CN114527107A - Candida albicans detection powder and preparation method thereof - Google Patents
Candida albicans detection powder and preparation method thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 239000000843 powder Substances 0.000 title claims abstract description 29
- 241000222122 Candida albicans Species 0.000 title claims abstract description 18
- 229940095731 candida albicans Drugs 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title abstract description 16
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims abstract description 56
- 238000004108 freeze drying Methods 0.000 claims abstract description 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 12
- 239000002994 raw material Substances 0.000 claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 8
- 239000003381 stabilizer Substances 0.000 claims abstract description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 33
- 238000007710 freezing Methods 0.000 claims description 28
- 230000008014 freezing Effects 0.000 claims description 22
- 229920000858 Cyclodextrin Polymers 0.000 claims description 13
- 239000001116 FEMA 4028 Substances 0.000 claims description 13
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 13
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 13
- 229960004853 betadex Drugs 0.000 claims description 13
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 11
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 238000000859 sublimation Methods 0.000 claims description 7
- 230000008022 sublimation Effects 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000003223 protective agent Substances 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- BTANRVKWQNVYAZ-UHFFFAOYSA-N butan-2-ol Chemical compound CCC(C)O BTANRVKWQNVYAZ-UHFFFAOYSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 108010090804 Streptavidin Proteins 0.000 claims description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 3
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- 229920002774 Maltodextrin Polymers 0.000 claims description 2
- 239000005913 Maltodextrin Substances 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 239000011668 ascorbic acid Substances 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 235000010323 ascorbic acid Nutrition 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
- 229960001375 lactose Drugs 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 229940035034 maltodextrin Drugs 0.000 claims description 2
- 235000002639 sodium chloride Nutrition 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 229960004793 sucrose Drugs 0.000 claims description 2
- 229940113088 dimethylacetamide Drugs 0.000 claims 3
- 230000001476 alcoholic effect Effects 0.000 claims 2
- 238000001816 cooling Methods 0.000 claims 2
- -1 fluorescein compound Chemical class 0.000 claims 2
- 238000010438 heat treatment Methods 0.000 claims 2
- 230000000149 penetrating effect Effects 0.000 claims 2
- 239000000853 adhesive Substances 0.000 claims 1
- 230000001070 adhesive effect Effects 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 238000003860 storage Methods 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 239000012535 impurity Substances 0.000 abstract description 2
- 230000007774 longterm Effects 0.000 abstract description 2
- 239000011814 protection agent Substances 0.000 abstract 1
- 239000000203 mixture Substances 0.000 description 7
- 241000233866 Fungi Species 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 102000012286 Chitinases Human genes 0.000 description 2
- 108010022172 Chitinases Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000011056 performance test Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000002407 reforming Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
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Abstract
本发明涉及检测制剂技术领域，尤其涉及一种白色念珠菌检测粉剂及其制备方法。所述检测粉剂由以下原料组成：粘附肽、链霉亲和素荧光素复合物、冻干保护剂、C2～C4的醇溶液、渗透剂、稳定剂、抗猝灭剂，所述C2～C4的醇溶液浓度为95％～98％。本发明通过使用叔丁醇溶液为整体的溶液，叔丁醇性质较为稳定，避免在冻干过程中产生杂质，从而影响荧光试剂的使用效果；其次，产品中含水量较低，有利于长期存放保存。The invention relates to the technical field of detection preparations, in particular to a Candida albicans detection powder and a preparation method thereof. The detection powder is composed of the following raw materials: adhesion peptide, streptavidin-fluorescein complex, freeze-drying protection agent, alcohol solution of C2～C4, penetrant, stabilizer, anti-quencher, the C2～C4 The concentration of the alcohol solution of C4 is 95% to 98%. By using the tert-butanol solution as a whole solution in the present invention, the properties of tert-butanol are relatively stable, and impurities are avoided in the freeze-drying process, thereby affecting the use effect of the fluorescent reagent; secondly, the water content in the product is low, which is conducive to long-term storage save.
Description
技术领域technical field
本发明涉及检测制剂技术领域，尤其涉及一种白色念珠菌检测粉剂及其制备方法。The invention relates to the technical field of detection preparations, in particular to a Candida albicans detection powder and a preparation method thereof.
背景技术Background technique
荧光检测法是一种已有较多应用的快速检测真菌方法。市场上流通的荧光检测真菌试剂盒，主要成分为荧光试剂、促溶剂和背景染料。目前常用的荧光试剂为CalfluorWhite(CFW),能与真菌细胞壁内的几丁质成分结合，在紫外的激发下发生蓝色荧光。但CFW特异性结合能力较差，可以与大部分真菌结合，需要另外人工判断菌属，同时淬灭时间短，能使背景组织发出明亮的荧光，影响真菌与背景的判断。所以CFW一般需要联合背景染料以去除背景干扰，利用促溶剂分解待测样品角质层。后来市场上出现利用重组几丁质酶标联的荧光素试剂盒检测真菌，此类方法提高了特异性和稳定性，但是生物酶较容易失活不易保存，而且几丁质酶检测法是广谱真菌检测方法，不能特异性识别白念珠菌。Fluorescence detection is a rapid detection method for fungi that has been widely used. The fluorescent detection fungus kits circulating in the market are mainly composed of fluorescent reagents, solubilizers and background dyes. The commonly used fluorescent reagent is CalfluorWhite (CFW), which can combine with the chitin component in the fungal cell wall and generate blue fluorescence under the excitation of ultraviolet light. However, CFW has poor specific binding ability and can bind to most fungi. It needs to manually determine the genus of bacteria. At the same time, the quenching time is short, which can make the background tissue emit bright fluorescence, which affects the judgment of fungi and background. Therefore, CFW generally needs to be combined with background dyes to remove background interference, and use solvotropes to decompose the stratum corneum of the sample to be tested. Later, the use of recombinant chitinase-labeled fluorescein kits to detect fungi appeared on the market. This method improves specificity and stability, but biological enzymes are easier to inactivate and difficult to preserve, and chitinase detection methods are widely used. Spectral fungal detection method cannot specifically identify Candida albicans.
其次，现有的的荧光检测试剂均为溶液形式，使用和存储过程容易发生降解，同时溶液形式的检测试剂也不适应长途运输，溶液剂比粉剂重量大，从而增了运输成本及风险。因此，将溶液剂制备成冻干粉剂可以有效解决上述问题，但是，现有的荧光检测液的冻干粉剂的制备存在几个方面的不足：冻干工艺不够成熟，在实际大规模冻干过程中，冻干粉剂合格率不高，对生产成本产生重要影响；2.冻干粉剂中水分难以去除，结构容易塌陷萎缩；3.冻干周期长，使得实际冻干产能较低，能耗过大。Secondly, the existing fluorescent detection reagents are all in the form of solutions, which are prone to degradation during use and storage, and are not suitable for long-distance transportation. Therefore, the above-mentioned problems can be effectively solved by preparing the solution into a freeze-dried powder, but the preparation of the freeze-dried powder of the existing fluorescent detection solution has several deficiencies: the freeze-drying process is not mature enough, and in the actual large-scale freeze-drying process Among them, the qualified rate of freeze-dried powder is not high, which has an important impact on the production cost; 2. The moisture in the freeze-dried powder is difficult to remove, and the structure is easy to collapse and shrink; big.
基于上述情况，本发明提出了一种白色念珠菌检测粉剂及其制备方法，可有效解决以上问题。Based on the above situation, the present invention proposes a Candida albicans detection powder and a preparation method thereof, which can effectively solve the above problems.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种白色念珠菌检测粉剂及其制备方法。The object of the present invention is to provide a Candida albicans detection powder and a preparation method thereof.
为实现上述目的，本发明提供了一种白色念珠菌检测粉剂，所述检测粉剂由以下原料组成：粘附肽、链霉亲和素荧光素复合物、冻干保护剂、C2～C4的醇溶液、渗透剂、稳定剂、抗猝灭剂，所述C2～C4的醇溶液浓度为95％～98％。In order to achieve the above purpose, the present invention provides a Candida albicans detection powder, the detection powder is composed of the following raw materials: adhesion peptide, streptavidin-fluorescein complex, freeze-drying protective agent, C2-C4 alcohol Solution, penetrant, stabilizer, anti-quenching agent, the concentration of the alcohol solution of C2-C4 is 95%-98%.
优选地，所述冻干保护剂包括蔗糖、麦芽糊精、乳糖、β-环糊精、食盐、淀粉的其中一种或两种以上的组合。Preferably, the lyoprotectant includes one or a combination of two or more of sucrose, maltodextrin, lactose, β-cyclodextrin, table salt, and starch.
优选地，所述冻干保护剂为β-环糊精。Preferably, the lyoprotectant is β-cyclodextrin.
优选地，所述C2或C4的醇溶液包括乙醇溶液、正丁醇溶液，异丁醇溶液，仲丁醇溶液，叔丁醇溶液的其中一种。Preferably, the alcohol solution of C2 or C4 includes one of ethanol solution, n-butanol solution, isobutanol solution, sec-butanol solution and tert-butanol solution.
优选地，所述荧光素为异硫氰酸荧光素、四甲基罗丹明异硫氰酸中的其中一种。Preferably, the fluorescein is one of fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.
优选地，所述渗透剂为二甲基亚砜、二甲基乙酰胺中的其中一种。Preferably, the penetrant is one of dimethyl sulfoxide and dimethylacetamide.
优选地，所述稳定剂为磷酸二氢钾、磷酸氢二钠中的至少一种。Preferably, the stabilizer is at least one of potassium dihydrogen phosphate and disodium hydrogen phosphate.
优选地，所述抗淬灭剂为丙三醇、抗坏血酸中的至少一种。Preferably, the anti-quenching agent is at least one of glycerol and ascorbic acid.
优选地，所述检测粉剂由以下重量份的原料组成：粘附肽5～15份、链霉亲和素异硫氰酸荧光素复合物80～90份、β-环糊精200～400份、98％叔丁醇溶液1000～1400份、二甲基乙酰胺200～300份、磷酸二氢钾120～160份、丙三醇50～150份。Preferably, the detection powder is composed of the following raw materials by weight: 5-15 parts of adhesion peptide, 80-90 parts of streptavidin fluorescein isothiocyanate complex, 200-400 parts of β-cyclodextrin , 1000-1400 parts of 98% tert-butanol solution, 200-300 parts of dimethylacetamide, 120-160 parts of potassium dihydrogen phosphate, and 50-150 parts of glycerol.
本发明还提供一种白色念珠菌检测粉剂的制备方法，所述制备方法包括以下步骤：The present invention also provides a preparation method of Candida albicans detection powder, the preparation method comprises the following steps:
(1)将粘附肽、98％叔丁醇溶液与链霉亲和素荧光素复合物混合，在室温下孵育30～40min，得到复合物的叔丁醇溶液；(1) Mix the adhesion peptide, 98% tert-butanol solution and streptavidin-fluorescein complex, and incubate at room temperature for 30-40 min to obtain a tert-butanol solution of the complex;
(2)将得到复合物的叔丁醇溶液与β-环糊精、二甲基乙酰胺、磷酸二氢钾、丙三醇混合均匀后，静置1～2h，去除不溶物，得到预冻液；(2) Mix the tert-butanol solution of the compound with β-cyclodextrin, dimethylacetamide, potassium dihydrogen phosphate, and glycerol, and let stand for 1-2 hours to remove insoluble matter to obtain pre-freezing liquid;
(3)预冻：将预冻液置于冻干柜中，在半小时内降温至5℃～-10℃，并在该温度区间维持30～40min；(3) Pre-freezing: place the pre-freezing solution in a freeze-drying cabinet, cool down to 5°C to -10°C within half an hour, and maintain this temperature range for 30 to 40 minutes;
(4)冻结及降压：在半小时内降温至-15℃～-25℃，并在该温度区间维持30～40min，随后调整真空度为5～10Pa，维持2～2.5h；(4) Freezing and depressurization: drop the temperature to -15℃～-25℃ within half an hour, and maintain this temperature range for 30～40min, then adjust the vacuum degree to 5～10Pa and maintain it for 2～2.5h;
(5)升华：升温至-5℃～-10℃，并在该温度区间维持2.5～3h；随后调整真空度为25～30Pa，维持2.5～3h；(5) Sublimation: raise the temperature to -5°C to -10°C, and maintain this temperature range for 2.5 to 3 hours; then adjust the vacuum to 25 to 30 Pa and maintain it for 2.5 to 3 hours;
(6)解析干燥：升温至20～25℃，调整真空度至15～20Pa，维持2～2.5h；负压压塞后，取出物料2～8℃低温保存。(6) Analytical drying: raise the temperature to 20-25°C, adjust the vacuum degree to 15-20Pa, and maintain for 2-2.5h; after the negative pressure is plugged, take out the material and store it at a low temperature of 2-8°C.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1.本发明通过使用叔丁醇溶液为整体的溶液，叔丁醇性质较为稳定，避免在冻干过程中产生杂质，从而影响荧光试剂的使用效果；其次，产品中含水量较低，有利于长期存放保存。1. The present invention uses the tert-butanol solution as a whole solution, the properties of the tert-butanol are relatively stable, and the generation of impurities in the freeze-drying process is avoided, thereby affecting the use effect of the fluorescent reagent; secondly, the water content in the product is low, which is conducive to Long-term storage preservation.
2.本发明通过使用β-环糊精作为冻干保护剂，相较于使用蔗糖作为保护剂，能大大加快产品的重新形成溶液剂的速度，且冻干效果好，成型率高。2. By using β-cyclodextrin as a freeze-drying protective agent in the present invention, compared with using sucrose as a protective agent, the speed of reforming the product into a solution can be greatly accelerated, and the freeze-drying effect is good and the molding rate is high.
3.本发明原材料在国内充足，价格适宜，使其规模化生产没有太高的成本限制；同时，制备方法简单，总体生产成本不高，有利于工业的大规模生产。3. The raw materials of the present invention are abundant in China and the price is suitable, so that the large-scale production does not have too high cost restrictions; at the same time, the preparation method is simple, the overall production cost is not high, and it is beneficial to industrial large-scale production.
具体实施方式Detailed ways
实施例1Example 1
按表1称量具体原料，其余步骤制备步骤如下：The specific raw materials are weighed according to Table 1, and the preparation steps of the remaining steps are as follows:
(1)将粘附肽、98％叔丁醇溶液与链霉亲和素荧光素复合物混合，在室温下孵育30min，得到复合物的叔丁醇溶液；(1) Mix the adhesion peptide, 98% tert-butanol solution and streptavidin-fluorescein complex, and incubate at room temperature for 30 min to obtain a tert-butanol solution of the complex;
(2)将得到复合物的叔丁醇溶液与β-环糊精、二甲基乙酰胺、磷酸二氢钾、丙三醇混合均匀后，静置1h，去除不溶物，得到预冻液；(2) after uniformly mixing the tert-butanol solution of the compound with β-cyclodextrin, dimethylacetamide, potassium dihydrogen phosphate and glycerol, let stand for 1 hour to remove insoluble matter to obtain a pre-freezing solution;
(3)预冻：将预冻液置于冻干柜中，在半小时内降温至5℃，并在该温度区间维持40min；(3) Pre-freezing: place the pre-freezing solution in a freeze-drying cabinet, cool down to 5°C within half an hour, and maintain this temperature range for 40 minutes;
(4)冻结及降压：在半小时内降温至-15℃，并在该温度区间维持40min，随后调整真空度为5Pa，维持2.5h；(4) Freezing and depressurization: cool down to -15°C within half an hour, and maintain this temperature range for 40min, then adjust the vacuum to 5Pa and maintain it for 2.5h;
(5)升华：升温至-5℃，并在该温度区间维持2.5h；随后调整真空度为25Pa，维持3h；(5) Sublimation: raise the temperature to -5°C and maintain this temperature range for 2.5h; then adjust the vacuum to 25Pa and maintain it for 3h;
(6)解析干燥：升温至20℃，调整真空度至15Pa，维持2.5h；负压压塞后，取出物料2～8℃低温保存。(6) Analytical drying: raise the temperature to 20°C, adjust the vacuum degree to 15Pa, and maintain for 2.5h; after the negative pressure is plugged, take out the material and store it at a low temperature of 2-8°C.
实施例2Example 2
按表1称量具体原料，其余步骤制备步骤如下：The specific raw materials are weighed according to Table 1, and the preparation steps of the remaining steps are as follows:
(1)将粘附肽、98％叔丁醇溶液与链霉亲和素荧光素复合物混合，在室温下孵育40min，得到复合物的叔丁醇溶液；(1) Mix the adhesion peptide, 98% tert-butanol solution and streptavidin-fluorescein complex, and incubate for 40 min at room temperature to obtain a tert-butanol solution of the complex;
(2)将得到复合物的叔丁醇溶液与β-环糊精、二甲基乙酰胺、磷酸二氢钾、丙三醇混合均匀后，静置2h，去除不溶物，得到预冻液；(2) after the tert-butanol solution of the obtained complex is mixed with β-cyclodextrin, dimethylacetamide, potassium dihydrogen phosphate, and glycerol, it is allowed to stand for 2 hours to remove insoluble matter to obtain a pre-freezing solution;
(3)预冻：将预冻液置于冻干柜中，在半小时内降温至-10℃，并在该温度区间维持30min；(3) Pre-freezing: place the pre-freezing solution in a freeze-drying cabinet, cool down to -10°C within half an hour, and maintain this temperature range for 30 minutes;
(4)冻结及降压：在半小时内降温至-25℃，并在该温度区间维持30min，随后调整真空度为10Pa，维持2h；(4) Freezing and depressurization: cool down to -25°C within half an hour, and maintain this temperature range for 30min, then adjust the vacuum to 10Pa and maintain it for 2h;
(5)升华：升温至-10℃，并在该温度区间维持3h；随后调整真空度为30Pa，维持2.5h；(5) Sublimation: raise the temperature to -10°C and maintain in this temperature range for 3h; then adjust the vacuum to 30Pa and maintain it for 2.5h;
(6)解析干燥：升温至25℃，调整真空度至20Pa，维持2h；负压压塞后，取出物料2～8℃低温保存。(6) Analytical drying: raise the temperature to 25°C, adjust the vacuum degree to 20Pa, and maintain for 2h; after the negative pressure is plugged, take out the material and store it at a low temperature of 2-8°C.
实施例3Example 3
按表1称量具体原料，其余步骤制备步骤如下：The specific raw materials are weighed according to Table 1, and the preparation steps of the remaining steps are as follows:
(1)将粘附肽、98％叔丁醇溶液与链霉亲和素荧光素复合物混合，在室温下孵育40min，得到复合物的叔丁醇溶液；(1) Mix the adhesion peptide, 98% tert-butanol solution and streptavidin-fluorescein complex, and incubate for 40 min at room temperature to obtain a tert-butanol solution of the complex;
(2)将得到复合物的叔丁醇溶液与β-环糊精、二甲基乙酰胺、磷酸二氢钾、丙三醇混合均匀后，静置2h，去除不溶物，得到预冻液；(2) after the tert-butanol solution of the obtained complex is mixed with β-cyclodextrin, dimethylacetamide, potassium dihydrogen phosphate, and glycerol, it is allowed to stand for 2 hours to remove insoluble matter to obtain a pre-freezing solution;
(3)预冻：将预冻液置于冻干柜中，在半小时内降温至-10℃，并在该温度区间维持40min；(3) Pre-freezing: place the pre-freezing solution in a freeze-drying cabinet, cool down to -10°C within half an hour, and maintain this temperature range for 40 minutes;
(4)冻结及降压：在半小时内降温至-25℃，并在该温度区间维持40min，随后调整真空度为10Pa，维持2.5h；(4) Freezing and depressurization: reduce the temperature to -25°C within half an hour, and maintain this temperature range for 40min, then adjust the vacuum to 10Pa and maintain it for 2.5h;
(5)升华：升温至-10℃，并在该温度区间维持3h；随后调整真空度为30Pa，维持3h；(5) Sublimation: raise the temperature to -10°C, and maintain this temperature range for 3h; then adjust the vacuum to 30Pa and maintain it for 3h;
(6)解析干燥：升温至25℃，调整真空度至20Pa，维持2.5h；负压压塞后，取出物料2～8℃低温保存。(6) Analytical drying: raise the temperature to 25°C, adjust the vacuum degree to 20Pa, and maintain for 2.5h; after the negative pressure is plugged, take out the material and store at 2-8°C at low temperature.
对比例1Comparative Example 1
按表1称量具体原料，其余步骤制备步骤如下：The specific raw materials are weighed according to Table 1, and the preparation steps of the remaining steps are as follows:
(1)将粘附肽、水与链霉亲和素荧光素复合物混合，在室温下孵育40min，得到复合物溶液；(1) Mix the adhesion peptide, water and streptavidin-fluorescein complex, and incubate at room temperature for 40min to obtain a complex solution;
(2)将得到复合物溶液与β-环糊精、二甲基乙酰胺、磷酸二氢钾、丙三醇混合均匀后，静置2h，去除不溶物，得到预冻液；(2) after uniformly mixing the obtained complex solution with β-cyclodextrin, dimethylacetamide, potassium dihydrogen phosphate and glycerol, let stand for 2 hours to remove insoluble matter to obtain a pre-freezing solution;
(3)预冻：将预冻液置于冻干柜中，在半小时内降温至-10℃，并在该温度区间维持40min；(3) Pre-freezing: place the pre-freezing solution in a freeze-drying cabinet, cool down to -10°C within half an hour, and maintain this temperature range for 40 minutes;
(4)冻结及降压：在半小时内降温至-25℃，并在该温度区间维持40min，随后调整真空度为10Pa，维持2.5h；(4) Freezing and depressurization: reduce the temperature to -25°C within half an hour, and maintain this temperature range for 40min, then adjust the vacuum to 10Pa and maintain it for 2.5h;
(5)升华：升温至-10℃，并在该温度区间维持3h；随后调整真空度为30Pa，维持3h；(5) Sublimation: raise the temperature to -10°C, and maintain this temperature range for 3h; then adjust the vacuum to 30Pa and maintain it for 3h;
(6)解析干燥：升温至25℃，调整真空度至20Pa，维持2.5h；负压压塞后，取出物料2～8℃低温保存。(6) Analytical drying: raise the temperature to 25°C, adjust the vacuum degree to 20Pa, and maintain for 2.5h; after the negative pressure is plugged, take out the material and store at 2-8°C at low temperature.
对比例2Comparative Example 2
按表1称量具体原料，其余步骤制备步骤如下：The specific raw materials are weighed according to Table 1, and the preparation steps of the remaining steps are as follows:
(1)将粘附肽、98％叔丁醇溶液与链霉亲和素荧光素复合物混合，在室温下孵育40min，得到复合物的叔丁醇溶液；(1) Mix the adhesion peptide, 98% tert-butanol solution and streptavidin-fluorescein complex, and incubate for 40 min at room temperature to obtain a tert-butanol solution of the complex;
(2)将得到复合物的叔丁醇溶液与蔗糖、二甲基乙酰胺、磷酸二氢钾、丙三醇混合均匀后，静置2h，去除不溶物，得到预冻液；(2) after uniformly mixing the tert-butanol solution of the compound with sucrose, dimethylacetamide, potassium dihydrogen phosphate and glycerol, let stand for 2h to remove insoluble matter to obtain a pre-freezing solution;
(3)预冻：将预冻液置于冻干柜中，在半小时内降温至-10℃，并在该温度区间维持40min；(3) Pre-freezing: place the pre-freezing solution in a freeze-drying cabinet, cool down to -10°C within half an hour, and maintain this temperature range for 40 minutes;
(4)冻结及降压：在半小时内降温至-25℃，并在该温度区间维持40min，随后调整真空度为10Pa，维持2.5h；(4) Freezing and depressurization: reduce the temperature to -25°C within half an hour, and maintain this temperature range for 40min, then adjust the vacuum to 10Pa and maintain it for 2.5h;
(5)升华：升温至-10℃，并在该温度区间维持3h；随后调整真空度为30Pa，维持3h；(5) Sublimation: raise the temperature to -10°C, and maintain this temperature range for 3h; then adjust the vacuum to 30Pa and maintain it for 3h;
(6)解析干燥：升温至25℃，调整真空度至20Pa，维持2.5h；负压压塞后，取出物料2～8℃低温保存。(6) Analytical drying: raise the temperature to 25°C, adjust the vacuum degree to 20Pa, and maintain for 2.5h; after the negative pressure is plugged, take out the material and store at 2-8°C at low temperature.
表1Table 1
实施例4性能测试Example 4 Performance Test
取出物料，观察冻干效果。测试结果见表2。Take out the material and observe the freeze-drying effect. The test results are shown in Table 2.
表2性能测试结果Table 2 Performance test results
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式，并且很显然，根据上述教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and illustration. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many changes and variations are possible in light of the above teachings. The exemplary embodiments were chosen and described for the purpose of explaining certain principles of the invention and their practical applications, to thereby enable others skilled in the art to make and utilize various exemplary embodiments and various different aspects of the invention. Choose and change. The scope of the invention is intended to be defined by the claims and their equivalents.
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