A reagent for detecting urinary monohydroxyphenolic metabolites and a method for preparing the same

Abstract

The application discloses a reagent for detecting monohydroxyphenol metabolites in urine and a preparation method thereof, and belongs to the technical field of urine detection reagents. The preparation method comprises the following steps: (1) preparing a mercurous nitrate solution, a mercury sulfate solution and a phosphomolybdic acid solution respectively; (2) mixing the three solutions and then laser soaking to prepare a composite system with stronger oxidation performance; (3) cross-linking after co-dissolving vanadate and nickel ions to form a complex three-dimensional hole cross-linking structure; and (4) ultrasonically mixing the first solution and a material containing nickel to obtain a simple, efficient and accurate urine monohydroxyphenol metabolite detection reagent. The reagent for detecting monohydroxyphenol metabolites in urine has the advantages of high sensitivity, simple operation, stable and reliable results and the like, and is suitable for clinical detection, scientific research and the like.

Description

Technical Field

[0001] This invention belongs to the field of urine test reagent technology, specifically relating to a reagent for detecting urinary monohydroxyphenol metabolites and its preparation method. Background Technology

[0002] Tumor grading and diagnosis aid in the early detection and treatment of cancer patients. Finding more convenient and rapid methods for tumor screening and diagnosis remains an important mission for many clinicians and laboratory staff. Using metabolites of tumor cells in the excrement (such as urine) of cancer patients for tumor screening offers advantages such as being non-invasive, painless, rapid, and convenient. Detecting monohydroxyphenol metabolites in patients' urine meets the requirements for rapid tumor screening. Urine monohydroxyphenol metabolite detection reagents react with monohydroxyphenol substances in urine, thereby determining the activity level of cancer cells and the body's anti-cancer immune status in the tested individual. This can serve as an auxiliary diagnostic indicator for early cancer during physical examinations and home self-examinations.

[0003] Monohydroxyphenol metabolites play important biological functions in the human body, and their levels are closely related to the occurrence and development of various diseases. Current methods for detecting urinary monohydroxyphenol metabolites suffer from problems such as complex operation, low sensitivity, and poor accuracy. Therefore, researching a simple, efficient, and accurate method for detecting urinary monohydroxyphenol metabolites is of significant clinical importance. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a reagent for detecting urinary monohydroxyphenol metabolites and a method for preparing the same. The prepared reagent has high sensitivity, rapid detection, and is simple and low-cost to operate.

[0005] In a first aspect, the present invention provides a method for preparing a reagent for detecting urinary monohydroxyphenol metabolites, comprising the following steps:

[0006] Prepare mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution respectively;

[0007] A mixture of mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution was then immersed under a laser to obtain the first solution.

[0008] Ammonium metavanadate and nickel nitrate were dissolved together in hot water, a water-soluble crosslinking agent was added and stirred continuously, and then freeze-dried to obtain the reactant;

[0009] The first solution is placed in deionized water and heated to prepare a suspension. Then, the reactants are added and ultrasonically vibrated to obtain the final product.

[0010] Furthermore, the concentrations of the mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution are 0.1–1.2 g / ml, 0.1–1.5 g / ml, and 0.05–0.5 g / ml, respectively.

[0011] Furthermore, the volume ratio of the mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution is 1–2.5:1:0.1–0.5.

[0012] Furthermore, the wavelength of the laser is 350–390 nm, and the immersion time is 30–120 min.

[0013] Furthermore, the freeze-drying method is as follows: first freeze at -10 to -20°C for 10 to 20 hours, and then vacuum dry at a pressure of 20 to 80 Pa for 1 to 2 hours.

[0014] Furthermore, the mass ratio of ammonium metavanadate, nickel nitrate, and water-soluble crosslinking agent is 400-500:100-150:10-80, and the water temperature is 70-90℃.

[0015] Furthermore, the water-soluble crosslinking agent is any one of butanetetracarboxylic acid, polyvinyl alcohol, or polyacrylic acid.

[0016] Furthermore, the mass ratio of the first solution to the reactants is 2.5–4:0.1–1, the heating temperature for preparing the suspension is 40–50°C, and the heating time is 30–60 min.

[0017] Furthermore, the ultrasonic oscillation has a power of 300–1000W, a frequency of 25–30KHz, and a duration of 15–60s.

[0018] Secondly, the present invention provides a reagent for detecting urinary monohydroxyphenol metabolites prepared by the above-described method for preparing such a reagent.

[0019] The technical solutions provided in this application have at least the following advantages compared with the prior art:

[0020] 1. This invention provides a reagent for detecting urinary monohydroxyphenol metabolites, which has the following technical features: high sensitivity, capable of accurately detecting low concentrations of monohydroxyphenol metabolites; simple operation, requiring no complex pretreatment steps; stable and reliable results with good repeatability; and wide applicability, applicable to multiple fields such as clinical testing and scientific research.

[0021] 2. This invention provides a method for preparing a reagent for detecting urinary monohydroxyphenol metabolites. Based on conventional chromogenic reagents, the morphology and structure of the reagent are adjusted through simple co-precipitation, cross-linking, and crystallization, giving it stronger oxidizing power for rapid urine detection. This also improves the reagent's specificity, reduces false positives, and increases sensitivity. Furthermore, the reagent is endowed with a certain adsorption effect, enabling even small amounts of reagent to exhibit high sensitivity and superior chromogenic performance. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0023] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0024] In a first aspect, the present invention provides a method for preparing a reagent for detecting urinary monohydroxyphenol metabolites, comprising the following steps:

[0025] Prepare mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution respectively;

[0026] A mixture of mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution was then immersed under a laser to obtain the first solution.

[0027] Ammonium metavanadate and nickel nitrate were dissolved together in hot water, a water-soluble crosslinking agent was added and stirred continuously, and then freeze-dried to obtain the reactant;

[0028] The first solution is placed in deionized water and heated to prepare a suspension. Then, the reactants are added and ultrasonically vibrated to obtain the final product.

[0029] In some optional embodiments of the present invention, the concentrations of the mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution are 0.1–1.2 g / ml, 0.1–1.5 g / ml, and 0.05–0.5 g / ml, respectively. Preferably, the concentrations of the mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution are 0.8 g / ml, 1.5 g / ml, and 0.1 g / ml, respectively. Mercurous nitrate and mercuric sulfate are both oxidizing agents; when mixed with phosphomolybdic acid, they form a composite system with stronger oxidizing power under laser irradiation. This composite system may more effectively oxidize monohydroxyphenolic metabolites in urine, thereby improving the sensitivity of detection.

[0030] Preferably, the volume ratio of the mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution is 1–2.5:1:0.1–0.5. More preferably, the volume ratio of the mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution is 2:1:0.5.

[0031] In some optional embodiments of the present invention, the wavelength of the laser is 350–390 nm, and the immersion time is 30–120 min. In the preferred embodiment of the present invention, the laser is 385 nm ultraviolet light, and the immersion time (i.e., ultraviolet irradiation) is 45 min. Sufficient irradiation with a certain laser can change the solubility, arrangement, and mixing degree of ions in the solution, resulting in a more stable first solution composite system and a better oxidation effect, allowing for rapid color development in small amounts as an oxidizing agent.

[0032] In some optional embodiments of the present invention, the freeze-drying method is as follows: first, freezing at -10 to -20°C for 10 to 20 hours, and then vacuum drying at a pressure of 20 to 80 Pa for 1 to 2 hours. Reactants obtained by freeze-drying have better stability, and the partially formed hydrogel structure can be further transformed into a partially formed aerogel structure, forming a three-dimensional porous interconnected structure. This also imparts certain adsorption properties to the reagents during subsequent preparation, improving the detection effect.

[0033] In some optional embodiments of the present invention, the mass ratio of ammonium metavanadate, nickel nitrate, and water-soluble crosslinking agent is 400–500:100–150:10–80, and the water temperature is 70–90°C. More preferably, the mass ratio of ammonium metavanadate, nickel nitrate, and water-soluble crosslinking agent is 500:120:30, and the water temperature is 75°C.

[0034] In some optional embodiments of the present invention, the water-soluble crosslinking agent is any one of butanetetracarboxylic acid, polyvinyl alcohol, or polyacrylic acid. The water-soluble crosslinking agent does not directly react chemically with nickel nitrate or nickel vanadate, but can form a stable suspension or gel with nickel nitrate or nickel vanadate and their products through physical action, thereby enhancing the crosslinking effect between materials.

[0035] In some optional embodiments of the present invention, the mass ratio of the first solution to the reactants is 2.5–4:0.1–1, the heating temperature for preparing the suspension is 40–50°C, and the heating time is 30–60 min. The ultrasonic oscillation power is 300–1000 W, the frequency is 25–30 kHz, and the time is 15–60 s. Through certain heating and ultrasonic oscillation treatments, the first solution and reactants can be mixed evenly, so that the mixture of the first solution can be uniformly dispersed in the reactants. The reagent prepared in this way will not have the colorimetric components settle due to storage, affecting the colorimetric effect, and can quickly react to obtain colorimetric results during colorimetric experiments, reducing waiting time.

[0036] Secondly, the present invention provides a reagent for detecting urinary monohydroxyphenol metabolites prepared by the above-mentioned method, which has the characteristics of high selectivity, high sensitivity, rapid response, convenient operation, strong anti-interference ability and low cost.

[0037] The method of using the reagent provided by this invention is as follows:

[0038] Collect 3 ml of fresh morning urine (without food or water, on an empty stomach), add 0.5 ml of the reagent of this invention, mix well, let stand for 2 minutes, and observe the reaction results. If a colorless to yellow or white precipitate appears, it is negative; if a pink, purple, red, brown, or darker color such as gray-black appears, it is positive; if any other color appears, it is invalid.

[0039] The principles and features of the present invention are described below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, specific conditions or manufacturer-recommended conditions should be followed in the embodiments. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0040] Example 1

[0041] This embodiment provides a method for preparing a reagent for detecting urinary monohydroxyphenol metabolites, comprising the following steps:

[0042] (1) Prepare a 0.8 g / ml mercurous nitrate solution, a 1.5 g / ml mercuric sulfate solution and a 0.1 g / ml phosphomolybdic acid solution respectively;

[0043] (2) Mix mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution in a volume ratio of 2:1:0.5, and then immerse in ultraviolet light at 385 nm for 45 min to obtain the first solution;

[0044] (3) Dissolve 50g ammonium metavanadate and 12g nickel nitrate in 1000ml hot water at 75℃, add 30g polyvinyl alcohol and stir continuously, freeze at -10℃ for 12h, and then dry under vacuum at 80Pa pressure for 1h to obtain the reactant.

[0045] (4) Place 40g of the first solution in 1000ml of deionized water and stir and heat for 30min to prepare a suspension at 40℃. Then, while it is still hot, add 1g of the reactant and sonicate to obtain the solution. The sonication power is 300W, the frequency is 25KHz, and the time is 30s.

[0046] Example 2

[0047] This embodiment provides a method for preparing a reagent for detecting urinary monohydroxyphenol metabolites, comprising the following steps:

[0048] (1) Prepare a 1.0 g / ml mercurous nitrate solution, a 1.0 g / ml mercuric sulfate solution and a 0.5 g / ml phosphomolybdic acid solution respectively;

[0049] (2) Mix mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution in a volume ratio of 1:1:0.1, and then immerse in ultraviolet light at 385 nm for 60 min to obtain the first solution;

[0050] (3) Dissolve 50g ammonium metavanadate and 12g nickel nitrate in 1000ml hot water at 75℃, add 10g polyvinyl alcohol and stir continuously, freeze at -10℃ for 12h, and then dry under vacuum at 80Pa pressure for 1h to obtain the reactant.

[0051] (4) Place 40g of the first solution in 1000ml of deionized water and stir and heat for 30min to prepare a suspension at 40℃. Then, while it is still hot, add 1g of the reactant and sonicate to obtain the solution. The sonication power is 500W, the frequency is 25KHz, and the time is 15s.

[0052] Example 3

[0053] This embodiment provides a method for preparing a reagent for detecting urinary monohydroxyphenol metabolites, comprising the following steps:

[0054] (1) Prepare 0.5 g / ml mercurous nitrate solution, 1.5 g / ml mercuric sulfate solution and 0.2 g / ml phosphomolybdic acid solution respectively;

[0055] (2) Mix mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution in a volume ratio of 2.5:1:0.25, and then immerse in ultraviolet light at 385 nm for 30 min to obtain the first solution;

[0056] (3) Dissolve 50g ammonium metavanadate and 12g nickel nitrate in 1000ml hot water at 80℃, add 30g polyvinyl alcohol and stir continuously, freeze at -10℃ for 12h, and then dry under vacuum at 80Pa pressure for 1h to obtain the reactant.

[0057] (4) Place 25g of the first solution in 1000ml of deionized water and stir and heat for 30min to prepare a suspension at 40℃. Then, while it is still hot, add 1g of the reactant and sonicate to obtain the solution. The sonication power is 300W, the frequency is 25KHz, and the time is 30s.

[0058] Comparative Example 1

[0059] This comparative example presents a preparation method different from that of Example 1, including the following steps:

[0060] (1) Prepare a 0.8 g / ml mercurous nitrate solution, a 1.5 g / ml mercuric sulfate solution and a 0.1 g / ml phosphomolybdic acid solution respectively;

[0061] (2) Mix mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution in a volume ratio of 2:1:0.5 to obtain the first solution;

[0062] (3) Dissolve 50g of ammonium metavanadate and 12g of nickel nitrate in 1000ml of hot water at 75℃, add 30g of polyvinyl alcohol and stir continuously to obtain the reactants;

[0063] (4) Place 40g of the first solution in deionized water, then add 1g of reactant, and sonicate after adding. The ultrasonic oscillation power is 300W, the frequency is 25KHz, and the time is 30s.

[0064] Comparative Example 2

[0065] This comparative example presents a preparation method different from that of Example 1, including the following steps:

[0066] (1) Prepare a 0.8 g / ml mercurous nitrate solution, a 1.5 g / ml mercuric sulfate solution and a 0.1 g / ml phosphomolybdic acid solution respectively;

[0067] (2) Mix mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution in a volume ratio of 2:1:0.5, and then immerse in 385nm ultraviolet light for 45min to obtain the first solution;

[0068] (3) Dissolve 50g of ammonium metavanadate and 12g of nickel nitrate in 1000ml of hot water at 75℃, add 30g of polyvinyl alcohol and stir continuously to obtain the reactant;

[0069] (4) Place 40g of the first solution in 1000ml of deionized water and stir and heat for 30min to prepare a suspension at 40℃. Then, while it is still hot, add 1g of the reactant and sonicate to obtain the solution. The sonication power is 300W, the frequency is 25KHz, and the time is 30s.

[0070] Comparative Example 3

[0071] (1) Prepare a 0.8 g / ml mercurous nitrate solution, a 1.5 g / ml mercuric sulfate solution and a 0.1 g / ml phosphomolybdic acid solution respectively;

[0072] (2) Mix mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution in a volume ratio of 2:1:0.5 to obtain the first solution;

[0073] (3) Dissolve 50g ammonium metavanadate and 12g nickel nitrate in 1000ml hot water at 75℃, add 30g polyvinyl alcohol and stir continuously, freeze at -10℃ for 12h, and then dry under vacuum at 80Pa pressure for 1h to obtain the reactant.

[0074] (4) Place 40g of the first solution in 1000ml of deionized water and stir and heat for 30min to prepare a suspension at 40℃. Then, while it is still hot, add 1g of the reactant and sonicate to obtain the solution. The sonication power is 300W, the frequency is 25KHz, and the time is 30s.

[0075] Comparative Example 4

[0076] This comparative example presents a preparation method different from that of Example 1, including the following steps:

[0077] (1) Prepare 0.8 g / ml mercurous nitrate solution, 1.5 g / ml mercuric sulfate solution, 0.1 g / ml phosphomolybdic acid solution and 1.0 g / ml nickel nitrate solution respectively;

[0078] (2) Prepare the reagent by mixing mercurous nitrate solution, mercuric sulfate solution, phosphomolybdic acid solution and nickel nitrate solution in a volume ratio of 2:1:0.5:1.

[0079] Test case

[0080] Experimental subjects: All subjects did not drink alcohol or take amino acid or hormone medications the day before the test. 3 ml of fresh morning urine (without food or water, collected on an empty stomach) was mixed with 0.5 ml of the reagent of this invention, allowed to stand for 2 minutes, and the reaction results were observed.

[0081] Group A comprised 325 patients clinically diagnosed with malignant tumors, aged 20-80, including 187 males and 138 females. The diagnosed cancers included thyroid cancer, liver cancer, stomach cancer, esophageal cancer, breast cancer, cervical cancer, and nasopharyngeal carcinoma.

[0082] Group B consisted of 200 healthy individuals aged 20-70 years, including 96 males and 104 females. All participants were healthy individuals without any history of malignant tumors.

[0083] The experimental reagents were those prepared in Example 1 and Comparative Examples 1-4. The experimental results are shown in Table 1.

[0084] Table 1

[0085]

[0086] As shown in Table 1, the detection reagent prepared using the method of this invention exhibits higher sensitivity to monohydroxyphenol metabolites in urine, enabling faster patient screening. This may be related to the special processing methods used in the preparation process, which may generate stronger oxidizing groups, resulting in selectivity for specific monohydroxyphenol metabolites. This helps improve the specificity of the detection and reduce false positive results.

[0087] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a reagent for detecting urinary monohydroxyphenol metabolites, characterized in that, Includes the following steps: Prepare mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution respectively; A mixture of mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution is placed under a laser for immersion to obtain a first solution; the wavelength of the laser is 350~390nm, and the immersion time is 30~120min. Ammonium metavanadate and nickel nitrate are dissolved together in hot water, a water-soluble crosslinking agent is added and stirred continuously, and then freeze-dried to obtain the reactant; the mass ratio of ammonium metavanadate, nickel nitrate and water-soluble crosslinking agent is 400~500:100~150:10~80, and the water temperature is 70~90℃; the water-soluble crosslinking agent is any one of butanetetracarboxylic acid, polyvinyl alcohol or polyacrylic acid; The first solution is placed in deionized water and heated to prepare a suspension. Then, the reactants are added and ultrasonically vibrated to obtain the final product.

2. The method for preparing the reagent for detecting urinary monohydroxyphenol metabolites according to claim 1, characterized in that, The concentrations of the mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution are 0.1~1.2 g / ml, 0.1~1.5 g / ml, and 0.05~0.5 g / ml, respectively.

3. The method for preparing the reagent for detecting urinary monohydroxyphenol metabolites according to claim 1 or 2, characterized in that, The volume ratio of the mercurous nitrate solution, mercuric sulfate solution, and phosphomolybdic acid solution is 1~2.5:1:0.1~0.

5.

4. The method for preparing the reagent for detecting urinary monohydroxyphenol metabolites according to claim 1, characterized in that, The freeze-drying method is as follows: first freeze at -10~-20℃ for 10~20h, then vacuum dry at 20~80Pa pressure for 1~2h.

5. The method for preparing the reagent for detecting urinary monohydroxyphenol metabolites according to claim 1, characterized in that, The mass ratio of the first solution to the reactants is 2.5~4:0.1~1, and the heating temperature for preparing the suspension is 40~50℃, and the heating time is 30~60min.

6. The method for preparing the reagent for detecting urinary monohydroxyphenol metabolites according to claim 1, characterized in that, The ultrasonic oscillation has a power of 300~1000W, a frequency of 25~30KHz, and a duration of 15~60s.

7. A reagent for detecting urinary monohydroxyphenol metabolites, characterized in that, It is prepared by the method for preparing a reagent for detecting urinary monohydroxyphenol metabolites as described in any one of claims 1 to 6.

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