Microfluidic enhanced colloidal gold immunodetection test strip and preparation method thereof

Abstract

The invention provides a microfluidic enhanced colloidal gold immunodetection test strip and a preparation method thereof, and belongs to the technical field of detection test strips. Comprising a sample pad, a colloidal gold pad, a reaction film and a water absorption pad which are sequentially overlapped and pasted on a back plate, and the reaction film is coated with a quality control line and a detection line; the sample pad is prepared from the following raw materials: a sample matrix plate and enhancement treatment liquid. According to the invention, bovine serum albumin and casein with a reasonable ratio are added into a modified treatment liquid, so that bovine serum albumin can be used as an enzyme inhibitor or regulator, and casein can provide necessary amino acids, stabilize target ions in a sample to a certain extent, effectively close non-specific binding sites on the surface of the sample pad, and improve the detection sensitivity of the sample pad. Meanwhile, the phosphate buffer solution provides a suitable acid-base environment for detection reaction, enzyme activity loss caused by pH fluctuation is prevented, interference of external factors on a detection result is reduced, and the accuracy of the detection result is ensured.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of test paper strips, and particularly relates to a microfluidic enhanced colloidal gold immunological test paper strip and a preparation method thereof. BACKGROUND

[0002] With the development of society, the detection task faced by inspection and quarantine, food safety and virus detection is getting heavier and heavier. The traditional detection method relying on instruments has the advantages of high detection sensitivity and good detection result stability. However, due to the high price of large instruments, the necessity of relevant professional knowledge of the operator, and the high detection cost, the detection method relying on large instruments cannot be widely used in various primary and on-site detection. The colloidal gold test paper strip detection technology is widely used because it does not need other complex instruments in the detection process and can determine the detection result of the test paper strip by naked eye observation.

[0003] In the structure of the colloidal gold immunological test paper strip, the sample pad is the starting area of sample drop and the first part in contact with the sample in the test paper strip detection system. The sample pad is not only easy to be interfered, but also the uneven sample delivery or the too fast or too slow speed can lead to insufficient or abnormal antigen and antibody reaction on the detection line, thereby affecting the accuracy of the detection result. SUMMARY

[0004] (1) Technical problem to be solved

[0005] In view of the defects of the prior art, the purpose of the present application is to provide a microfluidic enhanced colloidal gold immunological test paper strip and a preparation method thereof, so as to solve the problem that the sample pad of the existing colloidal gold immunological test paper strip is easy to be interfered, and the uneven sample delivery or the too fast or too slow speed can lead to insufficient or abnormal antigen and antibody reaction on the detection line, thereby affecting the accuracy of the detection result.

[0006] (2) Technical scheme

[0007] In order to solve the above technical problems, the present application provides a microfluidic enhanced colloidal gold immunological test paper strip, which comprises a sample pad, a colloidal gold pad, a reaction membrane and a water absorption pad which are sequentially and adhesively connected to a back plate, and the reaction membrane is coated with a quality control line and a detection line.

[0008] The preparation raw material of the sample pad comprises a sample matrix plate and an enhanced treatment liquid, and the matrix plate is selected from polyester fibers, and the polyester fibers are treated by the enhanced treatment liquid.

[0009] Preferably, a preparation method of a microfluidic enhanced colloidal gold immunological test paper strip comprises the following steps:

[0010] Step one, dip the modified treatment liquid on the sample pad, then dry the sample pad in the drying room or oven using 35-40℃;

[0011] Step two, spray the labeled gold complex on the colloidal gold pad, and dry the colloidal gold pad in the drying room or oven;

[0012] Step three, lay the sample pad, colloidal gold pad, reaction film and water absorption pad on the back plate in turn, then coat the quality control line and detection line to make the colloidal gold immune test paper;

[0013] Step four, cut the colloidal gold immune test paper into the test paper strip with uniform width and no indentation according to the width of the card slot of the test card.

[0014] Preferably, the modified treatment liquid in step one comprises the following raw materials: phosphate buffer, hydrophilic surfactant, sodium azide, polyacrylamide, complex protein and alginic acid;

[0015] The specific formula comprises the following raw materials (by mass fraction): 800-1000 parts of phosphate buffer, 3-5 parts of hydrophilic surfactant, 2-5 parts of sodium azide, 1-2 parts of polyacrylamide, 8-12 parts of complex protein and 5-8 parts of sodium alginate.

[0016] Preferably, the phosphate buffer is 900 parts, the hydrophilic surfactant is 4 parts, the sodium azide is 3 parts, the polyacrylamide is 1.5 parts, the complex protein is 10 parts and the sodium alginate is 6 parts.

[0017] Preferably, the pH value of the phosphate buffer is 7.2-8.0, and the concentration of the phosphate buffer is 30-180mM.

[0018] Preferably, the hydrophilic surfactant is a copolymer of polyoxyethylene and polyoxypropylene, and the HLB value of the hydrophilic surfactant is 15-18.

[0019] Preferably, the complex protein comprises bovine serum albumin and casein, and the mass ratio of the bovine serum albumin and casein is (1-2):(3-5).

[0020] Preferably, step one is specifically: completely immerse the sample pad in the modified treatment liquid, soak for 5-15 minutes, then take out and dehydrate for 3-5 minutes, then place on the screen and put into the oven at 37℃ for drying, and finally store the dried sample pad in the aluminum foil bag.

[0021] Preferably, the back plate is a polyvinyl chloride plate, and the colloidal gold pad is a glass fiber membrane.

[0022] Preferably, the length of the overlapping part between the sample pad, the colloidal gold pad, the reaction membrane and the water absorption pad is 2.5-3.5 mm.

[0023] (3) Beneficial effects

[0024] Compared with the prior art, the beneficial effects of the present application are that:

[0025] In the above scheme, by adding reasonable proportions of bovine serum albumin and casein in the modification treatment solution, the bovine serum albumin can be used as an inhibitor or regulator of the enzyme, and the casein can provide essential amino acids, to a certain extent, stabilize the target ions in the sample, effectively block the non-specific binding sites on the surface of the sample pad, prevent the non-specific adsorption of impurities, at the same time, the phosphate buffer provides a suitable acid-base environment for the detection reaction, prevents the loss of enzyme activity due to pH fluctuation, reduces the interference of external factors on the detection results, and ensures the accuracy of the detection results.

[0026] In the above scheme, the sodium alginate and polyacrylamide in the modification treatment solution can form an interpenetrating network structure, which can improve the drug loading capacity and sustained-release effect, and sodium azide not only effectively inhibits the growth and reproduction of microorganisms, avoids the contamination of microorganisms during storage and use, but also acts as a crosslinking agent to promote the formation of the network structure, thereby controlling the flow speed and diffusion range of the sample on the sample pad, making it uniformly react with the subsequent reagent, avoiding the detection errors caused by too fast or too slow sample flow, and optimizing the accuracy of the detection results. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Structure diagram of the microfluidic enhanced colloidal gold immunochromatographic test strip.

[0028] The labels in the drawings are: 1, back plate; 2, sample pad; 3, colloidal gold pad; 4, reaction membrane; 5, water absorption pad; 6, quality control line; 7, detection line. DETAILED DESCRIPTION

[0029] The present application of the embodiment provides a microfluidic enhanced colloidal gold immunochromatographic test strip, which comprises a sample pad 2, a colloidal gold pad 3, a reaction membrane 4 and a water absorption pad 5 sequentially and lap-jointingly pasted on a back plate 1, and the reaction membrane 4 is coated with a quality control line 6 and a detection line 7.

[0030] The raw materials for preparing the sample pad 2 include a sample matrix plate and an enhanced treatment solution, and the matrix plate is selected from polyester fibers, which are treated by the enhanced treatment solution.

[0031] The preparation method of the microfluidic enhanced colloidal gold immunochromatographic test strip of the embodiment comprises the following steps:

[0032] Step one, dip the modified treatment liquid on the sample pad 2, then dry the sample pad 2 in the drying room or oven using 35-40℃;

[0033] Step two, spray the labeled gold complex on the colloidal gold pad 3, dry the colloidal gold pad 3 in the drying room or oven;

[0034] Step three, lay the sample pad 2, colloidal gold pad 3, reaction film 4 and water absorption pad 5 on the back plate 1 in turn, then coat the quality control line 6 and detection line 7 to make the colloidal gold immune test paper;

[0035] Step four, cut the colloidal gold immune test paper into the test paper strip with uniform width and no indentation according to the width of the card slot of the test card.

[0036] The modified treatment liquid in step one of the embodiment includes the following raw materials: phosphate buffer, hydrophilic surfactant, sodium azide, polyacrylamide, complex protein and alginic acid;

[0037] The specific formula includes the following raw materials in mass fraction: phosphate buffer 800-1000 parts, hydrophilic surfactant 3-5 parts, sodium azide 2-5 parts, polyacrylamide 1-2 parts, complex protein 8-12 parts and sodium alginate 5-8 parts.

[0038] The phosphate buffer of the embodiment is 900 parts, the hydrophilic surfactant is 4 parts, the sodium azide is 3 parts, the polyacrylamide is 1.5 parts, the complex protein is 10 parts and the sodium alginate is 6 parts.

[0039] The pH value of the phosphate buffer of the embodiment is 7.2-8.0, and the concentration of the phosphate buffer is 30-180mM.

[0040] The hydrophilic surfactant of the embodiment is a copolymer of polyoxyethylene and polyoxypropylene, and the HLB value of the hydrophilic surfactant is 15-18.

[0041] The complex protein of the embodiment includes bovine serum albumin and casein, and the mass ratio of bovine serum albumin to casein is (1-2):(3-5).

[0042] The specific step one of the embodiment is: completely immerse the sample pad 2 in the modified treatment liquid, the immersion time is 5-15 minutes, then take out and dehydrate for 3-5 minutes, then place on the screen and put into the oven at 37℃ for drying, finally, pack the dried sample pad 2 in the aluminum foil bag.

[0043] The back plate 1 of the embodiment is a polyvinyl chloride plate, and the colloidal gold pad 3 is a glass fiber membrane.

[0044] The length of the overlapping part between the sample pad 2, the colloidal gold pad 3, the reaction membrane 4 and the water absorption pad 5 of the sample pad 2, the colloidal gold pad 3, the reaction membrane 4 and the water absorption pad 5 of the present embodiment is 2.5-3.5mm.

[0045] Embodiment 1.

[0046] The present embodiment provides a microfluidic enhanced colloidal gold immunoassay test strip, which comprises a sample pad 2, a colloidal gold pad 3, a reaction membrane 4 and a water absorption pad 5 sequentially and adhesively connected to a back plate 1, and the reaction membrane 4 is coated with a quality control line 6 and a detection line 7.

[0047] The preparation raw materials of the sample pad 2 include a sample matrix plate and an enhanced treatment liquid, and the matrix plate is selected from polyester fibers which are treated by the enhanced treatment liquid.

[0048] The preparation method of the microfluidic enhanced colloidal gold immunoassay test strip of the present embodiment comprises the following steps:

[0049] Step one, the modified treatment liquid is coated on the sample pad 2, and then the sample pad 2 is dried in a drying room or an oven;

[0050] Step two, the labeled gold complex is sprayed on the colloidal gold pad 3, and then the colloidal gold pad 3 is dried in a drying room or an oven;

[0051] Step three, the sample pad 2, the colloidal gold pad 3, the reaction membrane 4 and the water absorption pad 5 are sequentially and adhesively connected to the back plate 1, and then the quality control line 6 and the detection line 7 are coated to form the colloidal gold immunoassay test strip;

[0052] Step four, the colloidal gold immunoassay test strip is cut into colloidal gold immunoassay test strips with uniform width and no indentation according to the width of the card slot of the detection card.

[0053] The modified treatment liquid in step one of the present embodiment comprises the following raw materials: phosphate buffer, hydrophilic surfactant, sodium azide, polyacrylamide, complex protein and alginic acid;

[0054] The specific formula comprises the following raw materials in mass fraction: 800 parts of phosphate buffer, 3 parts of hydrophilic surfactant, 2 parts of sodium azide, 1 part of polyacrylamide, 8 parts of complex protein and 5 parts of sodium alginate.

[0055] The pH value of the phosphate buffer of the present embodiment is 7.2, and the concentration of the phosphate buffer is 30mM.

[0056] The hydrophilic surfactant of the present embodiment is a copolymer of polyoxyethylene and polyoxypropylene, and the HLB value of the hydrophilic surfactant is 15.

[0057] The complex protein of the present embodiment includes bovine serum albumin and casein, and the mass ratio of bovine serum albumin to casein is 1:3.

[0058] The step one of the embodiment is specifically: the sample pad 2 is completely soaked in the modified treatment liquid, the soaking time is 5 minutes, then it is taken out and dehydrated for 3 minutes, then it is placed on the screen, put into the oven at 37℃ for drying, finally the dried sample pad 2 is packed in the aluminum foil bag for storage.

[0059] The backboard 1 of the embodiment is a polyvinyl chloride plate, and the colloidal gold pad 3 is a glass fiber membrane.

[0060] The length of the overlapping part between the sample pad 2, the colloidal gold pad 3, the reaction membrane 4 and the water absorption pad 5 of the embodiment is 2.5 mm.

[0061] Embodiment 2.

[0062] The embodiment provides a microfluidic enhanced colloidal gold immunochromatographic test strip, which comprises a sample pad 2, a colloidal gold pad 3, a reaction membrane 4 and a water absorption pad 5 which are sequentially and adhesively connected to a backboard 1, and the reaction membrane 4 is coated with a quality control line 6 and a detection line 7.

[0063] The preparation raw material of the sample pad 2 comprises a sample matrix plate and an enhanced treatment liquid, and the matrix plate is selected from polyester fibers which are treated by the enhanced treatment liquid.

[0064] The preparation method of the microfluidic enhanced colloidal gold immunochromatographic test strip of the embodiment comprises the following steps:

[0065] Step one, the modified treatment liquid is coated on the sample pad 2, and then the sample pad 2 is dried in a drying room or an oven;

[0066] Step two, the labeled gold complex is sprayed on the colloidal gold pad 3, and then the colloidal gold pad 3 is dried in a drying room or an oven;

[0067] Step three, the sample pad 2, the colloidal gold pad 3, the reaction membrane 4 and the water absorption pad 5 are sequentially and adhesively connected to the backboard 1, and then the quality control line 6 and the detection line 7 are coated to form the colloidal gold immunochromatographic test strip;

[0068] Step four, the colloidal gold immunochromatographic test strip is cut into the colloidal gold immunochromatographic test strips with uniform width and no indentation according to the width of the card slot of the detection card.

[0069] The modified treatment liquid in step one of the embodiment comprises the following raw materials: phosphate buffer, hydrophilic surfactant, sodium azide, polyacrylamide, complex protein and alginic acid;

[0070] The specific formula comprises the following raw materials in parts by mass: 900 parts of phosphate buffer, 4 parts of hydrophilic surfactant, 3 parts of sodium azide, 1.5 parts of polyacrylamide, 10 parts of complex protein and 6 parts of sodium alginate.

[0071] The pH value of the phosphate buffer solution of the embodiment is 7.6, and the concentration of the phosphate buffer solution is 80 mM.

[0072] The hydrophilic surfactant of the embodiment is a copolymer of polyoxyethylene and polyoxypropylene, and the HLB value of the hydrophilic surfactant is 17.

[0073] The composite protein of the embodiment includes bovine serum albumin and casein, and the mass ratio of the bovine serum albumin and the casein is 1.5:4.

[0074] In step one of the embodiment, the sample pad 2 is completely soaked in the modified treatment solution for 7 minutes, then taken out and dehydrated for 4 minutes, then placed on a screen and dried in an oven at 37°C, and finally the dried sample pad 2 is stored in an aluminum foil bag.

[0075] The backboard 1 of the embodiment is a polyvinyl chloride plate, and the colloidal gold pad 3 is a glass fiber membrane.

[0076] The length of the overlapping part between the sample pad 2, the colloidal gold pad 3, the reaction membrane 4 and the water absorption pad 5 of the embodiment is 3 mm.

[0077] Embodiment 3.

[0078] The embodiment of the present application provides a microfluidic enhanced colloidal gold immunochromatographic test strip, which comprises a sample pad 2, a colloidal gold pad 3, a reaction membrane 4 and a water absorption pad 5 sequentially and adhesively connected on a backboard 1, and the reaction membrane 4 is coated with a quality control line 6 and a detection line 7.

[0079] The raw materials for preparing the sample pad 2 include a sample matrix plate and an enhanced treatment solution, and the matrix plate is selected from polyester fibers treated by the enhanced treatment solution.

[0080] The preparation method of the microfluidic enhanced colloidal gold immunochromatographic test strip of the embodiment comprises the following steps:

[0081] Step one, the modified treatment solution is coated on the sample pad 2, and then the sample pad 2 is dried in a drying room or an oven;

[0082] Step two, the labeled gold complex is sprayed on the colloidal gold pad 3, and then the colloidal gold pad 3 is dried in a drying room or an oven;

[0083] Step three, the sample pad 2, the colloidal gold pad 3, the reaction membrane 4 and the water absorption pad 5 are sequentially and adhesively connected on the backboard 1, and then the quality control line 6 and the detection line 7 are coated to form a colloidal gold immunochromatographic test paper;

[0084] Step four, the colloidal gold immunochromatographic test paper is cut into colloidal gold immunochromatographic test strips with uniform width and no indentation according to the width of the card slot of the detection card.

[0085] The modified treatment solution in step one of the embodiment comprises the following raw materials: phosphate buffer, hydrophilic surfactant, sodium azide, polyacrylamide, complex protein and alginic acid;

[0086] The specific formula of the embodiment comprises the following raw materials in mass fraction: 1000 parts of phosphate buffer, 5 parts of hydrophilic surfactant, 5 parts of sodium azide, 2 parts of polyacrylamide, 12 parts of complex protein and 8 parts of sodium alginate.

[0087] The pH value of the phosphate buffer of the embodiment is 8.0, and the concentration of the phosphate buffer is 180 mM.

[0088] The hydrophilic surfactant of the embodiment is a copolymer of polyoxyethylene and polyoxypropylene, and the HLB value of the hydrophilic surfactant is 18.

[0089] The complex protein of the embodiment comprises bovine serum albumin and casein, and the mass ratio of bovine serum albumin to casein is 2:5.

[0090] In step one of the embodiment, the sample pad 2 is completely soaked in the modified treatment solution for 15 minutes, then taken out and dehydrated for 5 minutes, then placed on a screen and put into a 37℃ oven for drying, and finally the dried sample pad 2 is packed in an aluminum foil bag for storage.

[0091] The back plate 1 of the embodiment is a polyvinyl chloride plate, and the colloidal gold pad 3 is a glass fiber membrane.

[0092] The length of the overlapping part between the sample pad 2, the colloidal gold pad 3, the reaction membrane 4 and the water absorption pad 5 of the embodiment is 3.5 mm.

[0093] Comparative Example

[0094] The difference between the embodiment 1-3 and the comparative example is that the sample pad of the immunodetection test strip is treated with a common treatment solution.

[0095] The products of the embodiment 1-3 and the comparative example are tested for performance;

[0096] 200 immunodetection test strips are made, and 200 samples confirmed as negative for influenza virus by a laboratory confirmed method are detected by nasal swab and throat swab, and the detection results are as follows.

[0097] Number of nasal swab samples Number of throat swab samples Negative concordance rate Example One 20 30 100% Example Two 25 25 100% Example Three 30 20 100% Comparative Example 20 30 96%

[0098] From the examples 1-3 and the comparative examples, it can be seen that by adding the reasonable proportion of bovine serum albumin and casein in the modification treatment liquid, the colloidal gold immunochromatographic test strip and the preparation method thereof can make the bovine serum albumin as the inhibitor or regulator of the enzyme, the casein can provide the necessary amino acids, to a certain extent, stabilize the target ions in the sample, effectively block the non-specific binding sites on the surface of the sample pad, prevent the non-specific adsorption of impurities, at the same time, the phosphate buffer provides a suitable acid-base environment for the detection reaction, prevents the loss of enzyme activity caused by pH fluctuation, reduces the interference of external factors on the detection result, and ensures the accuracy of the detection result.

[0099] All the technical features in the embodiment can be freely combined according to actual needs.

[0100] The above examples are the preferred implementation of the present application, in addition to this, the present application can be realized in other ways, without departing from the technical solution concept, any obvious replacement is within the protection scope of the present application.

Claims

1. A microfluidic-enhanced colloidal gold immunochromatographic test strip, characterized in that, The test paper comprises a sample pad (2), a colloidal gold pad (3), a reaction membrane (4) and a water absorption pad (5) which are sequentially pasted on a back plate (1), wherein the reaction membrane (4) is coated with a quality control line (6) and a detection line (7). The sample pad (2) is prepared from a sample matrix plate and a reinforcing treatment liquid, and the matrix plate is selected from polyester fibers which are treated by the reinforcing treatment liquid.

2. The preparation method of the microfluidic enhanced colloidal gold immunoassay test strip according to claim 1, characterized in that, The method comprises the following steps: Step one: the modified treatment liquid is coated on the sample pad (2), and then the sample pad (2) is dried in a drying room or an oven at 35-40℃; Step two: the labeled gold complex is sprayed on the colloidal gold pad (3), and then the colloidal gold pad (3) is dried in a drying room or an oven; Step three: the sample pad (2), the colloidal gold pad (3), the reaction membrane (4) and the water absorption pad (5) are sequentially pasted on the back plate (1), and then the quality control line (6) and the detection line (7) are coated to prepare the colloidal gold immune test paper; Step four: the colloidal gold immune test paper is cut into test paper strips with uniform width and no indentation according to the width of the card slot of the test card.

3. The method for preparing the microfluidic enhanced colloidal gold immune test paper strip according to claim 2, wherein the modified treatment liquid in step one comprises the following raw materials: phosphate buffer, hydrophilic surfactant, sodium azide, polyacrylamide, complex protein and alginic acid. The specific formula comprises the following raw materials (by mass fraction): 800-1000 parts of phosphate buffer, 3-5 parts of hydrophilic surfactant, 2-5 parts of sodium azide, 1-2 parts of polyacrylamide, 8-12 parts of complex protein and 5-8 parts of sodium alginate.

4. The method for preparing the microfluidic-enhanced colloidal gold immunoassay strip according to claim 3, characterized in that, The phosphate buffer is 900 parts, the hydrophilic surfactant is 4 parts, the sodium azide is 3 parts, the polyacrylamide is 1.5 parts, the complex protein is 10 parts and the sodium alginate is 6 parts.

5. The method for preparing the microfluidic-enhanced colloidal gold immunoassay strip according to claim 4, characterized in that, The pH value of the phosphate buffer is 7.2-8.0, and the concentration of the phosphate buffer is 30-180 mM. 6.The method for preparing a microfluidic-enhanced colloidal gold immunochromatographic test strip according to claim 5, characterized in that, The hydrophilic surfactant is a copolymer of polyoxyethylene and polyoxypropylene, and the HLB value of the hydrophilic surfactant is 15-18.

7. The method for preparing the microfluidic-enhanced colloidal gold immunoassay strip according to claim 6, characterized in that, The complex protein comprises bovine serum albumin and casein, and the mass ratio of the bovine serum albumin to the casein is (1-2):(3-5). 8.The method for preparing a microfluidic-enhanced colloidal gold immunochromatographic test strip according to claim 7, characterized in that, In step one, the sample pad (2) is completely soaked in the modified treatment liquid for 5-15 minutes, then taken out and dehydrated for 3-5 minutes, then placed on a screen and dried in a 37℃ oven, and finally the dried sample pad (2) is stored in an aluminum foil bag.

9. The method for preparing the microfluidic-enhanced colloidal gold immunoassay strip according to claim 8, characterized in that, The back plate (1) is a polyvinyl chloride plate, and the colloidal gold pad (3) is a glass fiber membrane. 10.The method for preparing a microfluidic-enhanced colloidal gold immunochromatographic test strip according to claim 9, characterized in that, The length of the overlapping part between the sample pad (2), the colloidal gold pad (3), the reaction membrane (4) and the water absorption pad (5) is 2.5-3.5 mm.

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