL of 37wt% formaldehyde solution, and add dropwise a 13wt% sodium hydroxide solution to adjust the pH value to 8.5, centrifuge at 15000r / min for 30min, discard the supernatant, wash repeatedly with distilled water until neutral, and dry to obtain antiseptic bioprotectant microcapsules.

[0097] Comparative Example 1: This comparative example is basically the same as Example 1, except that the trehalose and volatile oil extract are not processed.

[0098] Comparative Example 2: This comparative example is basically the same as Example 1, except that step 2) is omitted in the preparation of the antiseptic bioprotectant microcapsules.

[0099] Comparative Example 3: This comparative example is basically the same as Example 1, except that in the preparation of the antiseptic bioprotectant microcapsules, steps 1)-2) are omitted.

[0100] Comparative Example 4: This comparative example is basically the same as Example 1, except that step 4) is omitted in the preparation of the antiseptic bioprotectant microcapsules.

[0101] Comparative Example 5: This comparative example is basically the same as Example 1, except that in the preparation of the antiseptic bioprotectant microcapsules, steps 4)-5) are omitted.

[0102] Test experiment:

[0103] Taking immunoglobulin A calibrators and quality control products as examples, the composite stabilizers of Example 1 and Comparative Examples 1-5 were added to the existing immunoglobulin A calibrators and quality control products of Hunan Jefit Biotechnology Co., Ltd., and the added volume ratio was 0.05 ml / L, which were designated as calibrators 1-6 and quality control products 1-6, which were experimental groups 1-6; the calibrators and quality control products without stabilizers were used as the control group, designated as calibrators 7 and quality control products 7; the above-mentioned quality control products were at 2 levels, and the above-mentioned experimental groups 1-6 and the control group were placed at 2-8°C at the same time, and the stability after 0 month and 18 months was measured respectively. The relative deviation from the target value was calculated based on the measurement results, and the results are shown in Tables 1 to 6.

[0104] Among them, level 1 is 0.88g / L; level 2 is 2.04g / L.

[0105] Table 1

[0106]

[0107] Table 2

[0108]

[0109] Table 3

[0110]

[0111] Table 4

[0112]

[0113] Table 5

[0114]

[0115] Table 6

[0116]

[0117] It can be seen from Tables 1 to 6 that after the experimental group 1 was stored at 2-8°C for 18 months, the test results remained basically stable. After the experimental groups 2-6 and the control group were stored at 2-8°C for 18 months, the test results showed a downward trend, and the absolute value of the relative deviation from the target value was quite different. It can be seen that the composite stabilizer in the present invention helps to maintain the long-term stability of the in vitro diagnostic reagent.

[0118] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A composite stabilizer for colloidal gold in vitro diagnostic reagent, characterized in that: The composite stabilizer ingredients include polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate, preservative bioprotectant microcapsules and water; The molecular weight of the polyethyleneimine is 2000, and the content is 0.1-1% W / V; The content of bovine serum albumin is 20-35g / L; The content of Tween 20 is 10-13 g / L; The content of dithiothreitol is 0.02-0.05% W / V; The content of the xanthan gum is 1-10g / L; The content of sodium borate is 0.01-1g / L; The content of sodium tripolyphosphate is 0.05-0.5% W / V; The content of the antiseptic bioprotectant microcapsule is 1-3% W / V; the balance is distilled water.

2. The composite stabilizer for colloidal gold in vitro diagnostic reagent according to claim 1, characterized in that: The preparation method of the antiseptic bioprotectant microcapsule is as follows: 1) placing turnip seed powder in a container, adding distilled water, potassium hydrogen phthalate-sodium hydroxide buffer with a pH value of 7.0 and vitamin C, hydrolyzing in an ice bath at 40-43° C. for 2-3 hours, and then continuing distillation for 5-8 hours to obtain a volatile oil extract; 2) Immersing the porous nanomaterial into the volatile oil extract, stirring thoroughly, evacuating to 10-50 Pa, and maintaining under ultrasonic action for 20-30 minutes, after the treatment is completed, taking out the product to obtain the anticorrosive nanomaterial, 3) Add 56-85 mL of trehalose solution, 20-28 mL of distilled water and 3-7 g of antiseptic nanomaterial to 30-50 mL of gum arabic solution, stir thoroughly to obtain a mixed emulsion, then pour 60-90 mL of gelatin solution into the mixed emulsion while stirring, add acid solution dropwise to adjust the pH value to 4.0-4.5, maintain for 20-30 minutes, add 100-150 mL of distilled water at a temperature of 30-32° C. to dilute, place in an ice water bath, cool to 10-12° C., add 3-5 mL of 37 wt % formaldehyde solution, add alkali solution dropwise to adjust the pH value to 8.0-8.5, discard the supernatant after centrifugation, wash repeatedly with distilled water until neutral, and obtain antiseptic bioprotectant microcapsules after drying.

3. The composite stabilizer for colloidal gold in vitro diagnostic reagent according to claim 2, characterized in that: The dosage ratio of the turnip seed powder, distilled water, potassium hydrogen phthalate-sodium hydroxide buffer solution and vitamin C is (200-300) g: (100-200) mL: (400-700) mL: (0.4-0.8) g.

4. The composite stabilizer for colloidal gold in vitro diagnostic reagent according to claim 2, characterized in that: The solid-liquid ratio of the porous nanomaterial to the volatile oil extract is 1 g: (50-80) mL; The power of the ultrasonic action is 150-250W.

5. The composite stabilizer for colloidal gold in vitro diagnostic reagent according to claim 2, characterized in that: The concentration of the trehalose solution is 3-5wt%; The concentration of the gelatin solution is 2-3wt%; The concentration of the gum arabic solution is 2-3 wt %.

6. The composite stabilizer for colloidal gold in vitro diagnostic reagent according to claim 2, characterized in that: The acid solution is a hydrochloric acid solution with a concentration of 10-13wt%; The alkali solution is a sodium hydroxide solution with a concentration of 10-13wt%; The centrifugal speed is 10000-15000 r / min, and the processing time is 20-30 min.

7. The composite stabilizer for colloidal gold in vitro diagnostic reagent according to claim 2, characterized in that: The preparation method of the porous nanomaterial is as follows: 1) adding polyvinyl alcohol to deionized water, stirring in a water bath at 80-83° C. for 4-6 hours, cooling to room temperature, adding boric acid solution, continuing stirring for 4-6 hours, then adding polytetrafluoroethylene dispersion, continuing stirring for 12-15 hours to obtain spinning solution, and then performing electrospinning, drying the obtained product, placing it in a muffle furnace, heating it from room temperature to 280-300° C. at 3-5° C. / min, and keeping it warm for 2-3 hours, cooling it naturally to room temperature, heating it to 500-520° C. at 5-8° C. / min under argon protection, and then heating it to 900-920° C. at 3-5° C. / min and keeping it warm for 2-3 hours to obtain porous nanofibers; 2) Sodium molybdate, thiourea and 1-butyl-3-methylimidazolium tetrafluoroborate are dissolved in deionized water, and then the porous nanofibers are added, stirred at 800-1200 r / min for 30-50 min, and then transferred to a reactor, sealed, and reacted at 200-210° C. for 24-28 h under 100-160 W ultrasound. After the reaction is completed, the product is repeatedly washed with deionized water and ethanol at a low temperature, and the porous nanomaterial is obtained after drying.

8. The composite stabilizer for colloidal gold in vitro diagnostic reagent according to claim 7, characterized in that: The usage ratio of the polyvinyl alcohol, deionized water, boric acid and polytetrafluoroethylene dispersion is (2-5) g: (13-30) mL: (30-50) μL: (10-18) g; The concentration of the boric acid solution is 2.5-2.8wt%; The concentration of the polytetrafluoroethylene dispersion is 60-65wt%; The conditions for the electrospinning are a flow rate of 0.2-0.3 mL / h, a voltage of 18-20 kV, and a receiving distance of 15-20 cm.

9. The composite stabilizer for colloidal gold in vitro diagnostic reagent according to claim 7, characterized in that: The dosage ratio of the sodium molybdate, thiourea, 1-butyl-3-methylimidazolium tetrafluoroborate, deionized water and porous nanofibers is (1.5-2.0) mmol: (4.5-5.0) mmol: (7.5-8.2) mmol: (50-80) mL: (3-5) g.

10. The method for preparing a composite stabilizer for colloidal gold in vitro diagnostic reagent according to any one of claims 1 to 9, characterized in that: The specific steps include: According to the proportion, polyethyleneimine, bovine serum albumin, Tween 20, dithiothreitol, xanthan gum, sodium borate, sodium tripolyphosphate and preservative bioprotectant microcapsules are added to water in sequence, and shaken and stirred evenly.

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