Lateral flow detection method based on permeability changes of hydrogels to achieve naked eye direct reading distance quantification

Abstract

The present invention relates to a lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel, and utilizes the enzyme marker bound to the lateral flow test strip detection line to catalyze the hydrogel precursor solution to form a hydrogel, thereby directly reading the distance length of the matrix penetrating the hydrogel, and realizing naked eye direct reading distance quantification. In the process of hydrogel formation, the permeability of the hydrogel is related to the cross-linking degree of the hydrogel. The higher the cross-linking degree, the worse the permeability, that is, the shorter the penetration distance. The cross-linking degree of the hydrogel is significantly correlated with the concentration of the enzyme, and the concentration of the enzyme is in a fixed proportional relationship with the concentration of the target in the sample. Therefore, the naked eye directly reads the distance that the matrix solution penetrates the hydrogel, and the rapid quantitative determination of the target in the sample can be realized. Compared with the prior art, the method of the present invention is not limited to the detection target and the source of the sample, is easy to operate, has accurate results, and can be controlled by the reaction, and has a wide range of application prospects.

Description

Technical Field

[0001] The invention belongs to the field of rapid test strip product development and biological detection technology, and specifically relates to a lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel. Background Art

[0002] In the past few decades, point-of-care testing (POCT) has developed rapidly, providing the possibility of real-time diagnosis in home self-examination, primary care units and emergency situations. As one of the most widely used technologies in POCT, lateral flow testing (LFA) has great application prospects in resource-limited areas. However, LFA is currently mostly used for qualitative determinations with relatively low sensitivity, and less quantitative detection is provided. However, in some application scenarios, only providing qualitative results is not enough to meet the needs of users, such as clinical indicator detection. The quantitative analysis methods that have been implemented are mainly assisted by some instruments, such as digital scanners, cameras, chemiluminescence or electrochemistry. Although portable quantitative instruments have broad prospects for use, they may still have problems such as high cost, cumbersome operation, and high professional skills required of operators for some application scenarios, which is far from meeting the growing demand for point-of-care testing.

[0003] In recent years, new equipment-free detection methods, that is, directly reading LFA quantitative results by naked eyes, have been continuously reported. The LFA rapid naked eye quantitative methods reported in the literature mainly include color quantification, counting quantification, time quantification, text quantification and distance quantification. Among them, color quantification, counting quantification and text quantification are mainly carried out by setting the color development area, and the changes in color hue and / or intensity in the area are used to detect and quantify the target. Although the interpretation is straightforward, this detection method has limitations. On the one hand, there is a strong user subjectivity in distinguishing the reagent hue and tension changes. On the other hand, the test results are mostly semi-quantitative intervals rather than specific values. Although time quantification can obtain specific quantitative numerical results, it requires users to maintain a high degree of concentration during the detection process, and the operation is relatively cumbersome. Distance quantification uses distance length instead of color change for quantitative interpretation, which makes the analysis and measurement largely independent of the operator and greatly reduces the user's subjective error. Although the distance quantification method is easy to operate, it has the problems of low sensitivity and poor precision. Most of the hydrogel-based distance quantification methods reported so far are based on the viscosity change of hydrogels, and the interpretation method of the results is single and the scalability is limited.

[0004] Patent application 202110410562.3 discloses a method for detecting biological molecules and inhibitor molecules, comprising the following steps: mixing the analyte with the hydrogel to form a mixed solution, placing it at one end of the indicative test strip, and causing different amounts of target detection objects to cause different degrees of hydrolysis of the hydrogel. Therefore, the content of water released by the decomposition of the hydrogel is different, which will produce different distances of movement on the pH test strip, and the detection of the target detection object is achieved by the length of the moving distance. The commonly used biomolecule detection methods in the prior art have cumbersome operating steps, require professional personnel to be trained to operate related equipment, have long detection time and low efficiency, resulting in waste of resources and increased costs, and there are also environmental background factors such as matrix that affect the detection sensitivity. However, there are requirements for the type of analyte in this patent, and the type of analyte needs to match the type of hydrogel, which will greatly limit the range of options for the analyte. Summary of the invention

[0005] The purpose of the present invention is to provide a lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel in order to overcome the defects of the above-mentioned prior art, and utilize the enzyme marker combined on the lateral flow test strip detection line to catalyze the process of gel precursor solution forming hydrogel, and the permeability change of hydrogel caused, thereby directly reading the distance length of matrix penetration hydrogel, and realizing naked eye direct reading distance quantification. In the process of hydrogel formation, the permeability of hydrogel is related to the crosslinking degree of hydrogel, and the higher the crosslinking degree, the worse the permeability, that is, the shorter the penetration distance. At the same time, the crosslinking degree of hydrogel is significantly correlated with the concentration of enzyme, and the concentration of enzyme is in a fixed ratio relationship with the concentration of target in sample. Therefore, the distance that naked eye directly reads the matrix solution penetration hydrogel can realize the rapid quantitative determination of target in sample.

[0006] The purpose of the present invention can be achieved by the following technical scheme: a lateral flow detection method based on the permeability change of hydrogel to achieve naked eye direct reading distance quantification, using the enzyme marker bound to the test strip detection line to catalyze the hydrogel precursor solution to form a hydrogel, which triggers the permeability change of the hydrogel, thereby directly reading the distance length of the matrix penetrating the hydrogel, and achieving naked eye direct reading distance quantification. It is not limited to the detection target and sample source, and is used for the quantitative detection of macromolecular targets in biological samples such as whole blood, plasma, serum, urine and saliva.

[0007] Specifically, the method comprises the following steps:

[0008] (1) preparing a gel precursor material: modifying a hydrogel precursor with a phenol derivative to form a gel precursor material; the gel precursor solution prepared subsequently can be rapidly gelled under the catalysis of an enzyme in an environment of 18° C. to 35° C. within 1 to 10 minutes, preferably within 5 minutes at 25° C.;

[0009] (2) Preparing a detection solution: coupling the target recognition molecule 1 and the enzyme with a label carrier to prepare a detection solution; the detection solution contains a co-label of the target antibody 1 and the enzyme, which can be specifically paired with the target recognition molecule 2 to detect the target by a sandwich method. At the same time, the detection solution can also be prepared as needed to contain a solution containing a secondary antibody antigen-label carrier.

[0010] (3) preparing a test strip: the test strip is composed of at least a sample pad, a filter paper / nitrocellulose membrane, a water-absorbing pad and a backing plate, wherein the filter paper / nitrocellulose membrane is coated with a test line and a quality control line; the test line is coated with a target recognition molecule 2, and the quality control line is fixed with a secondary antibody;

[0011] (4) Performing sample testing: using the detection line to capture the target, thereby binding the target antibody 1-enzyme-label carrier complex;

[0012] (5) Complete distance quantification: Add a mixed solution of hydrogel precursor solution and enzyme catalytic substrate onto the detection line of the test strip, then add matrix solution onto the sample pad, and finally read the distance of matrix penetration into the hydrogel with naked eyes, thereby achieving rapid quantitative determination of the target in the sample.

[0013] Preferably, the gel precursor in step (1) includes sodium alginate and chitosan, which respectively have abundant carboxyl and amino groups that can be used for subsequent molecular modification, and the final mass concentration of the gel precursor is 0.2% to 2%;

[0014] Preferably, the phenol derivatives are tyramine and phloric acid, etc.; the mass ratio of the phenol derivatives in the gel precursor is 3% to 30%.

[0015] Preferably, the target recognition molecule 1, target recognition molecule 2 and secondary antibody in step (2) are all antibodies, and the high specificity and high affinity of antibodies can maximize the sensitivity of the lateral flow detection reaction. The sample can be derived from whole blood, plasma, serum, urine or saliva;

[0016] The enzyme can catalyze the generation of free radicals, thereby initiating the cross-linking of hydrogel precursors to form hydrogels. The enzyme includes horseradish peroxidase, catalase or peroxidase-like enzymes, and the enzyme concentration is 10U / mL to 100U / mL; horseradish peroxidase (HRP) is preferred, because of its specific and efficient catalytic performance and economical performance.

[0017] The carriers selected for the target recognition molecule 1-enzyme-label carrier complex and the secondary antibody antigen-label carrier may include one or more of nanomaterials, colored microspheres, quantum dots and fluorescent microspheres.

[0018] Furthermore, the label carriers in the target recognition molecule 1-enzyme-label carrier complex and the secondary antibody antigen-label carrier complex are preferably gold nanospheres. Gold nanospheres have the advantages of large specific surface area, simple synthesis, strong physical adsorption capacity, etc., which ensure the subsequent protein coupling modification.

[0019] In step (3), the sample pad, filter paper / nitrocellulose membrane and absorbent pad are sequentially placed side by side on the backing plate, the absorbent pad overlaps the filter paper / nitrocellulose membrane by 1 mm to 2 mm, and the sample pad overlaps the filter paper / nitrocellulose membrane by 1 mm to 2 mm;

[0020] Preferably, the sample pad is pure white filter paper made of pure plant fiber, with a length of 10 mm to 50 mm and a width of 1 mm to 8 mm.

[0021] Filter paper / nitrocellulose membrane is filter paper or nitrocellulose membrane;

[0022] The absorbent pad is a paper-based material made of natural or synthetic fibers, with a length of 10 mm to 60 mm and a width of 1 mm to 8 mm. Both the length and width can be optimized and adjusted according to the sensitivity and linear range required by the experiment.

[0023] Preferably, the target recognition molecule 2 coated with the test line is antibody 2, the concentration of antibody 2 is 1 mg / mL-3 mg / mL, and the line volume is 0.8 μL / cm;

[0024] Preferably, the secondary antibody fixed to the quality control line is goat anti-mouse antibody, the concentration of the goat anti-mouse antibody is 0.5 mg / mL-2 mg / mL, and the streaking volume is 0.8 μL / cm. The goat anti-mouse antibody can bind to antibody 1.

[0025] The enzyme catalytic substrate described in step (5) is preferably hydrogen peroxide (H2O2), and the final concentration of the enzyme catalytic substrate used in the test strip is 0.1mM to 10mM.

[0026] The matrix solution described in step (5) is preferably a phosphate buffer solution or pure water; the matrix solution is added at a distance of 5 mm to 20 mm from the bottom of the sample pad.

[0027] Preferably, the distance length in step (5) is directly measured using an absorbent pad printed with scale or using a precision ruler.

[0028] Compared with the prior art, the present invention has the following characteristics:

[0029] The present invention proposes a lateral flow detection method based on the permeability change of hydrogel to achieve naked eye direct reading distance quantification. This method is not limited to the detection target and sample source, and the sensitivity and linear range are adjustable. Compared with most of the current qualitative LFA tests, the method of the present invention can obtain accurate quantitative results. Secondly, compared with instrument-assisted quantification, this method uses the naked eye to directly read the quantitative results, which is low cost, easy to operate, and does not require professional personnel. In addition, unlike most of the current quantification that relies on the viscosity change of hydrogel, this method achieves the quantitative purpose based on the permeability change of hydrogel. In summary, the present invention has the advantages of simple operation, low cost and accurate results, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the principle of glycated hemoglobin detection according to Example 1 of the present invention.

[0031] Figure 2 This is a working curve for glycated hemoglobin detection according to Example 1 of the present invention.

[0032] Figure 3 This is a working curve for human serum albumin detection according to Example 2 of the present invention. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0034] Unless otherwise specified, all raw materials used in the present invention are commercially available products.

[0035] Example 1

[0036] The test strips are used for the quantitative detection of glycated hemoglobin in whole blood samples.

[0037] like Figure 1 As shown, this experiment uses the enzyme marker bound to the lateral flow test strip detection line to catalyze the hydrogel precursor solution to form a hydrogel, which triggers the change in hydrogel permeability, and directly reads the distance that the matrix solution penetrates the hydrogel, realizing naked eye direct reading distance quantification. In the process of hydrogel formation, the permeability of the hydrogel is related to the degree of cross-linking of the hydrogel. The higher the degree of cross-linking, the worse the permeability, that is, the shorter the penetration distance. At the same time, the degree of cross-linking of the hydrogel is significantly correlated with the concentration of the enzyme, and the concentration of the enzyme is in a fixed ratio with the proportion of glycated hemoglobin. Therefore, by directly reading the distance that the matrix solution penetrates the hydrogel with the naked eye, the rapid quantitative determination of glycated hemoglobin in the sample can be achieved. The higher the glycation ratio, the shorter the distance.

[0038] The specific steps are as follows:

[0039] (1) Sodium alginate (2.0 wt%) was dissolved in 0.1 M morpholineethanesulfonic acid (MES) buffer (pH 6.0) and stirred at 25°C overnight. Then, 5 mM of EDC hydrochloride and N-hydroxysuccinimide (NHS) were added to the above solution and stirred at 25°C for 4 h. Next, tyramine (10.0 wt%) was added and stirred at 25°C overnight. Excess unreacted tyramine molecules were removed by ultrafiltration and freeze-dried. A pure water solution was added to form a 1.0 wt% gel precursor solution. A gel precursor solution was formed.

[0040] (2) Prepare the detection probe solution (Probes), take a 50mL reactor and add 10mL of the purchased nano-gold ball solution, then add 60μL of potassium carbonate (K2CO3) solution (0.2M), stir for 30min, then add 60μg of glycosylated hemoglobin antibody (hemoglobin A1c, HbA1c) and 60μg of HRP, react at room temperature for 2h, then add 100μL of 5% bovine serum albumin (BSA) solution to block for 60min, then centrifuge the gold-labeled solution at 7000g for 10min at 4℃, then aspirate the supernatant and collect the precipitate. Centrifuge the supernatant solution again at 10000g for 30min at 4℃, aspirate the supernatant, combine and mix the precipitates from the two centrifuges, and store them in a refrigerator at 4℃ for use. The labeling process of rabbit immunoglobulin G (IgG) and nano-gold is the same as above, and then the two gold-labeled complexes are mixed to prepare the detection probe solution of OD8.

[0041] (3) Prepare a test strip, the test strip consists of a sample pad (such as Figure 1 Sample pad, pure white filter paper made of commercially available pure plant fiber), nitrocellulose membrane (commercially available), absorbent pad (such as Figure 1 The absorbent pad (using commercial absorbent paper) and the backing plate (using commercial PVC bottom plate) are composed in sequence. The length of the sample pad, nitrocellulose membrane and absorbent pad are all 4.0mm, and the widths are 2.2cm, 1.4cm and 2.8cm respectively, and they are all pasted on the backing plate. The test line and control line are hemoglobin (Hb Ab) and goat anti-rabbit antibody (GAR Ab), respectively, with a line volume of 0.8μL / cm and a concentration of 2.0mg / mL and 1.0mg / mL respectively.

[0042] (4) Sample testing was performed on hemoglobin samples with different glycated ratios prepared in 10 mM phosphate buffer solution. HbA1c Ab fully bound to HbA1c on the T line and did not bind to non-target hemoglobin (Nt Hb). A working curve was drawn based on the gray value of the T line and the glycated hemoglobin ratio, and then human whole blood samples were tested.

[0043] (5) To complete the distance quantification, first cut the test strip obtained in step (4) from the middle of the test line and the quality control line, attach a new 1.5mm x 50mm new absorbent pad to the upper end of the test line, then drip 5.0μL of a mixed solution of 0.5% gel precursor solution (prepared in step 1) and 0.5μL of 1mM H2O2 (enzyme catalytic substrate) on the test line, then add 70.0μL of 10mM phosphate buffer solution matrix (DPBS) at 10mm from the bottom of the sample pad, and finally use a ruler with a scale to measure the length of the matrix lateral flow. Figure 2 As shown, the lateral flow distance and the glycosylated hemoglobin ratio showed a good linear relationship.

[0044] Example 2

[0045] The test strips are used for the quantitative detection of human serum albumin in urine samples.

[0046] The specific steps are as follows:

[0047] (1) Chitosan (1.5 wt%) was dissolved in 0.1 M morpholineethanesulfonic acid (MES)-buffer (pH 6.0) and stirred at 25°C overnight. Then, 10 mM of EDC hydrochloride and N-hydroxysuccinimide (NHS) were added to the above solution and stirred at 25°C for 1 h. Next, phloric acid (5.0 wt%) was added and stirred at 25°C overnight. Excess unreacted phloric acid molecules were removed by ultrafiltration and freeze-dried. A pure aqueous solution was added to form a 1.5 wt% gel precursor solution.

[0048] (2) Prepare the binding pad. Take a 50mL reactor and add 10mL of the purchased nano-gold ball solution, then add 60μL of K2CO3 solution (0.2M), stir for 30min, then add 80μg of albumin antibody 1 (HSA Ab) and 80μg of HRP, react at room temperature for 2h, then add 100μL of 5% BSA solution to block for 60min, then centrifuge the gold-labeled solution at 7000g for 10min at 4℃, then aspirate the supernatant and collect the precipitate. Centrifuge the supernatant solution at 10000g for 30min at 4℃, aspirate the supernatant, combine and mix the precipitates from the two centrifugations, and store them in a refrigerator at 4℃ for later use. The labeling process of rabbit immunoglobulin G (IgG) and nano-gold is the same as above, and then the two gold-labeled complexes are mixed to prepare the OD5 detection probe solution.

[0049] (3) Prepare a test strip, the test strip consists of a sample pad (such as Figure 1 Sample pad, pure white filter paper made of commercially available pure plant fiber), nitrocellulose membrane (commercially available), absorbent pad (such as Figure 1 The absorbent pad (using commercial absorbent paper) and the backing plate (using commercial PVC bottom plate) are composed in sequence. The length of the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are all 3.8mm, and the widths are 1.9cm, 0.5cm, 2.5cm and 1.7cm respectively, and they are all pasted on the backing plate. The nitrocellulose membrane is coated with a detection line and a quality control line. The detection line and the quality control line are coated with albumin antibody 2 and goat anti-rabbit antibody, respectively, with a streaking volume of 0.8μL / cm and a concentration of 1.5mg / mL and 1.0mg / mL respectively.

[0050] (4) Performing sample testing: preparing albumin samples of different concentrations using 10 mM phosphate buffer solution, drawing a working curve, and then performing human urine sample testing.

[0051] (5) To complete the distance quantification, first drop 5.0 μL of a mixed solution of 1.0% gel precursor solution (prepared in step 1) and 0.5 μL of 5 mM H2O2 (enzyme catalysis substrate) on the test line, replace a new 4 mm x 40 mm absorbent pad with a scale, then add 80.0 μL of pure water matrix 10 mm from the bottom of the sample pad, and finally directly read the lateral flow length of pure water on the absorbent pad with a scale. Figure 3 As shown, the lateral flow distance showed a good linear relationship with the albumin concentration.

[0052] The method of the present invention is not limited to the detection target and the source of the sample. The key process conditions in the experiment are optimized, including the width of the absorbent pad, the labeling amount of the antibody and the enzyme, and the concentration of the precursor substance, so as to achieve the regulation of sensitivity and linear range. For example, by adjusting the width of the absorbent pad in Example 1 from 4 mm to 2 mm, the linear range can be widened from the original 6%-12% to 3%-15%, and the detection precision can be controlled below 5%. The method of the present invention can obtain accurate quantitative results, has the advantages of simple operation, low cost and accurate results, and has great application potential.

[0053] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A lateral flow detection method for quantitative naked eye direct reading distance based on the permeability change of hydrogel, characterized in that: By using the enzyme marker bound to the test strip detection line to catalyze the change in hydrogel permeability caused by the formation of hydrogel from the gel precursor solution, the distance the matrix penetrates the hydrogel can be directly read, realizing the direct reading of distance quantification with the naked eye.

2. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 1, characterized in that: The method specifically comprises the following steps: (1) Preparing a gel precursor: modifying a hydrogel precursor with a phenol derivative to form a gel precursor; (2) Preparing a detection solution: The detection solution contains at least a target recognition molecule 1-enzyme-labeling carrier complex; (3) Preparing a test strip: The test strip is composed of at least a sample pad, filter paper or nitrocellulose membrane, an absorbent pad and a backing plate, wherein the filter paper or nitrocellulose membrane is coated with a test line and a quality control line, and the test line is coated with a target recognition molecule 2; (4) Performing sample detection: using the detection line to capture the target, thereby combining the target recognition molecule 1-enzyme-label carrier complex; (5) Complete distance quantification: Add a mixed solution of gel precursor solution and enzyme-catalyzed substrate onto the detection line of the test strip, then add matrix solution to the sample pad, and finally read the distance the matrix penetrates the hydrogel with the naked eye to achieve rapid quantitative determination of the target in the sample.

3. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 2, characterized in that: The material source of the hydrogel precursor in step (1) includes sodium alginate or chitosan; the final mass concentration of the hydrogel precursor is 0.2% to 2%; The mass ratio of phenol derivatives in the gel precursor is between 3% and 30%.

4. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 2, characterized in that: The detection solution in step (2) is also prepared to contain a secondary antibody antigen-labeled carrier complex.

5. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 2 or 4, characterized in that: In step (2), the target recognition molecule 1 and the target recognition molecule 2 are antibodies; the sample is derived from whole blood, plasma, serum, urine or saliva; The enzyme includes horseradish peroxidase, catalase or peroxidase-like enzyme, and the enzyme concentration is 10 U / mL ~ 100 U / mL; The quality control line is fixed with a secondary antibody, which can bind to the target recognition molecule 1 or the secondary antibody antigen.

6. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 2, characterized in that: In step (3), the sample pad, filter paper or nitrocellulose membrane and absorbent pad are sequentially placed side by side on the backing plate, the absorbent pad overlaps the filter paper or nitrocellulose membrane by 1 mm to 2 mm, and the sample pad overlaps the filter paper or nitrocellulose membrane by 1 mm to 2 mm; The absorbent pad is a paper-based material made of natural or synthetic fibers, with a length of 10 mm to 60 mm and a width of 1 mm to 8 mm; The detection line is fixed with the target recognition molecule 2, with a concentration of 1 mg / mL -3 mg / mL and a streaking volume of 0.8 μL / cm; The quality control line was fixed with secondary antibody at a concentration of 0.5 mg / mL -2 mg / mL and a streaking volume of 0.8 μL / cm.

7. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 2, characterized in that: The final concentration of the enzyme-catalyzed substrate applied to the test strip in step (5) is between 0.1 mM and 10 mM.

8. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 2, characterized in that: The matrix solution described in step (5) is added 5 mm to 20 mm from the bottom of the sample pad.

9. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 2, characterized in that: When the temperature is between 18°C ​​and 35°C, the hydrogel precursor solution in step (5) rapidly gels within 1 min to 10 min.

10. The lateral flow detection method for realizing naked eye direct reading distance quantification based on the permeability change of hydrogel according to claim 2, characterized in that:Step (5) The distance length is measured directly using an absorbent pad with a printed scale or using a precision ruler.

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