CN112834741A - 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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- 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 method for preparing an ordered microstructure nitrocellulose membrane for lateral flow analysis, which comprises the following steps: step one, 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 a polymer casting solution; step two, preparing a monodisperse colloidal microsphere solution; step three, self-assembling the monodisperse colloidal microsphere solution on the substrate to obtain an ordered template; dripping the polymer casting solution on the ordered template, standing in a closed container to obtain a cured and molded sample; step five, etching or calcining the product obtained in the step four; and step six, soaking the substance obtained in the step five in a deionized water solution, and then drying to obtain the ordered microstructure nitrocellulose membrane. The invention has the advantages of easily obtained reaction raw materials, easily controlled reaction conditions and high preparation speed.
Description
Technical Field
The invention relates to a method for preparing an ordered membrane, in particular to a method for preparing a nitrocellulose membrane with an ordered microstructure for lateral flow analysis.
Background
The lateral flow immunoassay technology is widely applied to the field of biomedical detection due to the advantages of simple operation, high detection speed, convenience in carrying and the like. The nitrocellulose membrane is an important component of the lateral flow test strip, and the performance of the nitrocellulose membrane directly influences the accuracy and repeatability of a detection result. In addition, because of the advantages of convenient storage, good biocompatibility, environmental protection and the like, the nitrocellulose membrane is widely applied to analysis devices such as an immunodiafiltration vertical flow analysis device, an enzyme linked immunosorbent assay device, an electrophoresis gel device, an immunoblotting experiment device and the like as a detection membrane.
The commonly used preparation method of the nitrocellulose membrane is a phase separation method, a phase inversion method, an immersion 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 facility requirement, high cost and the like. In addition, the nitrocellulose prepared by the methods has the defects of nonuniform pore size distribution, poor light transmission, low mechanical property, easy chemical degradation and the like. The traditional cellulose nitrate detection membrane has nonuniform pore size, affects the uniformity of the distribution of colloidal gold or surface enhanced Raman nano-labels thereon, and has the defects of large fluctuation of detected signals, ubiquitous incapability of quantification, low sensitivity, large background signals and the like.
The nitrocellulose membrane with the ordered micro-nano structure is expected to replace the conventional nitrocellulose membrane with the disordered structure, so that the uniformity and the sensitivity of a detection result are improved. Currently, the common ordered porous membrane is difficult to be produced by one process, for example, the preparation process of the nanoscale ordered porous membrane can only be utilized in the preparation of the nanoscale ordered porous membrane; the preparation process of the micron-sized ordered porous membrane can only be utilized, and the preparation of the nano-sized porous membrane and the micron-sized porous membrane can not be realized through one preparation process. When the ordered porous membrane with different pore diameters is prepared, the production process needs to be frequently replaced, so that the production cost is increased and the influence of uncontrollable factors on the uniformity of the ordered membrane is increased. Therefore, a technical means with convenience, rapidness and low cost is urgently needed for preparing the nitrocellulose membrane with the lateral flow ordered micro-nano structure.
Disclosure of Invention
The purpose of the invention is as follows: 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 obtained 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:
step one, 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 a polymer casting solution;
step two, preparing a monodisperse colloidal microsphere solution;
step three, self-assembling the monodisperse colloidal microsphere solution on the substrate to obtain an ordered template;
dripping the polymer casting solution onto the ordered template, standing in a closed container with a stable state for 4-6 hours to obtain a cured and formed sample, wherein the stable state is that the relative humidity is 40-80%, and the temperature is 15-35 ℃;
etching or calcining the product obtained in the step four to remove the ordered template;
and step six, soaking the substance obtained in the step five in a 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 nitrocellulose, cellulose or nitrocellulose. The first solvent 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 blending treatment is at least one of stirring treatment, ultrasonic treatment, shaking 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 microspheres are polystyrene, titanium dioxide, silicon dioxide, polymethyl methacrylate, polyethylacrylate 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 needs to be cleaned of a surface oxide layer by acid or alkali and then subjected to hydrophilic treatment by a plasma technology. The monodisperse microsphere colloidal solution is sprayed and dropped on the substrate through 3 spray pipes, and in the process of spraying and dropping, the substrate is driven by a rolling wheel to move at the speed of 1cm/s-10cm/s, and self-assembly is carried out to form the ordered template.
Further, in the fifth step, etching is carried out for 4-6 hours by using hydrofluoric acid with the mass percent of 2-10 wt%, and the calcining temperature is 200-500 ℃.
Further, in the sixth step, the drying is performed for 10 to 30 minutes in a cool environment with the temperature of 25 to 35 ℃ and the relative humidity of 30 to 60%.
The thickness of the nitrocellulose membrane with the lateral flow ordered microstructure prepared by the preparation method is 100 nm-30 mu m.
The working principle is as follows: the side flow ordered microstructure nitrocellulose membrane has a photon forbidden band, and can slow down the group velocity of photons, so that the interaction between light and a substance can be increased.
Has the advantages 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 side flow ordered microstructure nitrocellulose membrane can conveniently control the aperture size of the ordered membrane by adjusting the particle size of the used colloidal microspheres, so that the prepared ordered membrane has light modulation characteristics;
3. the lateral flow ordered microstructure nitrocellulose membrane has an accurate electromagnetic field confinement effect, and can confine the energy of light with different frequencies to different positions in a membrane hole, so that a detection signal is enhanced;
4. the nitrocellulose membrane with the lateral flow ordered microstructure can enable nanoparticles to smoothly flow in the nitrocellulose membrane, is beneficial to the uniform distribution of the nanoparticles in the membrane, has a nano confinement effect, and can improve the reaction efficiency of immune biomolecule reaction in a local space, thereby improving the detection accuracy;
5. the nitrocellulose membrane with the lateral flow ordered microstructure has a large specific surface area, can be combined with more objects to be detected, is favorable for improving the linear detection range, and has potential application prospects in various fields such as 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 purchased and used as received. The substrate 3 is required to be subjected to acid or alkali cleaning of a surface oxidation layer and then to hydrophilic treatment by a plasma technique. The number of spray irrigation may be adjusted according to the size of the substrate 3 during the droplet spraying process.
Example 1
Referring to fig. 1, a method for preparing an ordered microstructured nitrocellulose membrane for lateral flow analysis, comprises the steps of:
s1, dissolving 5g of cellulose in dimethylformamide: acetone: carrying out ultrasonic treatment for 30 minutes at room temperature in a mixed solution of 31g of an organic solvent with ethanol ratio of 7:2:1, then stirring for 1 hour until the solution is completely dissolved, and then standing and defoaming to obtain a polymer casting solution 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 200 nm;
s3, spraying and dripping the monodisperse colloidal microsphere solution 2 on the glass slide substrate 3 through the microfluidic nozzle 7 to enable the monodisperse colloidal microsphere solution 2 to be self-assembled to obtain an ordered template 5, wherein in the dripping process, the substrate 3 is driven by the rolling wheel 4 to move at the speed of 1 cm/S;
s4, dripping the polymer casting solution 1 onto the ordered template 5, standing in a closed container with a stable state for 4 hours to obtain a solidified 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 by 2 wt% hydrofluoric acid, and removing the ordered template 5 to obtain a product 6 obtained by removing the ordered template;
s6, soaking the substance obtained in the step S5 (the substance 6 obtained by removing the ordered template) in a deionized water solution for 12 hours, and then drying the substance for 10 minutes in a shady and cool environment with the temperature of 25 ℃ and the relative humidity of 30% 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 a figure 2. As can be seen from FIG. 2, the prepared microstructure nitrocellulose membranes have uniform pore sizes and are arranged orderly.
Example 2
A method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis, comprising the steps of:
s1, 30g of nitrocotton was dissolved in dimethyl sulfoxide: dimethylformamide: carrying out ultrasonic treatment for 30 minutes at room temperature in a mixed solution of 90g of an organic solvent with ethanol ratio of 5:3:2, then stirring for 5 hours until the solution is completely dissolved, and then standing and defoaming to obtain a polymer casting solution 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 and dripping the monodisperse colloidal microsphere solution 2 on the silicon chip substrate 3 through the microfluidic nozzle 7 to enable the monodisperse colloidal microsphere solution 2 to be self-assembled to obtain an ordered template 5, wherein in the dripping process, the substrate 3 is driven by the rolling wheel 4 to move at the speed of 10 cm/S;
s4, dripping the polymer casting solution 1 onto the ordered template 5, standing in a closed container with a stable state for 6 hours to obtain a solidified and formed 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 at 300 ℃ for 4 hours, and removing the ordered template 5;
and S6, soaking the substance obtained in the step S5 in a deionized water solution for 24 hours, and drying the substance for 30 minutes in a shady and cool environment with the temperature of 35 ℃ and the relative humidity of 60% to obtain the 30-micrometer-thickness lateral flow ordered micro-nano structure nitrocellulose membrane.
Example 3
A method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis, comprising the steps of:
s1, dissolving 18g of nitrocotton and cellulose (mass ratio is 1:1) in dimethylformamide: acetone: in a mixed solution of 60g of an organic solvent with isopropanol of 6:2:2, carrying out ultrasonic treatment for 30 minutes at room temperature, then stirring for 3 hours until the solution is completely dissolved, and then standing and defoaming to obtain a polymer casting solution 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 and dripping the monodisperse colloidal microsphere solution 2 on the nylon membrane substrate 3 through the microfluidic nozzle 7 to enable the monodisperse colloidal microsphere solution 2 to be self-assembled to obtain an ordered template 5, wherein in the dripping process, the substrate 3 is driven by the rolling wheel 4 to move at the speed of 5 cm/S;
s4, dripping the polymer casting solution 1 onto the ordered template 5, standing in a closed container with a stable state for 5 hours to obtain a solidified and molded sample, wherein the stable state is that the relative humidity is 60% and the temperature is 25 ℃;
s5, etching the substance obtained in the step S4 for 5 hours by 6 weight percent of hydrofluoric acid, and removing the ordered template 5;
s6, soaking the substance obtained in the step S5 in a deionized water solution for 18 hours, and drying the substance for 20 minutes in a shady and cool environment with the temperature of 30 ℃ and the relative humidity of 45% to obtain the nitrocellulose membrane with the lateral flow ordered micro-nano structure and the thickness of 15 microns.
Example 4
A method of making 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: carrying out ultrasonic treatment for 30 minutes at room temperature in a mixed solution of 45g of an organic solvent with tetrahydrofuran being 5:2:1, then stirring for 4 hours until the solution is completely dissolved, and then standing and defoaming to obtain a polymer casting solution 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 and dripping the monodisperse colloidal microsphere solution 2 on the PVDF substrate 3 through the microfluidic nozzle 7 to enable the monodisperse colloidal microsphere solution 2 to be self-assembled to obtain an ordered template 5, wherein in the dripping spraying process, the substrate 3 is driven by the rolling wheel 4 to move at a speed of 3 cm/S;
s4, dripping the polymer casting solution 1 onto the ordered template 5, standing in a closed container with a stable state for 4.5 hours to obtain a solidified 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 S4 at 300 ℃ for 6 hours, and removing the ordered template 5;
and S6, soaking the substance obtained in the step S5 in a deionized water solution for 14 hours, and drying the substance for 150 minutes in a shady and cool environment with the temperature of 27 ℃ and the relative humidity of 40% to obtain the cellulose nitrate membrane with the lateral flow ordered micro-nano structure and the thickness of 500 nm.
Example 5
A method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis, comprising the steps of:
s1, dissolving 20g of nitrocellulose in chloroform: dimethylformamide: ethanol: carrying out ultrasonic treatment for 30 minutes at room temperature in a mixed solution of 70g of an organic solvent with isopropanol of 6:2:1:1, then stirring for 2 hours until the solution is completely dissolved, and then standing and defoaming to obtain a polymer casting solution 1;
s2, preparing a monodisperse colloidal microsphere solution 2 with the mass percentage concentration of 70%, wherein the monodisperse colloidal microsphere is polyethylacrylate or polyethylene with the particle size of 400 nm;
s3, spraying and dripping the monodisperse colloidal microsphere solution 2 on the polyester film substrate 3 through the microfluidic nozzle 7 to enable the monodisperse colloidal microsphere solution 2 to be self-assembled to obtain an ordered template 5, wherein in the dripping process, the substrate 3 is driven by the rolling wheel 4 to move at the speed of 8 cm/S;
s4, dripping the polymer casting solution 1 onto the ordered template 5, standing in a closed container with a stable state for 5.5 hours to obtain a solidified 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 S4 at 600 ℃ for 4 hours, and removing the ordered template 5;
and S6, soaking the substance obtained in the step S5 in a deionized water solution for 22 hours, and drying the substance for 25 minutes in a shady and cool environment with the temperature of 33 ℃ and the relative humidity of 50% to obtain the nitrocellulose membrane with the lateral flow ordered micro-nano structure and the thickness of 20 microns.
Comparative example
The ordered microstructure nitrocellulose membrane for lateral flow analysis prepared in example 3 was assembled into a lateral flow immunoassay to detect the inflammatory marker C-reactive protein, and compared with the detection results of the conventional lateral flow test strip based on the disordered nitrocellulose membrane. The result shows that the naked eye colorimetric minimum detection concentration is reduced to 1ng/mL from the original 10ng/mL, and the detection sensitivity is obviously improved. In addition, the flow speed of the sample on the ordered membrane is higher, the detection time is reduced from 15min of the traditional membrane to 10min, and the detection time is further saved. Therefore, the lateral flow immunoassay test strip based on the ordered micro-nano structure detection membrane is hopeful to be widely applied to the POCT detection field.
Claims (10)
1. A method for preparing an ordered microstructure nitrocellulose membrane for lateral flow analysis is characterized by comprising the following steps:
step one, 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 a polymer casting solution (1);
step two, preparing a monodisperse colloidal microsphere solution (2);
step three, self-assembling the monodisperse colloidal microsphere solution (2) on the substrate (3) to obtain an ordered template (5);
dripping the polymer casting solution (1) onto the ordered template (5), standing in a closed container with a stable state 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 template (5) to obtain a product (6);
and step six, soaking the product (6) obtained in the step five in a deionized water solution, and then drying to obtain the ordered microstructure nitrocellulose membrane.
2. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of claim 1, wherein: in the first step, the polymer is one or more of nitrocotton, cellulose or nitrocellulose.
3. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of claim 1, wherein: in the first step, the first solvent is one or more of toluene, acetone, chloroform, dimethylformamide or dimethyl sulfoxide.
4. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of claim 1, wherein: in the first step, the second solvent is one or more of ethanol, isopropanol and tetrahydrofuran.
5. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of claim 1, wherein: in the second step, the mass fraction of the monodisperse colloidal microsphere solution (2) is 20-80 wt%.
6. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of claim 1, wherein: in the second step, the monodisperse colloidal microspheres are polystyrene, titanium dioxide, silicon dioxide, polymethyl methacrylate, polyethylacrylate or polyethylene, and the particle size is 200-500 nm.
7. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of 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.
8. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of claim 1, wherein: in the fifth step, etching is performed by hydrofluoric acid with the mass percent of 2-10 wt%, and the calcining temperature is 200-500 ℃.
9. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of claim 1, wherein: in the sixth step, 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%.
10. The method of making an ordered microstructured nitrocellulose membrane for lateral flow analysis of claim 1, wherein: the thickness of the nitrocellulose membrane is 100 nm-30 μm.
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