CN110146693B - Nitrocellulose membrane with fluorescent dye and production method thereof - Google Patents
Nitrocellulose membrane with fluorescent dye and production method thereof Download PDFInfo
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- CN110146693B CN110146693B CN201910388410.0A CN201910388410A CN110146693B CN 110146693 B CN110146693 B CN 110146693B CN 201910388410 A CN201910388410 A CN 201910388410A CN 110146693 B CN110146693 B CN 110146693B
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- microporous membrane
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- cellulose nitrate
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- 239000000020 Nitrocellulose Substances 0.000 title claims abstract description 93
- 229920001220 nitrocellulos Polymers 0.000 title claims abstract description 93
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 62
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- 238000004519 manufacturing process Methods 0.000 title claims description 5
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 claims abstract description 92
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- IKYJCHYORFJFRR-UHFFFAOYSA-N Alexa Fluor 350 Chemical compound O=C1OC=2C=C(N)C(S(O)(=O)=O)=CC=2C(C)=C1CC(=O)ON1C(=O)CCC1=O IKYJCHYORFJFRR-UHFFFAOYSA-N 0.000 description 4
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention provides a nitrocellulose microporous membrane containing fluorescent dye and a preparation method thereof. The fluorescent dye providing donor fluorescent molecules is directly added into the membrane casting solution, and a fluorescent cellulose nitrate microporous membrane is directly produced through phase conversion, wherein the fluorescence intensity in the microporous membrane can be controlled and adjusted by adding different doses of fluorescein, and the microporous membrane is suitable for producing a fluorescence quenching immunochromatography device.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a nitrocellulose microporous membrane containing a fluorescent dye and a preparation method thereof.
Background
Since 1971, when Faulk and Taytor introduced colloidal gold into immunochemistry, the colloidal gold labeling technique has been increasingly widely used as a new immunological labeling technique in various fields of biomedicine, particularly in the field of immunological research. Becomes another important immunolabeling technology after fluorescent labeling, enzyme labeling and radioactive immunolabeling.
In 1990, BEGGS M and others organically combined various methods such as a colloidal gold labeling technology, an immunoassay technology, a chromatography analysis technology and the like for the first time, and established a colloidal Gold Immunochromatography (GICA) by taking an NC membrane as a reaction carrier, wherein the colloidal gold immunochromatography is used for detecting HCG in human urine and serum. Because the Gold Immunochromatography (GICA) has the characteristics of low development cost, simple and rapid use, relatively stable storage, wide application range and the like, the method is well known by people and is widely applied to the detection of other various samples. The GICA method becomes an economical and practical immunochromatography technology at the present stage, and is particularly suitable for the fields of instant examination, large-batch detection with tight time, large-area general investigation and the like. In 20 years thereafter, detection reagents for pathogens (CN 03115143.4), hormones (CN 200610014168.3), cardiac markers (CN 200410011165.5), tumor markers (CN 200510104796.6), autoimmune disease markers (CN 200410027291.X), and the like have been developed on the basis of GICA; meanwhile, the method is also applied to the fields of food, environment (CN 03116692.X), veterinarian (CN 0213972. X) and the like.
The GICA device is usually composed of a sample pad, a colloidal gold conjugate pad, a microporous chromatographic membrane, an absorbent pad, assembled on a backing plate by a pressure sensitive adhesive. During detection, a sample is added on a sample pad at the end of the test paper, and the analyte in the sample and the immune colloid on the binding pad interact with each other through capillary action and form a compound. The complex continues to move forward to the microporous chromatographic membrane detection line through capillary action, is specifically bound with the antibody (or antigen) on the detection line and is trapped, and is gathered on the detection line, and when the complex is accumulated to a certain amount, the red color with the characteristics of the colloidal gold is displayed. While the immuno-colloidal gold not bound to the analyte is not captured by the detection line, migrates to the quality control line and binds to the antibody thereon to show red color, achieving the purpose of separating from the complex, and indicating that the GICA device is working normally.
Although the chromatographic technique represented by colloidal gold is widely applied in many fields, the chromatographic technique can only be used for qualitative or semi-quantitative detection, and the requirement of the quantification of clinical detection indexes is difficult to meet; meanwhile, the detection result is judged by naked eyes, the sensitivity is low, and particularly when the detection result is weak positive, the artificial omission phenomenon is easily caused, and further development and perfection are needed.
Background fluorescence quenching-immunochromatography (bFQICA) is an analytical technique established on the basis of the Gold Immunochromatography (GICA). According to the technology, a layer of uniform fluorescent dye is added below a microporous chromatographic membrane of colloidal gold immunochromatography, background fluorescence is generated between a detection line (T line), a quality control line (C line) and the detection line (T line), colloidal gold marked by an antibody of an object to be detected is used as a fluorescence acceptor, fluorescein under the microporous chromatographic membrane provides donor fluorescent molecules, and according to the principle that the two molecules can generate Fluorescence Resonance Energy Transfer (FRET), namely quenching, within a 10nm range, when the antibody of the marked colloidal gold is combined at the T line, the colloidal gold carried by the antibody can quench the background fluorescence of the microporous chromatographic membrane at the T line, so that the fluorescence intensity is attenuated. If the relative fluorescence intensity of the background fluorescence and the detection line (T line) of the GICA device can be measured and compared, the aim of quantitative detection can be achieved.
However, since the market does not provide any microporous chromatographic membrane containing fluorescent dye, and there is no invention application in this respect, when background fluorescence quenching-immunochromatography (bFQICA) is used, fluorescent dye can only be attached below the microporous chromatographic membrane in the GICA apparatus by an additional apparatus, and the microporous chromatographic membrane not only shields the sensitivity of background fluorescence quenching-immunochromatography (bFQICA) interfering with the intensity of fluorescent dye, but also increases the labor cost of the apparatus, and also makes the uniformity of the product difficult to control. If the fluorescent dye providing donor fluorescent molecules can be directly added into the casting solution, and a fluorescent nitrocellulose microporous membrane can be directly produced through phase conversion, the problems of sensitivity, uniformity and the like in background fluorescence quenching-immunochromatography (bFQICA) can be overcome.
Disclosure of Invention
The inventor finds that, if a fluorescent dye providing donor fluorescent molecules can be directly added into a casting solution, and a fluorescent nitrocellulose microporous membrane can be directly produced through phase conversion, the problems of sensitivity, uniformity and the like in background fluorescence quenching-immunochromatography (bFQICA) can be overcome. The inventor further researches and discovers that fluorescent dye can be added into nitrocellulose membrane casting solution for direct coating, and then a nitrocellulose microporous membrane with fluorescence can be produced at one time through phase inversion, the fluorescence intensity in the microporous membrane can be controlled and adjusted by adding different dosages of fluorescein, and the microporous membrane is suitable for producing a fluorescence quenching immunochromatography device.
One aspect of the present invention provides a nitrocellulose microporous membrane containing a fluorescent dye.
In a preferred embodiment of the present invention, the fluorescent dye is selected from the group consisting of: any one or more of fluorescein dyes, rhodamine dyes, Cy series cyanine dyes or Alexa series dyes.
In another preferred embodiment of the present invention, the fluorescein-based dye is selected from: fluorescein Isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), 2-methoxyfluorescein, 4, 5-dimethoxyfluorescein, etc., and their analogues.
In another preferred embodiment provided by the present invention, the rhodamine-based dye is selected from: red Rhodamine (RBITC), Tetramethylrhodamine (TAMRA), rhodamine B (TRITC), rhodamine 6G, or rhodamine 123.
In another preferred embodiment of the present invention, the Cy series cyanine dye, is selected from the group consisting of: any one or more of Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7 and analogues thereof.
In another preferred embodiment provided by the present invention, the Alexa series dye is selected from: any one or combination of AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750.
In another preferred embodiment provided by the present invention, the nitrocellulose microporous membrane is provided with or without a backing.
In another preferred embodiment provided by the present invention, the pore size of the nitrocellulose microporous membrane is between 0.1 micron and 15 microns.
In another preferred embodiment provided by the present invention, the fluorescence intensity of the nitrocellulose microporous membrane is in the range of 500-10000.
The second aspect of the invention provides a preparation method of a nitrocellulose microporous membrane with a fluorescent dye, which comprises the following steps:
(1) preparing a cellulose nitrate membrane casting solution;
(2) preparing a fluorescent dye solution;
(3) mixing the prepared fluorescent dye solution into the prepared cellulose nitrate casting solution according to a required proportion, uniformly stirring, filtering and standing for later use;
(4) directly coating the liquid nitrocellulose casting solution mixed with the fluorescent dye on the surface of a stainless steel conveying belt or the surface of a transparent polyester membrane;
(5) and (3) running the coated casting film liquid layer to a drying tunnel for controlling temperature, humidity and air inlet and outlet through a steel belt to perform phase conversion reaction to form the nitrocellulose microporous film with a backing or without the backing and containing fluorescent dye.
In a preferred embodiment of the present invention, the cellulose nitrate casting solution in step (1) is prepared as follows:
a) dissolving a certain weight part of cellulose nitrate in a ketone compound or ester compound solvent, wherein the ketone compound or ester compound solvent is selected from the following components: any one or more of ethyl formate, methyl acetate, ethyl acetate, acetone or butanone.
b) Then adding a cosolvent, a non-solvent and a wetting agent in sequence, stirring uniformly at 15-30 ℃, defoaming, filtering, and curing to obtain a cellulose nitrate casting solution for later use; the cosolvent is selected from: any one or more of methanol and ethanol; the non-solvent is selected from: any one or more of n-propanol, isopropanol, n-butanol, isobutanol or deionized water; the wetting agent is selected from: any one or more of surfactants such as glycerol, triton X-100, tween 20, polyethylene glycol 400, alkyl sulfate, alkyl sulfonate and the like.
In another preferable scheme provided by the invention, the specific formula proportion of the cellulose nitrate casting solution is 2-20 parts by weight of cellulose nitrate, 15-60 parts by weight of solvent, 15-80 parts by weight of cosolvent, 2-25 parts by weight of non-solvent and 0.003-2 parts by weight of wetting agent.
In another preferred embodiment of the present invention, the fluorescent dye solution in step (2) is prepared as follows: dissolving a certain weight part of fluorescent dye into low-carbon alcohol with 1 to 6 carbon atoms, and mixing the low-carbon alcohol with a ketone compound or an ester compound according to a certain proportion to prepare a mixed solution; the fluorescent dye is selected from fluorescein dyes, including Fluorescein Isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET) and the like and analogues thereof; rhodamine dyes including red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), rhodamine 6G, rhodamine 123, and the like; cyanine dyes of the Cy series, which usually consist of two heterocyclic ring systems, including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7 and the like; alexa series dyes including Alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750. The lower alcohol with 1 to 6 carbon atoms is selected from: any one or more of methanol, ethanol and isopropanol; preferably ethanol; the ketone compound or ester compound is selected from: any one or the combination of more than one of methyl acetate, ethyl acetate, acetone and butanone; the preferred ketone compound is acetone and the preferred ester is methyl acetate.
In another preferred embodiment of the present invention, the fluorescent dye solution contains 1-50g, preferably 5-20g, and most preferably 10g of fluorescent dye per 500mL of fluorescent dye solution. The prepared fluorescent dye solution can be stored in a brown bottle and kept away from light at 4 ℃, and if the solution has precipitate, the solution can be filtered.
In another preferred embodiment provided by the present invention, the mixing ratio of the fluorescent dye solution and the nitrocellulose membrane casting solution in step (3) is 1:12000-200000 (V/V).
In another preferred embodiment provided by the present invention, the fluorescent dye solution in step (3) is slowly added to the previously prepared nitrocellulose membrane casting solution while stirring, until the fluorescent dye solution is completely dissolved in the solution, and the stirring time is preferably 2 to 3 hours. The well-stirred nitrocellulose membrane casting solution containing the fluorescent dye is preferably kept still for one day, and then the solution is filtered; and (3) further standing and curing the filtered membrane casting solution for a period of time to produce, wherein the standing time is preferably three days.
In another preferred embodiment provided by the present invention, the thickness of the nitrocellulose casting solution containing fluorescent dye coated in step (4) is 100-800 μm; the preferred coating thickness is 400-600 μm.
In another preferred embodiment provided by the present invention, in the phase-inversion reaction in step (5), the flow delay is performed, the temperature of the nitrocellulose casting solution containing the fluorescent dye is controlled within the range of 10 ℃ to 35 ℃, the contact treatment time of the air outside the drying tunnel and the nitrocellulose casting solution containing the fluorescent dye is controlled within the range of 10 seconds to 30 minutes, the temperature inside the drying tunnel is controlled within the range of 15 ℃ to 30 ℃, the relative humidity of the air is controlled within the range of 40% to 95%, and the air speed is controlled within the range of 0.1 m/s to 7 m/s.
In another preferred embodiment provided by the present invention, step (5) further comprises the following steps:
(6) immersing the formed nitrocellulose microporous membrane containing the fluorescent dye obtained in the step (5) into water to carry out cleaning of residual solvent and dust removal treatment on the surface of the membrane; followed by a continuous soaking treatment by immersion in a post-treatment solution containing a surfactant.
(7) After post-treatment, the nitrocellulose microporous membrane containing the fluorescent dye is dried by a heating roller and air blowing, and the drying temperature is controlled to be 30-85 ℃, so that the nitrocellulose microporous backing membrane containing the fluorescent dye or the microporous membrane without the backing is obtained.
The third aspect of the invention provides an application of a nitrocellulose microporous membrane containing a fluorescent dye, wherein the microporous membrane is suitable for producing a fluorescence quenching immunochromatography test strip.
Compared with the prior art, the invention has the following advantages:
(1) the preparation process is relatively simple and feasible.
(2) The fluorescence intensity of the membrane can be adjusted according to the addition of fluorescent dye solutions with different doses, is convenient and flexible, and can completely meet the test requirements of a background fluorescence quenching-immunochromatography (bFQICA).
Drawings
FIG. 1 is a flow chart of a process for producing nitrocellulose microporous membranes containing fluorescent dyes.
FIG. 2 is a schematic diagram of the production of nitrocellulose microporous membranes containing fluorescent dyes.
Figure 3 is a schematic diagram illustrating the production of nitrocellulose-backed microporous membranes containing fluorescent dyes.
FIG. 4 is a schematic of a test strip (GICA device) assembled with nitrocellulose microporous membrane.
FIG. 5 shows the standard curve prepared in example 4.
FIG. 6 shows the standard curve prepared in example 5.
FIG. 7 shows the standard curve prepared in example 6.
Detailed Description
Example 1
(1) Dissolving 10.3 parts by weight of cellulose nitrate in 50.8 parts by weight of acetone; then, 20.5 parts by weight of ethanol, 10.4 parts by weight of n-butanol, 7.98 parts by weight of deionized water and 0.02 part by weight of tween-20 are sequentially added and stirred uniformly at the temperature of 15-30 ℃, and after defoaming and filtering, the cellulose nitrate membrane casting solution is obtained for standby.
(2) Weighing 10G of fluorescent dye rhodamine 6G, dissolving in 500mL of acetone, storing in a brown bottle, and storing at 4 ℃ in a dark place for later use, wherein if the solution has a precipitate, the solution can be filtered.
(3) Adding the prepared fluorescent dye solution into the prepared nitrocellulose membrane casting solution according to the proportion of adding 30 mul of the membrane casting solution into 3000ml of the membrane casting solution, and uniformly stirring.
(4) Directly casting the prepared cellulose nitrate casting solution containing the fluorescent dye rhodamine 6G on the surface of a stainless steel belt (see figure 2), wherein the casting thickness of the casting solution is 450 mu m, and the contact time of the casting solution and air is controlled within the range of 10 seconds to 2 minutes.
(5) The cast film liquid layer after casting is immediately operated to a drying tunnel for controlling temperature, humidity and air inlet and outlet amount along with the operation of the stainless steel band to carry out phase conversion reaction, the temperature of the cast film liquid is controlled within the range of 20-23 ℃, the temperature in the drying tunnel is controlled within the range of 22-24 ℃, the relative humidity of air is controlled within the range of 55-70%, and the air speed is controlled within the range of 0.1-2.5 m/s, so that the cellulose nitrate microporous membrane is obtained.
(6) Immersing the nitrocellulose microporous membrane which leaves the drying tunnel into water to carry out the cleaning of residual solvent and the dust removal treatment of the membrane surface, and then immersing the nitrocellulose microporous membrane into post-treatment solution containing surfactant to carry out continuous immersion treatment.
(7) And (3) drying the post-treated cellulose nitrate microporous membrane by a heating roller and air blowing, wherein the drying temperature is controlled to be 35-65 ℃, and obtaining a finished product of the cellulose nitrate microporous membrane containing the fluorescent dye.
(8) Cutting a nitrocellulose microporous membrane containing fluorescent dye into samples of 25mm X5 mm, wetting with purified water, and placing into a fluorescence detector for detecting the intensity. The fluorescence intensity of the obtained nitrocellulose microporous membrane is in the range of 1000-3000.
Example 2
(1) Dissolving 9.2 parts by weight of cellulose nitrate in 47.8 parts by weight of methyl acetate; then, 21.6 parts by weight of ethanol, 12.3 parts by weight of isobutanol, 9 parts by weight of deionized water and 0.1 part by weight of glycerol are sequentially added and stirred uniformly at the temperature of 20-23 ℃, and after defoaming and filtering, the cellulose nitrate casting solution is obtained for later use.
(2) Weighing 10G of fluorescent dye rhodamine 6G, dissolving in 500mL of methyl acetate, storing in a brown bottle, and storing at 4 ℃ in a dark place for later use, wherein if the solution has a precipitate, the solution can be filtered.
(3) Adding the prepared fluorescent dye solution into the prepared nitrocellulose membrane casting solution according to the proportion of adding 15 mul of the membrane casting solution into 3000ml of the membrane casting solution, and uniformly stirring.
(4) The prepared fluorescent dye rhodamine 6G-containing cellulose nitrate casting solution is cast on a transparent polyester film (shown in figure 3) which is attached to the surface of a stainless steel belt and used as a backing material, and the casting thickness is 450 mu m. The contact time of the casting solution and the air is controlled within the range of 10 seconds to 2 minutes.
(5) The cast film liquid layer after casting and a transparent polyester film as a back lining material are immediately operated to a temperature control and humidity control drying tunnel for phase conversion reaction along with the operation of the stainless steel band, the temperature of the cast film liquid is controlled within the range of 20-23 ℃, the temperature in the drying tunnel is controlled within the range of 22-24 ℃, the relative humidity of air is controlled within the range of 55-70%, and the air speed is controlled within the range of 0.1-2.5 m/s, so that the cellulose nitrate microporous film with the back lining is obtained.
(6) Immersing the cellulose nitrate microporous membrane with the backing after leaving the drying tunnel into water for cleaning residual solvent and dedusting the surface of the membrane, and then immersing the cellulose nitrate microporous membrane into post-treatment solution containing surfactant for continuous immersion treatment.
(7) And (3) drying the post-treated cellulose nitrate microporous membrane with the backing by a heating roller and blowing, wherein the drying temperature is controlled to be 35-65 ℃, and thus obtaining a finished product of the fluorescent dye-containing cellulose nitrate microporous backing membrane.
(8) And cutting the microporous backing film containing the fluorescent dye cellulose nitrate into samples of 25mm X5 mm, wetting the samples by purified water, and then placing the samples into a fluorescence detector to detect the intensity. The fluorescence intensity of the nitrocellulose microporous membrane with the backing is in the range of 500-1000.
Example 3
(1) Dissolving 8.8 parts by weight of cellulose nitrate in 50.8 parts by weight of acetone; then adding 15.5 parts by weight of ethanol, 3.5 parts by weight of isopropanol, 11.0 parts by weight of isobutanol, 10.39 parts by weight of deionized water and 0.01 part by weight of triton X-100 in sequence, stirring uniformly at 15-30 ℃, defoaming and filtering to obtain the cellulose nitrate casting solution for later use.
(2) Weighing 10G of fluorescent dye rhodamine 6G, dissolving in 500mL of ethanol, storing in a brown bottle, and storing at 4 ℃ in a dark place for later use, wherein if the solution has a precipitate, the solution can be filtered.
(3) Adding the prepared fluorescent dye solution into the prepared nitrocellulose membrane casting solution according to the proportion of adding 250 mul of the membrane casting solution into 3000ml of the membrane casting solution, and uniformly stirring;
(4) and directly casting the prepared cellulose nitrate casting solution containing the fluorescent dye on the surface of the transparent polyester membrane, wherein the casting thickness is 450 mu m. The contact time of the casting solution and the air is controlled within the range of 10 seconds to 2 minutes.
(5) The cast film liquid layer after casting and a transparent polyester film as a back lining material are immediately operated to control the temperature, the humidity and the air inlet and outlet amount of a drying tunnel along with the operation of the stainless steel band to carry out phase conversion reaction, the temperature of the cast film liquid is controlled within the range of 20-23 ℃, the temperature in the drying tunnel is controlled within the range of 22-24 ℃, the relative humidity of air is controlled within the range of 55-70%, the air speed is controlled within the range of 0.1-2.5 m/s, and the contact treatment time of air and the cast film liquid is controlled within the range of 10-2 minutes, so that the cellulose nitrate microporous film with the back lining is obtained.
(6) Immersing the nitrocellulose microporous membrane with the backing after leaving the drying tunnel into water for cleaning residual solvent and dedusting the surface of the membrane, then immersing the nitrocellulose microporous membrane into post-treatment solution containing surfactant for continuous immersion treatment,
(7) and (3) drying the post-treated cellulose nitrate microporous membrane with the backing by a heating roller and air blowing, wherein the drying temperature is controlled to be 35-65 ℃, and thus obtaining a finished product of the cellulose nitrate microporous backing membrane containing the fluorescent dye.
(8) And cutting the nitrocellulose microporous membrane containing the fluorescent dye with a backing into samples of 25mm X5 mm, wetting the samples by using purified water, and then putting the samples into a fluorescence detector to detect the intensity. The fluorescence intensity of the nitrocellulose microporous membrane with the backing is within 5000-10000.
Example 4: detection of Procalcitonin (PCT) in serum (with nitrocellulose microporous membrane with backing having fluorescence intensity of 500-1000 as described in example 2)
1. Preparation of gold-labeled antibody
1.1 colloidal gold-antibody preservation solution
Dissolving in water, adjusting pH to 7.4 with 6N HCl, supplementing water to 250ml, filtering with 0.45 μm filter membrane, and storing at 4-8 deg.C.
1.2 working fluids
| Na 2 HPO 4 ·12H 2 O | 6.1g |
| NaCl | 8.5g |
| PVP40 | 5.0g |
| Boric acid | 2.1g |
| PEG | 1.0 |
| 10％BSA | 50ml |
| NaN 3 | 0.2g |
Dissolving in water, adjusting pH to 7.0-7.5 with 6N HCl, supplementing water to 1000ml, filtering with 0.45 μm filter membrane, and storing at 4-8 deg.C.
1.3 preparation of Gold-labeled antibody (Gold-Ab1)
1.3.1 taking 20ml of 20-30 nm particle colloidal gold solution, slowly adding 1.0ml (0.6mg/ml) of purified Ab1 antibody under magnetic stirring, and stirring at room temperature for 30 min;
1.3.2 adding 10% BSA 0.8ml (final concentration 0.4%), stirring at room temperature for 5 min;
1.3.3 adding 10% PEG 0.4ml (final concentration 0.2%), stirring at room temperature for 5 min;
1.3.412000-1500 r/min for 60-40 min, carefully sucking and centrifuging the supernatant, dissolving the precipitate in 0.5ml of preservation solution, wherein the optical density (O.D) of the gold-labeled antibody is about 100O.D, the concentration of Ab1 antibody is 1mg/ml, and preserving at 4 ℃ for later use;
biotin labeling Ab2
2.1Ab2 pretreatment
2.2 take 210. mu.L of the above pretreated Ab, add 25. mu.L of 1mg/ml NHSS-Biotin DMSO solution, mix well, react for 2 hours in the dark at 4 ℃ in a refrigerator, dialyze overnight for use.
SA dot film with uploading Gold-Ab1, biotin-Ab2
Taking 0.5 μ l of SA 1mg/ml phosphate buffer solution (pH7.2) to spot on the detection line position shown in figure 4, and drying in dark at room temperature;
Gold-Ab1, biotin-Ab20.5. mu.l was dropped onto the binding pad shown in FIG. 4.
4. Preparation of Standard Curve
4.1 preparing PCT series standard solution (the concentration is shown in a table 1) by using the working solution;
4.2 taking 7 test strips (shown in figure 4) assembled by nitrocellulose microporous membranes with backings and fluorescence intensities of 500-1000 described in example 2, horizontally placing, and respectively adding 50 mul of standard solutions with 7 concentrations on each sample pad;
4.310 min, measuring the fluorescence intensity F1 at 580nm of the detection line and the fluorescence intensity F2 at 580nm of the middle between the detection line and the quality control line on each test piece, calculating the data of F2/F1 and showing the standard curve in Table 1 and 5.
Table 1: PCT standard series concentration and corresponding fluorescence intensity and F2/F1 ratio
5. Sample detection
The standard series of solutions were replaced by serum samples, the steps 4.2 and 4.3 were repeated, F2/F1 was substituted into the standard curve, and PCT values of 6 samples were determined as shown in Table 2.
Table 2: comparing the detection result of the nitrocellulose microporous membrane with the backing with the fluorescence intensity of 500-1000 with the detection result of the Roche electrochemiluminescence method
The correlation between the detection result of the method and the detection result of the Roche electrochemical luminescence method is good, and R is 2 ＝0.9883。
Example 5: detection of Procalcitonin (PCT) in serum (with nitrocellulose microporous membrane with fluorescence intensity 1000-3000 as described in example 1)
The experiment in example 4 is repeated by using the nitrocellulose microporous membrane described in example 1, wherein the fluorescence intensity of the nitrocellulose microporous membrane is 1000-3000. Steps 1-3 are the same as in example 4.
4. Preparation of Standard Curve
4.1 preparing PCT series standard solution (the concentration is shown in a table 3) by using the working solution;
4.2 taking 7 test strips assembled by the nitrocellulose microporous membrane with the fluorescence intensity of 1000-3000 described in the embodiment 1, horizontally placing, and respectively adding 50 mul standard solutions with 7 concentrations on each sample pad;
4.310 min, measuring the fluorescence intensity F1 at 580nm of the detection line and the fluorescence intensity F2 at 580nm of the middle between the detection line and the quality control line on each test piece, calculating the data of F2/F1 and showing the standard curve in Table 3 and 6.
Table 3: PCT standard series concentration and corresponding fluorescence intensity and F2/F1 ratio
5. Sample detection
The standard series of solutions was replaced with serum samples, the steps 4.2 and 4.3 were repeated, F2/F1 was substituted into the standard curve, and PCT values for 6 samples were determined as shown in Table 4:
table 4: 1000-3000 fluorescence intensity detection result of nitrocellulose microporous membrane and Roche electrochemiluminescence detection result
| Sample number | The method of the invention (ng/ml) | Roche electrochemiluminescence method (ng/ml) |
| 1 | 19.5 | 19.17 |
| 2 | 3.64 | 3.00 |
| 3 | 0.46 | 0.40 |
| 4 | 1.53 | 1.80 |
| 5 | 0.60 | 0.54 |
| 6 | 0.05 | 0.07 |
The correlation between the detection result of the method and the detection result of the enzyme-linked immunosorbent assay (ELISA) methodGood, R is 2 ＝0.9985。
Example 6: detection of Procalcitonin (PCT) in serum (Nitrocellulose microporous membrane with backing with fluorescence intensity of 5000-10000)
The experiment in the example 4 is repeated by using the nitrocellulose microporous membrane with the backing, which is prepared in the example 3 and has the membrane surface fluorescence intensity of 5000-10000.
Steps 1-3 are the same as in example 4.
4. Preparation of Standard Curve
4.1 preparing PCT series standard solution (the concentration is shown in a table 5) by using the working solution;
4.2 taking 7 test strips assembled by the nitrocellulose microporous membrane with the backing and the fluorescence intensity of 5000-10000 in the embodiment 3, horizontally placing the test strips, and respectively adding 50 mul of standard solutions with 7 concentrations on each sample pad;
4.310 min, measuring the fluorescence intensity F1 at 580nm of the detection line and the fluorescence intensity F2 at 580nm of the middle between the detection line and the quality control line on each test piece, calculating the data of F2/F1 and showing the standard curve in Table 5 and 7.
Table 5: PCT standard series concentration and corresponding fluorescence intensity and F2/F1 ratio
5. Sample detection
The standard series of solutions was replaced with serum samples, the steps 4.2 and 4.3 were repeated, F2/F1 was substituted into the standard curve, and PCT values for 6 samples were determined as shown in Table 6:
table 6: 5000-10000 fluorescence intensity nitrocellulose microporous membrane detection result with backing and Roche electrochemiluminescence detection result
The correlation between the detection result of the method and the detection result of the Roche electrochemical luminescence method is good, and R is 2 ＝0.971。
Claims (3)
1. A nitrocellulose microporous membrane comprising a fluorescent dye, the nitrocellulose microporous membrane prepared by the following method:
(1) dissolving 10.3 parts by weight of cellulose nitrate in 50.8 parts by weight of acetone; then, sequentially adding 20.5 parts by weight of ethanol, 10.4 parts by weight of n-butanol, 7.98 parts by weight of deionized water and 0.02 part by weight of tween-20, uniformly stirring at 15-30 ℃, defoaming and filtering to obtain a cellulose nitrate membrane casting solution for later use;
(2) Weighing 10G of fluorescent dye rhodamine 6G, dissolving in 500mL of acetone, storing in a brown bottle, and storing at 4 ℃ in a dark place for later use, and if the solution has a precipitate, filtering;
(3) adding the prepared fluorescent dye solution into the prepared nitrocellulose membrane casting solution according to the proportion of adding 30 mul to 3000ml of membrane casting solution and uniformly stirring;
(4) directly casting the prepared cellulose nitrate casting solution containing the fluorescent dye rhodamine 6G on the surface of a stainless steel belt, wherein the casting thickness of the casting solution is 450 mu m, and the contact time of the casting solution and air is controlled within the range of 10 seconds to 2 minutes;
(5) the cast film liquid layer after casting is immediately operated to a drying tunnel for controlling temperature, humidity and air inlet and outlet amount along with the operation of the stainless steel band to carry out phase-transition reaction, the temperature of the cast film liquid is controlled within the range of 20-23 ℃, the temperature in the drying tunnel is controlled within the range of 22-24 ℃, the relative humidity of air is controlled within the range of 55-70%, and the air speed is controlled within the range of 0.1-2.5 m/s, so that the cellulose nitrate microporous membrane is obtained;
(6) immersing the cellulose nitrate microporous membrane after leaving the drying tunnel into water to perform cleaning of residual solvent and membrane surface dust removal treatment, and then immersing the cellulose nitrate microporous membrane into an after-treatment solution containing a surfactant to perform continuous immersion treatment;
(7) And (3) drying the post-treated cellulose nitrate microporous membrane by a heating roller and air blowing, wherein the drying temperature is controlled to be 35-65 ℃, and obtaining a finished product of the cellulose nitrate microporous membrane containing the fluorescent dye.
2. Use of the nitrocellulose microporous membrane of claim 1 in the production of a fluorescence quenching immunochromatographic test strip.
3. The use according to claim 2, wherein the fluorescence quenching immunochromatographic test strip is a test strip for detecting procalcitonin in serum.
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| TA01 | Transfer of patent application right |
Effective date of registration: 20201230 Address after: 515063 Lu Rongsheng Science Park, University of Guangdong, Shantou Applicant after: RUNBIO BIOTECH Co.,Ltd. Address before: 515063 1st floor, neirunhe biomedical technology (Shantou) Co., Ltd., Rongsheng Science Park, University Road, Shantou City, Guangdong Province Applicant before: SHANTOU EALON MEMBRANE Co.,Ltd. |
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| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Room 116, Floor 1, Transportation Bureau Building, No. 95, Yingbin Avenue, Huacheng Street, Huadu District, Guangzhou, Guangdong Province, 510801 Patentee after: Guangzhou Iconas Biomedical Technology Co.,Ltd. Address before: Room 116, Floor 1, Transportation Bureau Building, No. 95, Yingbin Avenue, Huacheng Street, Huadu District, Guangzhou, Guangdong Province, 510801 Patentee before: Guangzhou Iconas Biomedical Technology Co.,Ltd. |
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| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: Room 116, Floor 1, Transportation Bureau Building, No. 95, Yingbin Avenue, Huacheng Street, Huadu District, Guangzhou, Guangdong Province, 510801 Patentee after: Guangzhou Iconas Biomedical Technology Co.,Ltd. Address before: 515063 Lu Rongsheng Science Park, University of Guangdong, Shantou Patentee before: RUNBIO BIOTECH Co.,Ltd. |
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| CI03 | Correction of invention patent | ||
| CI03 | Correction of invention patent |
Correction item: Patentee|Address Correct: RUNBIO BIOTECH Co.,Ltd.|515063 Lu Rongsheng Science Park, University of Guangdong, Shantou False: Guangzhou Iconas Biomedical Technology Co.,Ltd.|Room 116, Floor 1, Transportation Bureau Building, No. 95, Yingbin Avenue, Huacheng Street, Huadu District, Guangzhou, Guangdong Province, 510801 Number: 10-02 Volume: 39 |
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| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Room 116, Floor 1, Transportation Bureau Building, No. 95, Yingbin Avenue, Huacheng Street, Huadu District, Guangzhou, Guangdong Province, 510801 Patentee after: Guangdong Iconas Biomedical Technology Co.,Ltd. Address before: Room 116, Floor 1, Transportation Bureau Building, No. 95, Yingbin Avenue, Huacheng Street, Huadu District, Guangzhou, Guangdong Province, 510801 Patentee before: Guangdong Iconas Biomedical Technology Co.,Ltd. |
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| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: Room 116, Floor 1, Transportation Bureau Building, No. 95, Yingbin Avenue, Huacheng Street, Huadu District, Guangzhou, Guangdong Province, 510801 Patentee after: Guangdong Iconas Biomedical Technology Co.,Ltd. Address before: 515063 Lu Rongsheng Science Park, University of Guangdong, Shantou Patentee before: RUNBIO BIOTECH Co.,Ltd. |