CN112834741B - Preparation method of ordered microstructure nitrocellulose membrane for lateral flow analysis - Google Patents
Preparation method of ordered microstructure nitrocellulose membrane for lateral flow analysis Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title abstract description 19
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000005507 spraying Methods 0.000 claims description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 claims description 8
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- 239000007921 spray Substances 0.000 claims description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 7
- 238000005096 rolling process Methods 0.000 claims description 7
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 6
- 229920002678 cellulose Polymers 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 235000012239 silicon dioxide Nutrition 0.000 claims description 4
- 239000002033 PVDF binder Substances 0.000 claims description 3
- 239000004698 Polyethylene Substances 0.000 claims description 3
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 3
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- 239000003513 alkali Substances 0.000 claims description 3
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- -1 polyethylene Polymers 0.000 claims description 3
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- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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Abstract
The invention discloses a preparation method of an ordered microstructure nitrocellulose membrane for lateral flow analysis, which comprises the following steps: dissolving 5-30 parts of polymer in 30-80 parts of solvent I, adding 1-10 parts of solvent II, uniformly mixing, standing and defoaming to obtain polymer casting solution; step two, preparing a monodisperse colloidal microsphere solution; step three, self-assembling the monodisperse colloidal microsphere solution on a substrate to obtain an ordered template; step four, dripping the polymer casting film onto the ordered templates, and standing in a closed container to obtain a solidified and molded sample; step five, etching or calcining the product obtained in the step four; and step six, soaking the product obtained in the step five in deionized water solution, and drying to obtain the ordered microstructure nitrocellulose membrane. The invention has the advantages of easily available reaction raw materials, easily controlled reaction conditions and high preparation speed.
Description
Technical Field
The invention relates to a preparation method of an ordered membrane, in particular to a preparation method of an ordered microstructure nitrocellulose membrane for lateral flow analysis.
Background
The lateral flow immunoassay technology is widely applied in the biomedical detection field due to the advantages of simple operation, high detection speed, portability and the like. The nitrocellulose membrane is used as an important component of the lateral flow test strip, and the performance of the nitrocellulose membrane directly influences the accuracy and repeatability of the detection result. In addition, the nitrocellulose membrane has the advantages of convenient storage, good biocompatibility, environmental protection and the like, and is widely applied to the analysis devices such as immunofiltration vertical flow analysis, enzyme-linked immunosorbent assay, electrophoresis gel, immunoblotting assay and the like as a detection membrane.
Common preparation methods of the nitrocellulose membrane are a phase separation method, a phase inversion method, a submerged coating method, a plasma polymerization method, an interfacial polymerization method or an ionic liquid nano-welding method, but the methods have the defects of complex preparation process, higher requirements on facilities, high cost and the like. In addition, the nitrocellulose prepared by the method has the defects of nonuniform pore size distribution, poor light transmittance, low mechanical property, easiness in chemical degradation and the like. The traditional nitrocellulose detection membrane has the defects of uneven pore size, influence on the uniformity of distribution of colloidal gold or surface-enhanced Raman nano labels on the membrane, large fluctuation of detected signals, general existence of incapability of quantification, low sensitivity, large background signals and the like.
It is desirable to replace the conventional nitrocellulose membrane of disordered structure with the nitrocellulose membrane of ordered micro-nano structure to improve uniformity and sensitivity of detection results. Currently, it is generally difficult to produce an ordered porous membrane by a process, for example, a process for preparing an ordered porous membrane of a nano-scale size can only use a preparation process of a nano-scale porous membrane; the preparation of the ordered porous membrane with the micron level can only utilize the preparation process of the porous membrane with the micron level, and the preparation of the porous membrane with the nanometer level and the micron level is difficult to simultaneously realize by one preparation process. When preparing ordered porous membranes with different pore diameters, the production process needs to be replaced frequently, so that the production cost and the influence of uncontrollable factors on the uniformity of the ordered membranes are increased. Therefore, a convenient, quick and low-cost technical means is urgently needed for preparing the lateral flow ordered micro-nano structure nitrocellulose membrane.
Disclosure of Invention
The invention aims to: in order to overcome the defects in the prior art, the invention aims to provide the preparation method of the lateral flow ordered microstructure nitrocellulose membrane, which has the advantages of easily available raw materials, simple control conditions and high preparation speed.
The technical scheme is as follows: the invention relates to a preparation method of an ordered microstructure nitrocellulose membrane for lateral flow analysis, which comprises the following steps:
Dissolving 5-30 parts of polymer in 30-80 parts of solvent I, adding 1-10 parts of solvent II, uniformly mixing, standing and defoaming to obtain polymer casting solution;
Step two, preparing a monodisperse colloidal microsphere solution;
step three, self-assembling the monodisperse colloidal microsphere solution on a substrate to obtain an ordered template;
Dripping the polymer casting film onto the ordered templates, and standing in a closed container with a stable state for 4-6 hours to obtain a solidified and molded sample, wherein the stable state is that the relative humidity is 40-80%, and the temperature is 15-35 ℃;
Step five, etching or calcining the product obtained in the step four to remove the ordered templates;
And step six, soaking the product obtained in the step five in deionized water solution for 12-24 hours, and then drying to obtain the ordered micro-nano nitrocellulose membrane.
Further, in the first step, the polymer is one or more of nitrocotton, cellulose or nitrocellulose. The solvent I is one or more of toluene, acetone, chloroform, dimethylformamide or dimethyl sulfoxide. The second solvent is one or more of ethanol, isopropanol and tetrahydrofuran. The mixing treatment is at least one of stirring treatment, ultrasonic treatment, vibration treatment or vibration treatment.
Further, in the second step, the mass fraction of the monodisperse colloidal microsphere solution is 20-80 wt%. The monodisperse colloidal microsphere is polystyrene, titanium dioxide, silicon dioxide, polymethyl methacrylate, polyethyl acrylate or polyethylene, and the particle size is 200-500 nm.
Further, in the third step, the substrate is a glass slide, a silicon wafer, a nylon film, PVDF or a polyester film. The substrate is subjected to an acid or alkali cleaning surface oxide layer and then to a hydrophilic treatment by a plasma technique. The monodisperse microsphere colloid solution is sprayed and dripped on the substrate through 3 spray pipes, and the substrate moves at a speed of 1cm/s-10cm/s under the drive of a rolling wheel in the process of spraying and dripping, so that the substrate is self-assembled into an ordered template.
In the fifth step, etching is performed for 4-6 hours by hydrofluoric acid with the mass percentage of 2-10wt%, and the calcining temperature is 200-500 ℃.
In the sixth step, the drying is carried out in a cool environment with the temperature of 25-35 ℃ and the relative humidity of 30-60% for 10-30 minutes.
The thickness of the lateral flow ordered microstructure nitrocellulose membrane prepared by the preparation method is 100 nm-30 mu m.
Working principle: the lateral flow ordered microstructure nitrocellulose membrane has photon forbidden bands, can slow down the group velocity of photons, and can increase the interaction of light and substances.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable characteristics:
1. The reaction raw materials are easy to obtain, the reaction conditions are easy to control, and the preparation speed is high;
2. The size of the aperture of the ordered membrane can be conveniently controlled by adjusting the particle size of the colloidal microspheres, so that the prepared ordered membrane has light modulation property;
3. the lateral flow ordered microstructure nitrocellulose membrane has an accurate electromagnetic field limiting effect, and can limit the energy of light with different frequencies to different positions in a membrane hole, so that a detection signal is enhanced;
4. The lateral flow ordered microstructure nitrocellulose membrane can enable the nano particles to smoothly flow in the nitrocellulose membrane, is favorable for the uniform distribution of the nano particles in the membrane, has a nano finite field effect, and can improve the reaction efficiency of immune biomolecule reaction in a local space, thereby improving the detection accuracy;
5. The lateral flow ordered microstructure nitrocellulose membrane has large specific surface area, can be combined with more objects to be detected, is beneficial to improving the linear detection range, and has potential application prospects in various fields of biomedical target detection, food safety detection, environmental pollutant monitoring, customs quarantine and the like.
Drawings
FIG. 1 is a flow chart of the preparation of the present invention;
Fig. 2 is a scanning electron microscope image of the present invention.
Detailed Description
The raw materials in the following examples were all used as purchased. The substrate 3 is subjected to an acid or alkali cleaning surface oxide layer and then to a hydrophilic treatment by a plasma technique. The number of spray jets can be adjusted according to the size of the substrate 3 during the spray-dripping process.
Example 1
As shown in fig. 1, a method for preparing an ordered microstructure nitrocellulose membrane for lateral flow analysis, comprising the steps of:
s1, dissolving 5g of cellulose in dimethylformamide: acetone: in a mixed solution of 31g of organic solvent of ethanol=7:2:1, firstly carrying out ultrasonic treatment for 30 minutes at room temperature, then stirring for 1 hour, completely dissolving, and then standing and defoaming to obtain polymer casting film liquid 1;
S2, preparing a monodisperse colloidal microsphere solution 2 with the mass percentage concentration of 20%, wherein the monodisperse colloidal microsphere is silicon dioxide with the particle size of 200nm;
s3, spraying the monodisperse colloidal microsphere solution 2 on a slide substrate 3 through a microfluidic spray pipe 7, so that the monodisperse colloidal microsphere solution 2 is self-assembled to obtain an ordered template 5, and in the spraying process, the substrate 3 moves at a speed of 1cm/S under the drive of a rolling wheel 4;
s4, dropwise adding the polymer casting solution 1 onto an ordered template 5, and standing in a closed container with a stable state for 4 hours to obtain a cured and molded sample, wherein the stable state is that the relative humidity is 40%, and the temperature is 15 ℃;
S5, etching the product obtained in the step S4 for 6 hours through 2wt% hydrofluoric acid, and removing the ordered templates 5 to obtain a product 6 from which the ordered templates are removed;
S6, soaking the obtained product (the obtained product 6 with the ordered templates removed) in deionized water solution for 12 hours, and drying in a shady environment with the relative humidity of 30% at 25 ℃ for 10 minutes to obtain the lateral flow ordered microstructure nitrocellulose membrane with the thickness of 100nm, wherein a scanning electron microscope image of the nitrocellulose membrane is shown in figure 2. As can be seen from fig. 2, the prepared microstructure nitrocellulose membrane has uniform pore size and ordered arrangement.
Example 2
A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis, comprising the steps of:
s1, dissolving 30g of nitrocotton in dimethyl maple: dimethylformamide: in a mixed solution of 90g of organic solvent of ethanol=5:3:2, firstly carrying out ultrasonic treatment for 30 minutes at room temperature, then stirring for 5 hours, completely dissolving, and then standing and defoaming to obtain polymer casting film liquid 1;
s2, preparing a monodisperse colloidal microsphere solution 2 with the mass percentage concentration of 80%, wherein the monodisperse colloidal microsphere is titanium dioxide with the particle size of 500 nm;
S3, spraying a monodisperse colloidal microsphere solution 2 on a silicon wafer substrate 3 through a microfluidic spray pipe 7, so that the monodisperse colloidal microsphere solution 2 is self-assembled to obtain an ordered template 5, wherein the substrate 3 moves at a speed of 10cm/S under the drive of a rolling wheel 4 in the process of spraying;
S4, dropwise adding the polymer casting solution 1 onto the ordered templates 5, and standing in a closed container with a stable state for 6 hours to obtain a solidified and molded sample, wherein the stable state is that the relative humidity is 80%, and the temperature is 35 ℃;
s5, calcining the product obtained in the step S4 for 4 hours at the temperature of 300 ℃ to remove the ordered template 5;
S6, soaking the product obtained in the step S5 in deionized water solution for 24 hours, and drying the product in a shady environment with the temperature of 35 ℃ and the relative humidity of 60% for 30 minutes to obtain the lateral flow ordered micro-nano structure nitrocellulose membrane with the thickness of 30 mu m.
Example 3
A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis, comprising the steps of:
S1, dissolving 18g of nitrocotton and cellulose (mass ratio 1:1) in dimethylformamide: acetone: in a mixed solution of 60g of organic solvent of isopropanol=6:2:2, firstly carrying out ultrasonic treatment for 30 minutes at room temperature, then stirring for 3 hours, completely dissolving, and then standing and defoaming to obtain polymer casting film liquid 1;
S2, preparing a monodisperse colloidal microsphere solution 2 with the mass percentage concentration of 50%, wherein the monodisperse colloidal microsphere is silicon dioxide with the particle size of 350 nm;
S3, spraying monodisperse colloidal microsphere solution 2 on nylon membrane substrate 3 through microfluidic spray pipe 7, so that monodisperse colloidal microsphere solution 2 is self-assembled to obtain ordered template 5, wherein substrate 3 moves at 5cm/S under the drive of rolling wheel 4 during spraying;
s4, dropwise adding the polymer casting solution 1 onto the ordered templates 5, and standing in a closed container with a stable state for 5 hours to obtain a cured and molded sample, wherein the stable state is that the relative humidity is 60%, and the temperature is 25 ℃;
s5, etching the product obtained in the step S4 for 5 hours through 6wt% hydrofluoric acid, and removing the ordered templates 5;
s6, soaking the product obtained in the step S5 in deionized water solution for 18 hours, and drying the product in a shady environment with the temperature of 30 ℃ and the relative humidity of 45% for 20 minutes to obtain the lateral flow ordered micro-nano structure nitrocellulose membrane with the thickness of 15 mu m.
Example 4
A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis, comprising the steps of:
S1, dissolving 10g of cellulose and nitrocotton (3:2) in toluene: trichloromethane: in a mixed solution of 45g of organic solvent of tetrahydrofuran=5:2:1, firstly carrying out ultrasonic treatment for 30 minutes at room temperature, then stirring for 4 hours, completely dissolving, and then standing and defoaming to obtain polymer casting film liquid 1;
s2, preparing a monodisperse colloidal microsphere solution 2 with the mass percentage concentration of 30%, wherein the monodisperse colloidal microsphere is polymethyl methacrylate with the particle size of 300 nm;
S3, spraying a monodisperse colloidal microsphere solution 2 on a PVDF substrate 3 through a microfluidic spray pipe 7, so that the monodisperse colloidal microsphere solution 2 is self-assembled to obtain an ordered template 5, wherein the substrate 3 moves at a speed of 3cm/S under the drive of a rolling wheel 4 in the spraying process;
S4, dropwise adding the polymer casting solution 1 onto an ordered template 5, and standing in a closed container with a stable state for 4.5 hours to obtain a cured and molded sample, wherein the stable state is that the relative humidity is 50%, and the temperature is 20 ℃;
S5, calcining the product obtained in the step S4 at 300 ℃ for 6 hours, and removing the ordered template 5;
S6, soaking the product obtained in the step S5 in deionized water solution for 14 hours, and drying the product in a shady environment with the temperature of 27 ℃ and the relative humidity of 40% for 150 minutes to obtain the lateral flow ordered micro-nano structure nitrocellulose membrane with the thickness of 500 nm.
Example 5
A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis, comprising the steps of:
s1, dissolving 20g of nitrocellulose in chloroform: dimethylformamide: ethanol: in a mixed solution of 70g of organic solvent of isopropanol=6:2:1:1, firstly performing ultrasonic treatment for 30 minutes at room temperature, then stirring for 2 hours, completely dissolving, and then standing and defoaming to obtain polymer casting film liquid 1;
S2, preparing a monodisperse colloidal microsphere solution 2 with the mass percentage concentration of 70%, wherein the monodisperse colloidal microsphere is polyethyl acrylate or polyethylene with the particle size of 400 nm;
s3, spraying a monodisperse colloidal microsphere solution 2 on a polyester film substrate 3 through a microfluidic spray pipe 7, so that the monodisperse colloidal microsphere solution 2 is self-assembled to obtain an ordered template 5, and moving the substrate 3 at a speed of 8cm/S under the drive of a rolling wheel 4 in the process of spraying;
s4, dropwise adding the polymer casting solution 1 onto the ordered template 5, and standing in a closed container with a stable state for 5.5 hours to obtain a cured and molded sample, wherein the stable state is that the relative humidity is 70%, and the temperature is 30 ℃;
s5, calcining the product obtained in the step S4 at 600 ℃ for 4 hours, and removing the ordered template 5;
S6, soaking the product obtained in the step S5 in deionized water solution for 22 hours, and drying the product in a shady environment at 33 ℃ and with relative humidity of 50% for 25 minutes to obtain the lateral flow ordered micro-nano structure nitrocellulose membrane with the thickness of 20 mu m.
Comparative example
The ordered microstructure nitrocellulose membrane for lateral flow analysis prepared in example 3 is assembled into lateral flow immunoassay, detection of inflammatory marker C reactive protein is carried out, and comparison is carried out with the detection result of the conventional lateral flow test strip based on disordered nitrocellulose membrane. The result shows that the macroscopic colorimetric minimum detection concentration is reduced from the original 10ng/mL to 1ng/mL, and the detection sensitivity is obviously improved. In addition, the flowing speed of the sample on the ordered membrane is faster, the detection time is reduced from 15min of the traditional membrane to 10min, and the detection time is saved more. Therefore, the lateral flow immunoassay test strip based on the ordered micro-nano structure detection membrane is hopefully widely applied to the POCT detection field.
Claims (7)
1. A method for preparing an ordered microstructure nitrocellulose membrane for lateral flow analysis, comprising the steps of:
dissolving 5-30 parts of polymer in 30-80 parts of a first solvent, adding 1-10 parts of a second solvent, uniformly mixing, standing and defoaming to obtain polymer casting solution (1);
Step two, preparing a monodisperse colloidal microsphere solution (2); the mass fraction of the monodisperse colloidal microsphere solution (2) is 20-80wt%; the monodisperse colloidal microspheres are polystyrene, titanium dioxide, silicon dioxide, polymethyl methacrylate, polyethyl acrylate or polyethylene, and the particle size is 200-500nm;
Thirdly, self-assembling the monodisperse colloidal microsphere solution (2) on the substrate (3) to obtain an ordered template (5); the substrate (3) is subjected to acid or alkali cleaning surface oxidation layer and then is subjected to plasma hydrophilic treatment; the monodisperse colloidal microsphere solution (2) is sprayed and dripped on the substrate (3) through 3 spray pipes, and in the process of spraying and dripping, the substrate (3) moves at the speed of 1cm/s-10cm/s under the drive of a rolling wheel, and self-assembly is carried out to form an ordered template (5);
step four, dropwise adding the polymer casting solution (1) onto an ordered template (5), and standing in a closed container with a stable state, so as to obtain a solidified and molded sample, wherein the stable state is that the relative humidity is 40-80%, and the temperature is 15-35 ℃;
Step five, etching or calcining the product obtained in the step four, and removing the ordered templates (5) to obtain a product (6);
step six, soaking the substance (6) obtained in the step five in deionized water solution, and drying to obtain the ordered microstructure nitrocellulose membrane; the thickness of the nitrocellulose membrane is 100 nm-30 mu m;
The pore size of the ordered membrane can be controlled by adjusting the particle size of the monodisperse colloidal microspheres, so that the prepared ordered membrane has light modulation property.
2. A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis according to claim 1, wherein: in the first step, the polymer is one or more of nitrocotton, cellulose or nitrocellulose.
3. A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis according to claim 1, wherein: in the first step, the first solvent is one or more of toluene, acetone, chloroform, dimethylformamide or dimethyl sulfoxide.
4. A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis according to claim 1, wherein: in the first step, the second solvent is one or more of ethanol, isopropanol and tetrahydrofuran.
5. A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis according to claim 1, wherein: in the third step, the substrate (3) is a glass slide, a silicon wafer, a nylon film, PVDF or a polyester film.
6. A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis according to claim 1, wherein: in the fifth step, etching is performed by hydrofluoric acid with the mass percentage of 2-10wt%, and the calcining temperature is 200-500 ℃.
7. A method of preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis according to claim 1, wherein: in the step six, the drying is carried out for 10-30 minutes in a cool environment with the temperature of 25-35 ℃ and the relative humidity of 30-60%.
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