Preparation method of high-sensitivity and high-linear-range urine albumin test paper

Abstract

The present application relates to a kind of preparation methods of high sensitivity, high linear range urine albumin test paper, comprising the following steps: step 1, preparation albumin reagent solution;Step 2, with the albumin reagent solution obtained in step 1 as raw material, by processing to filter paper, the test paper for detection is obtained.The beneficial effects of the present application are: 1.it realizes the target of simultaneously detecting trace albumin and albumin on the same test paper block;2.it improves production efficiency, reduces production cost, reduces detection time, solves the problems of complex operation, the need for professional equipment and experienced operators in the prior art;3.it overcomes the problems of detection linearity and sensitivity in the prior art, and improves the application range of detection;4.it is not only suitable for rapid screening in medical institutions, but also very convenient for personal daily health monitoring.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic urine analysis technology, specifically to a method for preparing a high-sensitivity, high-linear-range urine albumin test strip. Background Technology

[0002] Protein is one of the most important items in urine chemical analysis. Due to the filtration function of the glomerular filtration membrane and the reabsorption function of the renal tubules, only trace amounts of albumin are excreted in urine under physiological conditions. Urinary albumin <30mg / 24h is considered normal albuminuria. Under certain pathological conditions, albumin excreted by the kidneys will be >30mg / 24h. When 24-hour urinary albumin is 30~300 mg, it is called microalbuminuria (UMA), and when 24-hour urinary albumin >300 mg, it is called macroalbuminuria. UMA is a sensitive indicator for diagnosing early or mild kidney damage and is one of the indicators commonly used to monitor diabetic nephropathy and hypertensive nephropathy. It not only reflects the damage to glomerular and tubular function, but is also an important marker of systemic vascular endothelial damage [2-4]. UMA is also increased in patients with metabolic chronic low-grade inflammatory diseases such as atherosclerosis and obesity, as well as some immune diseases (rheumatoid arthritis), and is also considered an indicator of chronic low-grade inflammation [5]. It is the most important influencing factor for coronary heart disease in patients with type 2 diabetes [6]. All clinical guidelines and consensus recommend that patients with type 1 diabetes and diagnosed type 2 diabetes should routinely undergo screening for diabetic nephropathy every year. Once kidney damage occurs in diabetic patients, their drug metabolism capacity will decrease. Therefore, the treatment plan should be adjusted in time to avoid increasing the burden on the kidneys. Urinary protein is protein in urine. The content in normal urine is low and difficult to monitor. If the content is high, it is easy to monitor. When there is kidney disease, the protein content in urine will increase. Therefore, the detection of urinary protein can predict kidney disease. Therefore, albumin and microalbumin have important clinical value in professional medical institutions and in individuals' daily disease and health monitoring.

[0003] Currently, methods for detecting urinary albumin include dry chemistry, radioimmunoassay, immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and time-resolved fluorescence assay (TRFI). Different methodologies exhibit varying sensitivity and specificity. Dry chemistry and immunoassay are the most commonly used methods for detecting urinary albumin due to their speed and simplicity. The linear range for dry chemistry is 0–5000 mg / L, while the linear range for UMA immunoassay is 5–300 mg / L. Furthermore, discrepancies between the two results for the same sample are frequently observed. Analysis has revealed that when urinary protein concentrations are high, immunoassay exhibits a hook effect, leading to results that do not correlate with clinical findings. Dry chemistry, employing a colorimetric method to detect protein errors, has a detection limit of 100–300 mg / L, but this method is also affected by urine pH. Radioimmunoassay, HPLC, and TRFI are more complex to operate, requiring specialized equipment and experienced operators. Therefore, there is no test kit on the market that can simultaneously detect urinary albumin and urinary microalbumin, and that is simple to use, fast to detect, sensitive and accurate in its results. It can be used by professional medical institutions and is also suitable for individuals to perform daily self-testing.

[0004] To address these issues, researchers have been exploring new detection technologies and methods. One potential solution is the use of a dry chemistry multi-indicator method combined with cellulose derivatives. This method simultaneously detects albumin and microalbumin using multiple indicators. The addition of cellulose derivatives improves the sensitivity and accuracy of the detection (i.e., achieving a detection limit of 10 mg / L and a linear range of 0-20000 mg / L on the same reagent block). Furthermore, the method is simple to operate, provides rapid and easily interpretable results, and is suitable for various applications. However, selecting appropriate indicator combinations, overcoming the susceptibility to interference inherent in dry chemistry, and simplifying the operational process to improve production efficiency remain key research focuses and challenges.

[0005] Several invention patents have been granted regarding solutions to the problem of improving urine protein sensitivity:

[0006] For example, CN106153947A discloses a rapid urine protein detection reagent, its preparation method, and its application. This patent uses sodium polyacrylate and water in a weight ratio of 90-95:5-10 to obtain superabsorbent polymer particles. A protein indicator is dissolved in a sodium dihydrogen phosphate buffer solution at pH 7.4±0.2 to obtain a protein indicator buffer solution. The superabsorbent polymer particles are then mixed with the protein indicator buffer solution, causing the particles to swell into superabsorbent polymer beads with a diameter of 20mm±2mm, thus obtaining the rapid urine protein detection reagent. However, this patent still has issues regarding the selection of the protein indicator and the need for further optimization of the detection method.

[0007] CN103901034B discloses a test strip for detecting trace albumin in urine, comprising a substrate and filter paper. The filter paper is attached to the substrate and is sequentially immersed in solutions A and B. Solution A contains a buffered aqueous solution of sodium citrate, citric acid, and concentrated hydrochloric acid; solution B contains a methanol or ethanol solution of tetrahydrofuran, polypropylene glycol, and sulfophthalein dye. The test strip prepared by this patent allows the sulfophthalein dye to react rapidly with trace albumin in urine under acidic conditions. When the trace albumin concentration is in the range of 0–200 mg / L, the test strip exhibits a distinct color gradation from colorless to deep blue, which is easy to distinguish visually and read by machine. The test results are accurate and unaffected by interference from other ions in the urine, demonstrating stable performance. However, this patent still has shortcomings, including insufficient linear range and sensitivity of the test strip, the inability to simultaneously detect trace albumin and albumin concentration ranges, and the need for further simplification. Summary of the Invention

[0008] In summary, to overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a method for preparing a high-sensitivity, high-linear-range urine albumin test strip, which achieves linear detection of albumin and trace albumin on the same test strip by using a composite colorimetric agent, adding cellulose derivatives and improving the process.

[0009] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing a high-sensitivity, high-linear-range urine albumin test strip, comprising the following steps:

[0010] Step 1: Prepare albumin reagent solution;

[0011] Step 2: Using the albumin reagent solution obtained in Step 1 as raw material, filter paper is processed to obtain test paper for detection.

[0012] Based on the above technical solution, the present invention can be further improved as follows:

[0013] Furthermore, step 1 specifically includes:

[0014] Step 1.1: Dissolve 0.1 g / L-10 g / L of the mixed color developer in one or more organic solvents such as methanol, ethanol, and acetone, and add 1 g / L-10 g / L of nonionic surfactant to form a first mixed solution.

[0015] Step 1.2: Dissolve 0.1 g / L-10 g / L of water-soluble cellulose in a buffer solution containing 0.01 M-0.5 M to form a second mixed solution;

[0016] Step 1.3: Mix the first mixed solution and the second mixed solution according to the set mixing volume ratio of 1:9-9:1 to obtain albumin reagent solution.

[0017] Furthermore, the mixed colorimetric agent in step 1.1 is prepared by mixing an indicator with a wide linear range with a highly sensitive indicator.

[0018] Furthermore, the indicator with a wide linear range is one or a combination of more than one of tetrabromophenol blue, bromophenol blue, and bromocresol green.

[0019] Furthermore, the highly sensitive indicator is one or a combination of two of 5',5''-dinitro-3',3''-diiodo-3,4,5,6-tetrabromophenolsulfonphthalein and malachite green oxalate.

[0020] Further, the surfactant in step 1.1 is one or a combination of one or more of Triton X-100, OP emulsifier, Tween 20, Tween 40, Tween 60, Tween 80 and polyethylene glycol.

[0021] Further, the cellulose-based substance mentioned in step 1.2 is one or a combination of one or more of methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate, carboxymethylcellulose, and hydroxypropylmethylcellulose acetate succinate.

[0022] Furthermore, the buffer solution in step 1.2 is one or a combination of one or more of the following: phosphate buffer, citrate buffer, borate buffer, tartaric acid, malic acid, benzoic acid buffer, and hydrochloric acid-TRIS buffer.

[0023] Furthermore, step 2 specifically includes:

[0024] Step 2.1: Immerse the filter paper in the albumin reagent solution;

[0025] Step 2.2: Drain and rotate the filter paper to distribute it evenly, and then use an electric heating drying oven to dry, cut, and seal the filter paper laid flat on the drying tray to obtain the albumin test strip.

[0026] Furthermore, the filter paper is cotton fiber filter paper or glass fiber filter paper.

[0027] The beneficial effects of this invention are:

[0028] 1. By using tetrabromophenol blue and DIDNTB (5',5''-dinitro-3',3''-diiodo-3,4,5,6-tetrabromophenolsulfonylphthalein) as mixed indicators, the goal of simultaneously detecting trace albumin and albumin on the same test strip was achieved;

[0029] 2. The traditional two-step process has been improved into one step, which significantly simplifies the operation process, improves production efficiency, reduces production costs, and reduces testing time. This solves the problems of complex operation, the need for specialized equipment, and the need to train experienced operators in the existing technology.

[0030] 3. It has a wide detection linear range and high sensitivity, and can accurately determine the urinary protein content in the range of 0-20000 mg / L, with a sensitivity of 10 mg / L. It overcomes the problems of detection linearity and sensitivity in the existing technology and improves the applicability of the detection.

[0031] 4. Simultaneous detection of microalbumin and albumin within 30 seconds is not only suitable for rapid screening in medical institutions, but also very convenient for personal daily health monitoring. It has high value in evaluating the treatment effect, especially for patients with chronic kidney disease and diabetic nephropathy. Attached Figure Description

[0032] Figure 1 This is a curve fitting graph for the urine albumin test. Detailed Implementation

[0033] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0034] A method for preparing a highly sensitive, high-linear-range urine albumin test strip includes the following steps:

[0035] Step 1, Prepare albumin reagent solution:

[0036] Step 1.1: Dissolve 0.1 g / L-10 g / L of the mixed colorimetric reagent in one or more organic solvents such as methanol, ethanol, and acetone, and add 1 g / L-10 g / L of a nonionic surfactant to form a first mixed solution. The mixed colorimetric reagent is a mixture of an indicator with a wide linear range and a highly sensitive indicator. Preferably, the indicator with a wide linear range is one or more of tetrabromophenol blue, bromophenol blue, and bromocresol green; the highly sensitive indicator is one or more of 5',5''-dinitro-3',3''-diiodo-3,4,5,6-tetrabromophenol sulfonyl phthalate and malachite green oxalate; and the surfactant is one or more of Triton X-100, OP emulsifier, Tween 20, Tween 40, Tween 60, Tween 80, and polyethylene glycol.

[0037] Technical solutions employing indicator mixing or novel indicators can improve the linear range and detection limit of the test. Currently, most albumin test strips use indicators such as tetrabromophenol blue, bromophenol blue, and bromocresol green. At pH ≈ 3, these indicators will cause protein-free solutions to turn yellow. For solutions containing protein, the presence of protein causes the buffer dye to impart green or blue color, with a linear range of 0-20000 mg / L. However, its detection sensitivity is low, only detecting albumin concentrations of 100-300 mg / L.

[0038] 5',5''-dinitro-3',3''-diiodo-3,4,5,6-tetrabromophenolsulfonphthalein (DIDNTB), a malachite green oxalate indicator, exhibits high chemosensitivity and specificity to albumin, with a detection limit of approximately 10 mg / L, far superior to traditional dyes such as tetrabromophenol blue (TBPB) and bromophenol blue (BPB). However, its linear detection range is limited to 0-200 mg / L.

[0039] Therefore, this invention employs a mixed indicator, specifically a combination of indicators with a wide linear range (one or more of tetrabromophenol blue, bromophenol blue, bromocresol green, etc.) and highly sensitive indicators (one or more of 5',5''-dinitro-3',3''-diiodo-3,4,5,6-tetrabromophenol sulfonphthalein (DIDNTB), malachite green oxalate-based indicators). This addresses the shortcomings in sensitivity and linear range of traditional dry chemical methods for detecting urinary albumin (currently available products have a linear range of 0-5000 mg / L and a sensitivity of 100-300 mg / L). Using the albumin test strip obtained by this invention, a detection limit of 10 mg / L and a linear range of 0-20000 mg / L can be achieved on the same reagent block.

[0040] Step 1.2: Dissolve 0.1 g / L-10 g / L of water-soluble cellulose in a buffer solution containing 0.01 M-0.5 M to form a second mixed solution. Preferably, the cellulose is one or a combination of methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate, carboxymethylcellulose, and hydroxypropylmethylcellulose acetate succinate; and the buffer solution is one or a combination of phosphate buffer, citrate buffer, borate buffer, tartaric acid, malic acid, benzoic acid buffer, and hydrochloric acid-TRIS buffer.

[0041] Existing methods for detecting urinary microalbumin (such as immunogold labeling, immunofluorescence, and enzyme-linked immunosorbent assays) generally suffer from insufficient linear range, high-dose hook effect, long operation time (generally 5-15 minutes or longer), and low automation, making it difficult to meet the needs of rapid outpatient testing. Currently, the methods used in the market to detect urinary albumin and microalbumin on the same test strip involve either using a dry chemistry method to prepare two separate test strips for each test, or using one dry chemistry method and the other lateral immunochromatography method, with separate sample drops for detection. In personal application, these methods present challenges when microalbumin and urinary albumin results are inconsistent, as individuals lacking professional clinical skills may be unable to interpret the results, leading to psychological stress. Furthermore, it is difficult to simultaneously detect microalbumin and albumin on a single test strip within 30 seconds, limiting the comprehensiveness and accuracy of the detection.

[0042] In aqueous systems, complex weak interactions exist between cellulose and proteins, such as electrostatic interactions, van der Waals forces, hydrogen bonds, and hydrophobic interactions, with electrostatic and hydrophobic interactions being the most important. Hydrophobic interactions occur because the hydrophobic groups of the polymer repel water, leading to a tendency for the polymer to aggregate. This generally occurs between proteins. Cellulose derivatives such as carboxymethyl cellulose (CMC), methyl cellulose (MC), and ethyl cellulose (EC) exhibit enhanced hydrophobicity due to the introduction of methyl and ethyl groups, thereby increasing their binding to proteins. Therefore, when using cotton or glass fiber filter paper as the substrate for urine test strips, the addition of cellulose derivatives increases protein adsorption, thus improving the detection sensitivity of urinary albumin. These mainly include one or more cellulose ether derivatives such as methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, cellulose acetate trimellitate, hydroxypropyl methyl cellulose phthalate, carboxymethyl cellulose, and hydroxypropyl methyl cellulose acetate succinate, used in combination.

[0043] In summary, the albumin test strip obtained by this invention has a wide detection linear range and high sensitivity, accurately determining urinary protein levels from 0 to 20000 mg / L, with a sensitivity of 10 mg / L. This overcomes the problems of detection linearity and sensitivity in existing technologies, and improves the applicability of the detection. Its colorimetric range is shown in Table 1.

[0044] Table 1. Colorimetric properties of albumin test strips in the range of 0-20000 mg / L

[0045]

[0046] As shown in Table 1, the albumin test strip obtained by this invention has high color range resolution, making it very suitable for personal testing.

[0047] Step 1.3: Mix the first mixed solution and the second mixed solution according to the set mixing volume ratio of 1:9-9:1 to obtain albumin reagent solution.

[0048] Step 2: Using the albumin reagent solution obtained in Step 1 as raw material, the filter paper is processed to obtain the test paper for detection. Preferably, the filter paper is cotton fiber filter paper or glass fiber filter paper.

[0049] Step 2.1: Immerse the filter paper in the albumin reagent solution.

[0050] Step 2.2: Drain and rotate the filter paper to distribute the liquid evenly. Then, use an electric heating drying oven to dry the filter paper laid flat on a drying tray, cut it, and seal it for storage to obtain the albumin test strip. Specifically: Hold one end of the filter paper and immerse it in the prepared albumin reagent solution. After holding it in the solution for about 2 seconds, remove it and allow the reagent solution to drain naturally into droplets. Rotate the filter paper 270° to distribute the albumin solution evenly on the filter paper. Then, lay the filter paper flat on the drying tray with the marked side facing up, ensuring it is flat and free of air bubbles. After laying it flat, place it stably in an electric heating drying oven at (65±5)℃ for (20-25) minutes. After drying, cut it into albumin test strips, attach them to a substrate, and cut them into strips to obtain the albumin test strip. Store the strips in a sealed container at room temperature (2℃-30℃).

[0051] Preparation of standard colorimetric cards

[0052] Using the albumin test strip obtained in this invention as a chromogenic carrier, combined with an artificially prepared albumin standard solution, a standard colorimetric card for result comparison is finally obtained through color development, color selection, and verification. Details are as follows:

[0053] Preparation of albumin standard solutions: Using negative urine as the preparation matrix, standard solutions of 10 concentrations were prepared with human serum albumin, with concentrations of 0 mg / L, 10 mg / L, 30 mg / L, 80 mg / L, 150 mg / L, 300 mg / L, 1000 mg / L, 3000 mg / L, 10000 mg / L, and 20000 mg / L, as shown in Table 2.

[0054] Table 2 Human serum albumin standard solution

[0055]

[0056] Test strip color development and standard color selection: Add albumin standard solutions of various concentrations to the albumin test strips prepared in this invention and wait for them to develop color; have 3-5 people visually inspect the color development results and compare them with the standard color chart to select the standard colors corresponding to each concentration.

[0057] Color matching card determination: The selected standard colors are printed, and then repeatedly verified and adjusted to finally determine the standard color matching card.

[0058] Clinical application testing

[0059] Method 1. Visual inspection

[0060] Place the rapid urine albumin detection reagent prepared according to the present invention into the sample to be tested, and observe the test results for 30 seconds using the standard colorimetric card.

[0061] Option 2. Machine testing

[0062] The urine analyzer was used for testing according to the operating procedures, and the results are given directly. This albumin test strip was tested with the Guilin Ulite UC-1800 fully automated urine analyzer, and the results are shown in Table 3:

[0063] Table 3. Reflectance results of albumin test strips on a urine analyzer.

[0064]

[0065] The curves were plotted using the results obtained in Table 3, as follows: Figure 1 As shown. (Through) Figure 1 The following conclusions can be drawn: The rapid urine albumin test strip prepared by this invention has the advantages of high sensitivity, wide linear range, and is simple and easy to use. It can be used for semi-quantitative or quantitative detection with high accuracy. It is suitable for screening urine albumin and meets the requirements of screening experiments for early damage to the kidneys. Currently, the color differentiation provided by commercially available test strips is insufficient to accurately determine the protein content in urine from 0 mg / L to approximately 20,000 mg / L. With the rapid urine albumin test strip prepared by this invention, individuals can easily determine the protein content of their urine sample from the range of 0 mg / L to approximately 20,000 mg / L visually. Therefore, a more accurate understanding of an individual's urine protein content is clinically important. This simple and easy-to-distinguish test strip simultaneously detects trace albumin and albumin within 30 seconds, making it not only suitable for rapid screening in medical institutions but also very convenient for daily personal health monitoring, especially for patients with chronic kidney disease and diabetic nephropathy, where it has high value in evaluating the effectiveness of treatment.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a highly sensitive, high-linear-range urine albumin test strip, characterized in that, The steps include the following: Step 1: Prepare albumin reagent solution; Step 2: Using the albumin reagent solution obtained in Step 1 as raw material, filter paper is processed to obtain test paper for detection.

2. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to claim 1, characterized in that, Step 1 specifically involves: Step 1.1: Dissolve 0.1 g / L-10 g / L of the mixed color developer in one or more organic solvents such as methanol, ethanol, and acetone, and add 1 g / L-10 g / L of nonionic surfactant to form a first mixed solution. Step 1.2: Dissolve 0.1 g / L-10 g / L of water-soluble cellulose in a buffer solution containing 0.01 M-0.5 M to form a second mixed solution; Step 1.3: Mix the first mixed solution and the second mixed solution according to the set mixing volume ratio of 1:9-9:1 to obtain albumin reagent solution.

3. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to claim 2, characterized in that, The mixed colorimetric agent in step 1.1 is prepared by mixing an indicator with a wide linear range and a highly sensitive indicator.

4. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to claim 3, characterized in that, The indicator with a wide linear range is one or a combination of tetrabromophenol blue, bromophenol blue, and bromocresol green.

5. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to claim 3, characterized in that, The highly sensitive indicator is one or a combination of two of 5',5''-dinitro-3',3''-diiodo-3,4,5,6-tetrabromophenolsulfonphthalein and malachite green oxalate.

6. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to claim 2, characterized in that, The surfactant mentioned in step 1.1 is one or more of Triton X-100, OP emulsifier, Tween 20, Tween 40, Tween 60, Tween 80 and polyethylene glycol.

7. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to claim 2, characterized in that, The cellulose-based substance mentioned in step 1.2 is one or a combination of one or more of methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate, carboxymethylcellulose, and hydroxypropylmethylcellulose acetate succinate.

8. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to claim 2, characterized in that, The buffer solution mentioned in step 1.2 is one or a combination of one or more of phosphate buffer, citrate buffer, borate buffer, tartaric acid, malic acid, benzoic acid buffer and hydrochloric acid-TRIS buffer.

9. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to any one of claims 1 to 8, characterized in that, Step 2 specifically involves: Step 2.1: Immerse the filter paper in the albumin reagent solution; Step 2.2: Drain and rotate the filter paper to distribute it evenly, and then use an electric heating drying oven to dry, cut, and seal the filter paper laid flat on the drying tray to obtain the albumin test strip.

10. The method for preparing the high-sensitivity, high-linear-range urine albumin test strip according to any one of claims 1 to 8, characterized in that, The filter paper is cotton fiber filter paper or glass fiber filter paper.

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