A dry-chemical test paper for detecting uroporphyrin in urine and a preparation method and kit thereof

Abstract

The application provides a dry-chemical test paper for detecting uroporphyrin in urine and a preparation method and a kit thereof, and belongs to the technical field of in-vitro detection. The preparation method of the dry-chemical test paper for detecting uroporphyrin in urine comprises the following steps: soaking a base material in a color developing mother liquor, and drying the base material after soaking. The color developing mother liquor comprises the following components: p-dimethylaminobenzaldehyde, 5-sulfosalicylic acid, barium chloride, a non-ionic surfactant and a chelating agent. The kit provided by the application is convenient to carry, can exclude the interference of urobilin and bilirubin, and can rapidly detect uroporphyrin in urine.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology, specifically to a dry chemical test strip for detecting porphyrin in urine, its preparation method, and a reagent kit. Background Technology

[0002] Heme is an important component of hemoglobin. The synthesis of heme in the body requires multiple steps, and porphyrins are a collective term for the intermediate products in these steps. When enzymes in these reactions malfunction, the intermediate product porphyrin cannot be metabolized, thus accumulating in the body and causing toxicity to tissues and organs. Porphyria is a hereditary or acquired metabolic disease caused by a deficiency of enzyme activity in the heme biosynthesis pathway, leading to an abnormally high concentration of porphyrins or their precursors, which accumulate in tissues and cause cell damage. It easily causes damage to tissues and organs (especially the skin, liver, and nervous system). Porphyrins include type I and III uroporphyrinogen and uroporphyrin, type I and III coproporphyrinogen and coproporphyrin, and the porphyrin precursor porphyrobilinogen (PBG). Detection typically uses urine, feces, and red blood cells as samples, and qualitative or quantitative analysis is performed using ultraviolet fluorescence reactions.

[0003] Qualitative detection of porphyrin (PBG) typically employs the Hoesch method or the classic p-dimethylaminobenzaldehyde test (Watson-Schwartz test). The Hoesch method simply requires the addition of p-dimethylaminobenzaldehyde under acidic conditions; a pinkish-red color indicates a positive result. While this technique is simple and quick, it has the drawback of showing a negative result even with high porphyrin concentrations due to a faint color development. The Watson-Schwartz method… The test is a simple and reliable method for checking PBG. The principle is that PBG reacts with p-dimethylaminobenzaldehyde (Ehrlich's aldehyde reagent) under acidic conditions and turns deep red. Although urobilinogen or indole also produce a red color with this reagent, the red color of these two substances is removed by the solvent after shaking with chloroform, while the red color of PBG remains in the aqueous layer. The residual red color can be used to determine a positive result. Although the Watson-Schwartz test can eliminate interference from substances such as urobilinogen, it is complicated to operate, the use of chloroform is harmful to human health and the environment, and there are many reagents, making it inconvenient to carry. If the reagent detection of porphyrin is changed to test strip detection, although it is easier to carry, the interference of urobilinogen cannot be eliminated. Summary of the Invention

[0004] In view of this, the present invention provides a dry chemical test strip for detecting porphyrin in urine, its preparation method and reagent kit, which is easy to carry, eliminates interference from urobilinogen and bilirubin, and enables rapid detection of porphyrin in urine.

[0005] To achieve the above objectives, the present invention provides a method for preparing a dry chemical test strip for detecting porphyrin in urine, comprising the following steps: immersing a substrate in a colorimetric mother solution, and then removing and drying the substrate; the colorimetric mother solution comprises the following components: p-dimethylaminobenzaldehyde, 5-sulfosalicylic acid, barium chloride, nonionic surfactant, and chelating agent.

[0006] The technical solution provided by this invention does not react with barium chloride and 5-sulfosalicylic acid under acidic conditions. 5-sulfosalicylic acid can form an acidic buffer system within the test paper system, stably maintaining the strongly acidic environment of the test paper (pH≈1.5) through an acid-base balance reaction. This avoids the risk of corrosion to the test paper material due to excessive acidity and solves the problem of insufficient color development and weak signal caused by excessively low acidity, overcoming the industry pain point of difficulty in maintaining a stable acidic environment for test paper.

[0007] In addition to its conventional protein precipitation function, 5-sulfosalicylic acid in this invention also acts as a porphyrin precursor molecule stabilizer: under acidic conditions, its sulfonic acid groups form hydrogen bonds with porphyrin molecules, preventing porphyrin from spontaneously polymerizing under acidic conditions; it forms a hydrogen-bonded complex with p-dimethylaminobenzaldehyde, improving the stability of the colorimetric reagent and extending the shelf life of the test strip; and it enhances the absorbance of the colorimetric product, improving the detection sensitivity.

[0008] In this invention, barium chloride eliminates the interference of urinary bilirubin on detection results. Barium chloride reacts with sulfate ions in urine to form barium sulfate (BaSO4) microcrystalline precipitate in situ. The newly formed barium sulfate precipitate has a very large specific surface area, which can adsorb and encapsulate conjugated bilirubin in urine like a sponge. A pretreatment layer is incorporated into the design of the test substrate. The bilirubin is immobilized in the precipitate and cannot diffuse into the subsequent colorimetric area, thus removing the interference of urinary bilirubin.

[0009] Optionally, each liter of the colorimetric mother liquor comprises the following components: 4-6 g p-dimethylaminobenzaldehyde, 180-220 g 5-sulfosalicylic acid, 90-110 g barium chloride, 3-5 mL nonionic surfactant, and 0.4-0.6 g chelating agent.

[0010] Optionally, the nonionic surfactant is Tween-20.

[0011] In this invention, by precisely controlling the amount of Tween-20 (HLB value 16.7) added, p-dimethylaminobenzaldehyde molecules form an ordered monolayer arrangement on the surface of the test paper fibers. This arrangement optimizes the spatial distribution of reagent molecules, effectively reducing the activation energy of the Schiff base reaction between porphyrin and p-dimethylaminobenzaldehyde, and significantly accelerating the colorimetric reaction rate. Under high humidity conditions (>80% RH), Tween-20 molecules can form a stable self-assembled structure in the gaps between the test paper fibers, constructing a uniform micro-water channel network. This avoids reagent diffusion and edge smudging caused by excessive moisture absorption of the test paper, resulting in a colorimetric edge clarity improvement of more than 35% compared to the control group, and significantly improving the stability of interpretation.

[0012] Optionally, the chelating agent is sodium ethylenediaminetetraacetate.

[0013] The system of this invention maintains a strongly acidic environment at pH 1.5. Sodium ethylenediaminetetraacetate can effectively complex trace amounts of divalent metal impurities such as zinc ions mixed in the sample and test strip substrate, eliminating the catalytic degradation and quenching effect of metal ions on the colorimetric products, and improving the colorimetric signal intensity. The molar absorptivity of the system was increased by 28% compared with the conventional system. At the same time, sodium ethylenediaminetetraacetate and 5-sulfosalicylic acid form a synergistic protective system, which captures stray metal ions on the one hand and stabilizes the colorimetric reagent loaded on the test strip on the other hand, effectively delaying the decay of the colorimetric products and extending the effective reading time window of the test strip to 15 minutes, while the effective reading time of the conventional test strip system is less than 5 minutes.

[0014] Optionally, the soaking time is 5 to 10 minutes.

[0015] Optionally, the pH value of the colorimetric mother liquor is 1.2 to 1.7.

[0016] The present invention also provides a dry chemical test strip for detecting porphyrin in urine, comprising a substrate and a colorimetric mother solution, wherein the colorimetric mother solution comprises the following components: p-dimethylaminobenzaldehyde, 5-sulfosalicylic acid, barium chloride, nonionic surfactant, and chelating agent.

[0017] The dry chemical test strips of this invention are easy to carry and use.

[0018] Optionally, the substrate is filter paper for adsorbing colorimetric mother liquor or a PVC base plate with filter paper on its surface.

[0019] The present invention also provides a kit for detecting porphyrin in urine, comprising dry chemical test strips for detecting porphyrin in urine and urine pretreatment reagent, wherein the urine pretreatment reagent comprises the following components: ammonium sulfate and an acidic reagent.

[0020] This invention uses acidic reagents to stably control the test paper system in a strongly acidic environment. This acidity allows for precise protonation of the nitrogen atom on the pyrrole ring of porchocyanin, effectively regulating the electron distribution of the pyrrole ring, significantly enhancing the nucleophilic activity of the α-carbon atom of porchocyanin, reducing the energy barrier for its nucleophilic addition-dehydration condensation reaction with the aldehyde group of p-dimethylaminobenzaldehyde, efficiently promoting the rapid and stable formation of the Schiff base colorimetric product, and greatly improving the colorimetric sensitivity and reaction sufficiency.

[0021] Optionally, the acidic reagent is one of sulfuric acid, metaphosphoric acid, and hydrochloric acid.

[0022] Optionally, the sulfuric acid has a mass percentage of 95-98%; the metaphosphoric acid has a concentration of 90-200 g / L; and the hydrochloric acid has a volume fraction of 45-55%.

[0023] To achieve the above objectives, the present invention also provides an application of a kit for detecting porphyrin in urine. A urine pretreatment reagent is added to the urine to be tested and mixed well. The substrate is then immersed in the treated urine to be tested. After removal, the substrate is allowed to stand. If the substrate turns red, it is determined that the urine contains porphyrin.

[0024] In this invention, ammonium sulfate is not only used as a common salting-out agent, but also specifically eliminates urobilinogen interference through a unique two-site recognition and precipitation mechanism.

[0025] High concentrations of urobilinogen in urine are the core interfering substance in porphyrin colorimetric detection. High concentrations of urobilinogen in strongly acidic colorimetric systems easily undergo non-specific oxidation, disordered molecular aggregation, and non-specific adsorption on the test strip membrane, leading to severe false-positive signals and significantly reducing detection accuracy. This invention achieves precise anti-interference by using a urine pretreatment reagent: its ammonium sulfate dissociates into NH4+ in the system. - and SO4 2- NH4 - and SO4 2- They possess extremely strong hydration capabilities, "stealing" large amounts of free water molecules from the solution. This disrupts the hydration layer on the surface of urobilinogen molecules, leading to denaturation or aggregation and precipitation of urobilinogen. Simultaneously, this system specifically confines only the interfering urobilinogen, without affecting the protonation activation process of the pyrrole ring of porphyrin or interfering with the characteristic condensation colorimetric reaction of porphyrin with p-dimethylaminobenzaldehyde. Therefore, it can effectively reduce false positives while completely preserving the detection sensitivity for low concentrations of porphyrin.

[0026] Specificity experiments showed that under strong interference conditions, such as a urobilinogen concentration as high as 30 mg / dL in urine, conventional detection systems could not shield against urobilinogen interference, resulting in a false positive rate as high as 87%. Using the urine pretreatment reagent of this invention, non-specific color development caused by high concentrations of urobilinogen was effectively suppressed, reducing the false positive rate to below 2.3%. Simultaneously, detection verification was performed on a 10 μmol / L low-concentration porphyrin sample. The color development response of this invention was essentially consistent with that of the conventional system, with no attenuation in detection sensitivity, achieving a balance between high anti-interference capability and high detection sensitivity.

[0027] Optionally, the urine pretreatment reagent includes the following components: ammonium sulfate and an acidic reagent; the mass of ammonium sulfate added per mL of urine to be tested is 0.68~0.76 g; the volume of the acidic reagent added per mL of urine to be tested is 0.35~0.45 mL.

[0028] Optionally, the soaking time is 2-3 seconds, and the standing time is 30 seconds to 2 minutes.

[0029] The above-described technical solution of the present invention has at least the following beneficial effects: 1. The test strip provided by this invention is simple and quick to operate. The test result can be determined as positive or negative based on the color change in about 30 seconds to 2 minutes.

[0030] 2. The reagent kit of the present invention contains barium chloride and ammonium sulfate, which reduces the interference of bilirubin and urobilinogen on the detection of porphyrin.

[0031] 3. The reagent kit of the present invention pre-treats the sample with acid, which ensures the acidic environment required for the reaction without corroding the test paper.

[0032] 4. This invention is simple, quick, has little interference, low cost, and greatly reduces the harm to operators and the environment. Attached Figure Description

[0033] Figure 1 This is the colorimetric image of the dry chemical test strip in Example 5 of the present invention; Figure 2 This is a comparison diagram showing the elimination of interference between barium chloride and ammonium sulfate in this invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figures 1-2 The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0035] Example 1 A method for preparing a dry chemical test strip for detecting porphyrin in urine includes the following steps: Preparation of the colorimetric stock solution: Each liter of the colorimetric stock solution contains the following components: 4g p-dimethylaminobenzaldehyde, 220g 5-sulfosalicylic acid, 90g barium chloride, 5mL Tween-20, 0.6g sodium ethylenediaminetetraoxate, with the remainder being water. The pH of the colorimetric stock solution is approximately 1.5.

[0036] Immerse the filter paper in the colorimetric mother liquor for 5 minutes, then remove it and place it in a drying oven at 90°C for 10 minutes to dry. Use a strip cutter to evenly cut the filter paper into 6 pieces. 6mm 2 The square shape is used to form dry chemical test strips for later use.

[0037] Example 2 A method for preparing a dry chemical test strip for detecting porphyrin in urine includes the following steps: Preparation of the colorimetric stock solution: Each liter of the colorimetric stock solution contains the following components: 6g p-dimethylaminobenzaldehyde, 180g 5-sulfosalicylic acid, 110g barium chloride, 3mL Tween-20, 0.4g sodium ethylenediaminetetraoxate, with the volume balance being water. The pH of the colorimetric stock solution is approximately 1.5.

[0038] Immerse the PVC substrate with filter paper on its surface in the color developing solution for 10 minutes, then remove it and dry it in a drying oven at 90°C for 10 minutes. Use a strip cutter to evenly cut the substrate into 6 pieces. 6mm 2 The square shape is used to form dry chemical test strips for later use.

[0039] Example 3 A method for preparing a dry chemical test strip for detecting porphyrin in urine includes the following steps: Preparation of the colorimetric stock solution: Each liter of the colorimetric stock solution consists of the following components: 5g p-dimethylaminobenzaldehyde, 200g 5-sulfosalicylic acid, 100g barium chloride, 4mL Tween-20, 0.5g sodium ethylenediaminetetraoxate, with the volume balance being water. The pH of the colorimetric stock solution is approximately 1.5.

[0040] The substrate was immersed in the color developing mother liquor for 8 minutes, then removed and dried in a drying oven at 90°C for 10 minutes. The filter paper was then evenly cut into 6-piece strips using a strip cutter. 6mm 2 A square, and glued on 6mm. A test substrate is formed on a 5cm PVC substrate for later use.

[0041] Example 4 A method for preparing a dry chemical test strip for detecting porphobilinogen in urine, comprising the following steps: Prepare a chromogenic mother liquor: Each liter of the chromogenic mother liquor consists of the following components: 5 g of p-dimethylaminobenzaldehyde, 200 g of 5-sulfosalicylic acid, 100 g of barium chloride, 4 mL of Tween-20, 0.5 g of sodium ethylenediaminetetraacetate, and the balance is water. The pH value of the chromogenic mother liquor is about 1.5.

[0042] Soak the filter paper in the chromogenic mother liquor, take out the filter paper after soaking for 8 minutes, and place it in a drying oven at 90 °C for 10 minutes to dry. Use a strip cutter to evenly cut the filter paper into strips to form a dry chemical test strip for use.

[0043] A kit for detecting porphobilinogen in urine, comprising the above dry chemical test strip for detecting porphobilinogen in urine, and also comprising a urine pretreatment reagent. The urine pretreatment reagent includes the following components: ammonium sulfate and sulfuric acid. The added mass of ammonium sulfate in each mL of the urine to be detected is 0.72 g; the added volume of the sulfuric acid in each mL of the urine to be detected is 0.4 mL, and the mass percentage of the sulfuric acid is 98%.

[0044] Example 5 Based on the kit prepared in Example 4, use a pipette or a disposable graduated plastic straw to suck 1 ml of fresh urine and add it to a 2 ml EP tube with a treatment agent. Vortex and mix quickly on a vortex mixer for 1 minute to completely dissolve the treatment agent in the EP tube (a little remaining particles are normal).

[0045] Take the test strip prepared in Example 4, hold one end by hand, immerse the section with the test paper piece in the treated urine, take it out after 2 - 3 seconds, and read the result within 30 s to 2 minutes of standing. Figure 1 .

[0046] It can be seen from Figure 1 that the kit provided by the present invention for detecting porphobilinogen in urine has fast detection speed, is convenient to carry, and does not require reagents with strong external hazards such as chloroform.

[0047] Resistance interference comparison experiment 1 The kit prepared in Example 4 is denoted as the treatment group, and the kit in Example 4 without adding barium chloride and ammonium sulfate reagents is denoted as the untreated group. Four comparative experiments are carried out between the treatment group and the untreated group, and the experimental steps and methods are all the same, and Figure 2 ,["0]] Figure 2 In [ ], "untreated" represents the untreated group, and "treatment" represents the treatment group. ,["4]]

[0048] It can be seen from Figure 2It can be seen that the untreated group showed a weak positive color (light pink) due to interference from bilirubin and urobilinogen, while the treated group showed a negative color (light yellow) because the added barium chloride and ammonium sulfate reagents, combined with other reagents, eliminated the interference from bilirubin and urobilinogen. Therefore, the kit prepared in this invention effectively removes the interference from bilirubin and urobilinogen compared to the kit without added barium chloride and ammonium sulfate.

[0049] Anti-interference comparison experiment 2 1. Experimental instruments and reagents Experimental test strips: Conventional acidic colorimetric test strips (control group, without urine pretreatment reagents), and the optimized test strips of this invention (experimental group, containing urine pretreatment reagents with a specified ratio). Standard samples: 30 mg / dL high-concentration urobilinogen negative interfering urine, 10 μmol / L low-concentration porphyrin positive standard urine, and blank negative urine; Testing equipment: fully automatic test strip grayscale analysis system, ultraviolet spectrophotometer; experimental environment: temperature 25℃, relative humidity 45%~55%.

[0050] 2. Experimental Grouping and Operating Procedures The experiment was set up with a control group (conventional test strip system) and an experimental group (optimized system of this invention), with 20 parallel samples in each group to eliminate random error.

[0051] (1) High concentration urobilinogen anti-interference test: 30 mg / dL high concentration urobilinogen negative interference sample was taken and added to the control group and experimental group test strips in turn. After reacting in the dark and at constant temperature for 5 min, the color development image of the test strip was collected, the false positive color development was determined, and the false positive rate of the two groups was counted. (2) Low concentration sensitivity verification experiment: 10 μmol / L porphyrin standard positive sample was taken and the two groups of test strips were tested under the same conditions. After the reaction was completed, the colorimetric absorbance value of the test strip was measured and the difference between the colorimetric intensity and detection sensitivity of the two groups was compared. (3) Summarize all parallel experimental data, calculate the mean and error, and evaluate the system's anti-interference ability and sensitivity stability. The test results are shown in Table 1.

[0052] Table 1. Results of Anti-interference Test

[0053] High concentrations of urobilinogen are the main source of interference in the rapid detection of porphyrin. Conventional test strip systems cannot effectively shield against its non-specific colorimetric interference, resulting in a serious false positive problem. As shown in Table 1, the present invention, through a urine pretreatment reagent anti-interference system, can effectively constrain the non-specific reaction of high concentrations of urobilinogen and shield against interference from urine matrix impurities, significantly reducing the false positive rate. Moreover, this composite system does not participate in or interfere with the specific colorimetric reaction of porphyrin, and has no negative impact on the detection sensitivity of low concentrations of the target analyte. This greatly improves the accuracy, specificity, and stability of the test strip detection, solving the industry pain points of traditional test strips being susceptible to urobilinogen interference and having low detection accuracy.

[0054] Experiment on the effect of nonionic surfactants on reading stability Experimental conditions Control group: Regular test strips without Tween-20 Experimental group: The test paper of this invention with an optimized amount of Tween-20 (HLB=16.7) added. Test conditions: Temperature 25℃, relative humidity >85% RH, test performed after 24 hours of storage. Evaluation method: Image analysis software was used to perform gray-level gradient analysis on the edges of the colored bands, and the edge gray-level change rate was used as a quantification of sharpness. The experimental results are shown in Table 2.

[0055] Table 2. Effect of the addition of nonionic surfactants on interpretation stability.

[0056] As shown in Table 2, the test strips with added Tween-20 showed significantly improved edge clarity, 35% higher grayscale change rate, and significantly reduced band width deviation in high humidity conditions compared to the control group. This effectively solved the problems of reagent diffusion and edge smudging under high humidity and improved the stability of interpretation.

[0057] Experiment on the effect of chelating agents on interpretation time Experimental conditions Control group: pH 1.5 system, without sodium ethylenediaminetetraacetate, with only 5-sulfosalicylic acid added; Experimental group: pH 1.5 system, with the addition of sodium ethylenediaminetetraacetate + 5-sulfosalicylic acid (formulation of this invention); Test conditions: ambient temperature 25 ℃, relative humidity 50% RH; substrate was an equal amount of standard porphyrin sample; detection equipment: spectrophotometer + test strip chromatography imaging system.

[0058] Detection indicators: molar absorptivity, retention rate of chromogenic product, and effective interpretation time window. Detection results are shown in Table 3.

[0059] Table 3. Experimental Results on the Effect of Chelating Agents on Reading Time

[0060] Table 3 shows that in a strongly acidic system with pH 1.5, the addition of sodium ethylenediaminetetraacetate can complex trace amounts of Zn²⁺ in the system. + Metal impurities such as sodium ethylenediaminetetraacetate (EDTA) inhibit the destruction of colorimetric products, and the colorimetric molar absorptivity is increased by 28% compared with the control group. The synergistic effect of sodium ethylenediaminetetraacetate (EDTA) and 5-sulfosalicylic acid significantly delays the decay of colorimetric products, extending the effective reading time of the test strip from less than 5 minutes to 15 minutes, greatly improving the timeliness and performance of the test strip.

[0061] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a dry chemical test strip for detecting porphyrin in urine, characterized in that, Includes the following steps: The substrate is immersed in the color developing mother liquor, and then removed and dried. The color developing mother liquor includes the following components: p-dimethylaminobenzaldehyde, 5-sulfosalicylic acid, barium chloride, nonionic surfactant, and chelating agent.

2. The method for preparing the dry chemical test strip for detecting porphyrin in urine according to claim 1, characterized in that, Each liter of the colorimetric mother liquor comprises the following components: 4-6g p-dimethylaminobenzaldehyde, 180-220g 5-sulfosalicylic acid, 90-110g barium chloride, 3-5mL nonionic surfactant, and 0.4-0.6g chelating agent.

3. The method for preparing the dry chemical test strip for detecting porphyrin in urine according to claim 1, characterized in that, The soaking time is 5-10 minutes.

4. The method for preparing the dry chemical test strip for detecting porphyrin in urine according to claim 1, characterized in that, The pH value of the colorimetric mother liquor is 1.2~1.

7.

5. A dry chemical test strip for detecting porphyrin in urine, characterized in that, It includes a substrate and a color developing mother liquor, wherein the color developing mother liquor comprises the following components: p-dimethylaminobenzaldehyde, 5-sulfosalicylic acid, barium chloride, nonionic surfactant, and chelating agent.

6. A kit for detecting porphyrin in urine, characterized in that, The invention includes dry chemical test strips for detecting porphyrin in urine and urine pretreatment reagents, the urine pretreatment reagents comprising the following components: ammonium sulfate and an acidic reagent.

7. The kit for detecting porphyrin in urine according to claim 6, characterized in that, The acidic reagent is one of sulfuric acid, metaphosphoric acid, and hydrochloric acid.

8. The application of a kit for detecting porphyrin in urine as described in any one of claims 6-7 in the detection of porphyrin in urine, characterized in that, Add urine pretreatment reagent to the urine sample to be tested and mix well. Soak the substrate in the pretreated urine sample, remove it and let it stand. If it turns red, it indicates that the urine contains porchogen.

9. The application of the kit for detecting porphyrin in urine according to claim 8 in the detection of porphyrin in urine, characterized in that, The urine pretreatment reagent comprises the following components: ammonium sulfate and an acidic reagent; the mass of ammonium sulfate added per mL of urine to be tested is 0.68~0.76 g; the volume of the acidic reagent added per mL of urine to be tested is 0.35~0.45 mL.

10. The application of the kit for detecting porphyrin in urine according to claim 8, characterized in that, The soaking time is 2-3 seconds, and the standing time is 30 seconds to 2 minutes.

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