Reagent capable of detecting monohydroxyphenol metabolites in urine and preparation method thereof

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical field of urine detection reagents, and in particular relates to a reagent capable of detecting urine monohydric phenol metabolites and a preparation method thereof. Background Art

[0002] Tumor grading diagnosis provides assistance for early detection and treatment of cancer patients. Finding more convenient and faster means of tumor screening and diagnosis remains an important mission for many clinicians and laboratory staff. Using the metabolites of tumor cells in the excretions (such as urine) of tumor patients to screen for tumors has the advantages of being non-invasive, painless, rapid and convenient. Detecting monohydric phenol metabolites in patient urine meets the requirements for rapid tumor screening. The urine monohydric phenol metabolite detection reagent can react with monohydric phenol substances in urine to determine the activity of cancer cells in the body of the tested person and the body's anti-cancer immunity status. It can be used as an auxiliary diagnostic indicator for early cancer during physical screening and family self-examination.

[0003] Monohydroxyphenol metabolites have important biological functions in the human body, and changes in their content are closely related to the occurrence and development of a variety of diseases. At present, the methods for detecting monohydroxyphenol metabolites in urine have problems such as complex operation, low sensitivity, and poor accuracy. Therefore, it is of great clinical significance to study a simple, efficient, and accurate method for detecting monohydroxyphenol metabolites in urine. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a reagent capable of detecting urine monohydroxyphenol metabolites and a preparation method thereof. The prepared reagent has high sensitivity, rapid detection, simple operation and low cost.

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

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

[0007] Mixing mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution, and then immersing under laser to obtain a first solution;

[0008] Dissolving ammonium metavanadate and nickel nitrate in hot water, adding a water-soluble cross-linking agent, stirring continuously, and freeze-drying to obtain a reactant;

[0009] The first solution is placed in deionized water and heated to form a suspension, and then the reactants are added and ultrasonically vibrated to obtain the 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 mixing volume ratio of the mercurous nitrate solution, the mercuric sulfate solution and the 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: 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.

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

[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 reactant is 2.5-4:0.1-1, the heating temperature when preparing the suspension is 40-50° C., and the heating time is 30-60 min.

[0017] Furthermore, the power of the ultrasonic oscillation is 300-1000W, the frequency is 25-30KHz, and the time is 15-60s.

[0018] In a second aspect, the present invention provides a reagent capable of detecting urinary monohydric phenol metabolites prepared by using the above-mentioned method for preparing a reagent capable of detecting urinary monohydric phenol metabolites.

[0019] The above technical solution provided by the embodiment of the present application has at least the following advantages compared with the prior art:

[0020] 1. The present invention provides a reagent for detecting monohydric phenol metabolites in urine, which has the following technical characteristics: high sensitivity, capable of accurately detecting low concentrations of monohydric phenol metabolites; simple operation, no need for complicated pre-treatment steps; stable and reliable results, good repeatability; wide range of application, can be used in clinical testing, scientific research and other fields.

[0021] 2. The present invention provides a method for preparing a reagent capable of detecting urine monohydroxyphenol metabolites. On the basis of conventional color developing agents, the morphology and structure of the detection reagent are adjusted by simple coprecipitation, cross-linking and crystallization, so that the reagent has a stronger oxidation ability and can quickly detect urine, and the specificity of the reagent is improved, false positive results are reduced, and sensitivity is improved. The reagent is also endowed with a certain adsorption effect, so that a small amount of reagent also has a higher sensitivity and exhibits a more excellent color development effect. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0023] Unless otherwise specified, various 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 capable of detecting urine monohydroxyphenol metabolites, comprising the following steps:

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

[0026] Mixing mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution, and then immersing under laser to obtain a first solution;

[0027] Dissolving ammonium metavanadate and nickel nitrate in hot water, adding a water-soluble cross-linking agent, stirring continuously, and freeze-drying to obtain a reactant;

[0028] The first solution is placed in deionized water and heated to form a suspension, and then the reactants are added and ultrasonically vibrated to obtain the 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. Among them, mercurous nitrate and mercuric sulfate are both oxidants, which, after mixing with phosphomolybdic acid, form a composite system with stronger oxidizing ability under laser irradiation. This composite system may more effectively oxidize monohydroxyphenol metabolites in urine, thereby improving the sensitivity of detection.

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

[0031] In some optional embodiments of the present invention, the wavelength of the laser is 350-390nm, and the immersion time is 30-120min. In the best embodiment of the present invention, the laser is 385nm ultraviolet light, and the immersion (i.e. ultraviolet light irradiation) time is 45min. Sufficient light treatment by 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, and as an oxidant, a small amount but fast color development can be achieved.

[0032] In some optional embodiments of the present invention, the freeze-drying method is: first freeze at -10 to -20°C for 10 to 20 hours, and then vacuum dry at 20 to 80 Pa for 1 to 2 hours. The reactant obtained by freeze-drying has better stability, and the formed part of the hydrogel structure can also be converted into a part of the aerogel structure to form a three-dimensional porous interconnected structure, which can also give the reagent a certain adsorption performance when it is prepared into a reagent later, thereby 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 temperature of hot water 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 temperature of hot water is 75° C.

[0034] In some optional embodiments of the present invention, the water-soluble cross-linking agent is any one of butanetetracarboxylic acid, polyvinyl alcohol or polyacrylic acid. The water-soluble cross-linking agent will 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, which can enhance the cross-linking effect between materials.

[0035] In some optional embodiments of the present invention, the mass ratio of the first solution to the reactant is 2.5-4:0.1-1, the heating temperature when configuring the suspension is 40-50°C, and the heating time is 30-60min. The power of the ultrasonic oscillation is 300-1000W, the frequency is 25-30KHz, and the time is 15-60s. Through certain heating and ultrasonic oscillation treatment, the first solution and the reactant can be mixed evenly, so that the mixed system of the first solution can be evenly dispersed in the reactant, and the reagent prepared in this way will not cause the color development component to sink due to storage, affecting the color development effect, and can quickly react to obtain the color development result when performing the color development experiment, reducing the waiting time.

[0036] In the second aspect, the present invention provides a reagent for detecting urine monohydric phenol metabolites prepared by the above-mentioned method for preparing the reagent for detecting urine monohydric phenol metabolites, which has the characteristics of high selectivity, high sensitivity, rapid response, convenient operation, strong anti-interference and low cost.

[0037] The method of using the reagent provided by the present invention is:

[0038] Take 3 ml of fresh morning urine (do not eat or drink water, collect urine on an empty stomach), take 0.5 ml of the reagent of the present invention, mix well, let stand for 2 minutes, and observe the reaction result. If it is a colorless to yellow precipitate or a white precipitate, it is negative; if it is a pink, purple, red, brown, or a darker color such as gray-black, it is positive; if it is other colors, it is invalid.

[0039] The principles and features of the present invention are described below in conjunction with the examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0040] Example 1

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

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

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

[0044] (3) 50 g of ammonium metavanadate and 12 g of nickel nitrate were dissolved in 1000 ml of hot water at 75° C., 30 g of polyvinyl alcohol was added and stirred continuously, and the mixture was first frozen at -10° C. for 12 h, and then vacuum dried at 80 Pa pressure for 1 h to obtain a reactant;

[0045] (4) 40 g of the first solution was placed in 1000 ml of deionized water, stirred and heated for 30 min to prepare a suspension at 40° C., and then 1 g of the reactant was added while hot and ultrasonically oscillated to obtain the product. The power of the ultrasonic oscillation was 300 W, the frequency was 25 KHz, and the time was 30 s.

[0046] Example 2

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

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

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

[0050] (3) 50 g of ammonium metavanadate and 12 g of nickel nitrate were dissolved in 1000 ml of hot water at 75° C., 10 g of polyvinyl alcohol was added and stirred continuously, and the mixture was first frozen at -10° C. for 12 h, and then vacuum dried at 80 Pa pressure for 1 h to obtain a reactant;

[0051] (4) 40 g of the first solution was placed in 1000 ml of deionized water, stirred and heated for 30 min to prepare a suspension at 40° C., and then 1 g of the reactant was added while hot and ultrasonically oscillated to obtain the product. The power of the ultrasonic oscillation was 500 W, the frequency was 25 KHz, and the time was 15 s.

[0052] Example 3

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

[0054] (1) preparing 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) mixing a mercurous nitrate solution, a mercuric sulfate solution, and a phosphomolybdic acid solution in a volume ratio of 2.5:1:0.25, and then immersing the mixture in a 385 nm ultraviolet light for 30 min to obtain a first solution;

[0056] (3) 50 g of ammonium metavanadate and 12 g of nickel nitrate were dissolved in 1000 ml of hot water at 80° C., 30 g of polyvinyl alcohol was added and stirred continuously, and the mixture was first frozen at -10° C. for 12 h, and then vacuum dried at 80 Pa for 1 h to obtain a reactant;

[0057] (4) 25 g of the first solution was placed in 1000 ml of deionized water, stirred and heated for 30 min to prepare a suspension at 40° C., and then 1 g of the reactant was added while hot and ultrasonically oscillated to obtain the product. The power of the ultrasonic oscillation was 300 W, the frequency was 25 KHz, and the time was 30 s.

[0058] Comparative Example 1

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

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

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

[0062] (3) dissolving 50 g of ammonium metavanadate and 12 g of nickel nitrate in 1000 ml of hot water at 75° C., adding 30 g of polyvinyl alcohol and stirring continuously to obtain a reactant;

[0063] (4) 40 g of the first solution is placed in deionized water, and then 1 g of the reactant is added, followed by ultrasonic oscillation. The ultrasonic oscillation has a power of 300 W, a frequency of 25 KHz, and a time of 30 s.

[0064] Comparative Example 2

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

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

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

[0068] (3) 50 g of ammonium metavanadate and 12 g of nickel nitrate were dissolved in 1000 ml of hot water at 75° C., and 30 g of polyvinyl alcohol was added and stirred continuously to obtain a reactant;

[0069] (4) 40 g of the first solution was placed in 1000 ml of deionized water, stirred and heated for 30 min to prepare a suspension at 40° C., and then 1 g of the reactant was added while hot and ultrasonically oscillated to obtain the product. The power of the ultrasonic oscillation was 300 W, the frequency was 25 KHz, and the time was 30 s.

[0070] Comparative Example 3

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

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

[0073] (3) 50 g of ammonium metavanadate and 12 g of nickel nitrate were dissolved in 1000 ml of hot water at 75° C., 30 g of polyvinyl alcohol was added and stirred continuously, and the mixture was first frozen at -10° C. for 12 h, and then vacuum dried at 80 Pa pressure for 1 h to obtain a reactant;

[0074] (4) 40 g of the first solution was placed in 1000 ml of deionized water, stirred and heated for 30 min to prepare a suspension at 40° C., and then 1 g of the reactant was added while hot and ultrasonically oscillated to obtain the product. The power of the ultrasonic oscillation was 300 W, the frequency was 25 KHz, and the time was 30 s.

[0075] Comparative Example 4

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

[0077] (1) preparing 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) Mercurous nitrate solution, mercuric sulfate solution, phosphomolybdic acid solution and nickel nitrate solution are mixed in a volume ratio of 2:1:0.5:1 to prepare a reagent.

[0079] Test example

[0080] Experimental subjects: All subjects did not drink alcohol the day before the test, and did not take amino acids, hormones and other drugs. Take 3 ml of fresh morning urine (no food, no water, fasting urine), take 0.5 ml of the reagent of the present invention, mix well, let stand for 2 minutes, and observe the reaction results.

[0081] Among them, group A consisted of 325 patients diagnosed with malignant tumors by clinical diagnosis, aged 20 to 80, 187 males and 138 females. The cancers diagnosed included thyroid cancer, liver cancer, stomach cancer, esophageal cancer, breast cancer, cervical cancer, nasopharyngeal cancer, etc.

[0082] Among them, group B is a healthy group with a total of 200 people, aged 20 to 70 years old, 96 males and 104 females. All of them are healthy people without malignant tumors.

[0083] The experimental reagents are respectively the reagents prepared in Example 1 and Comparative Examples 1 to 4. The experimental results are shown in Table 1.

[0084] Table 1

[0085]

[0086] As can be seen from Table 1, the detection reagent improved by the preparation method of the present invention has a higher sensitivity to monohydroxyphenol metabolites in urine and can screen out patients more quickly. This may be related to the special processing method in the preparation process, which may produce stronger oxidized groups in the process, resulting in selectivity for specific monohydroxyphenol metabolites, which helps to improve the specificity of the test and reduce false positive results.

[0087] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for preparing a reagent capable of detecting urine monohydroxyphenol metabolites, characterized in that: The following steps are involved: preparing mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution respectively; Mixing mercurous nitrate solution, mercuric sulfate solution and phosphomolybdic acid solution, and then immersing under laser to obtain a first solution; Dissolving ammonium metavanadate and nickel nitrate in hot water, adding a water-soluble cross-linking agent, stirring continuously, and freeze-drying to obtain a reactant; The first solution is placed in deionized water and heated to form a suspension, and then the reactants are added and ultrasonically vibrated to obtain the product.

2. The method for preparing a reagent capable of detecting urine monohydric phenol 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 a reagent capable of detecting urine monohydric phenol metabolites according to claim 1 or 2, characterized in that: The mixing volume ratio of the mercurous nitrate solution, the mercuric sulfate solution and the phosphomolybdic acid solution is 1-2.5:1:0.1-0.

5.

4. The method for preparing a reagent capable of detecting urine monohydric phenol metabolites according to claim 1, characterized in that: The wavelength of the laser is 350-390 nm, and the soaking time is 30-120 min.

5. The method for preparing a reagent capable of detecting urine monohydric phenol metabolites according to claim 1, characterized in that: The freeze-drying method is: 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.

6. The method for preparing a reagent capable of detecting urine monohydric phenol metabolites according to claim 1, characterized in that: The mass ratio of the ammonium metavanadate, nickel nitrate and water-soluble cross-linking agent is 400-500:100-150:10-80, and the water temperature of the hot water is 70-90°C.

7. The method for preparing a reagent capable of detecting urine monohydric phenol metabolites according to claim 1, characterized in that: The water-soluble crosslinking agent is any one of butanetetracarboxylic acid, polyvinyl alcohol or polyacrylic acid.

8. The method for preparing a reagent capable of detecting urine monohydric phenol metabolites according to claim 1, characterized in that: The mass ratio of the first solution to the reactant is 2.5-4:0.1-1, the heating temperature when preparing the suspension is 40-50° C., and the heating time is 30-60 min.

9. The method for preparing a reagent capable of detecting urine monohydric phenol metabolites according to claim 1, characterized in that: The power of the ultrasonic oscillation is 300-1000W, the frequency is 25-30KHz, and the time is 15-60s.

10. A reagent capable of detecting monohydroxyphenol metabolites in urine, characterized in that: The method for preparing a reagent capable of detecting urine monohydroxyphenol metabolites according to any one of claims 1 to 9 is used to prepare the reagent.

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