阅读说明：本技术 快速响应细微酸碱度变化的指示剂及其制备方法和应用 (Indicator capable of rapidly responding to slight pH value change and preparation method and application thereof ) 是由 袁萌萌 张蕾 曹洋 张梦鸽 张琼琼 朱云珊 朴学娇 汪大为 武玮 于 2021-08-30 设计创作，主要内容包括：本发明提供了一种快速响应细微酸碱度变化的指示剂,包括酸碱指示剂、等电点为3.0-10.0、分子量为10kDa-200kDa的稳定性蛋白质和缓冲溶液。本发明的指示剂的颜色突变区间可以控制在0.1-0.2之间,变色明显,可以使用含有该指示剂的固态基质对不同人体代谢物进行快速检测。(The invention provides an indicator capable of rapidly responding to slight pH value change, which comprises an acid-base indicator, stable protein with isoelectric point of 3.0-10.0 and molecular weight of 10-200 kDa and buffer solution. The color mutation interval of the indicator can be controlled between 0.1 and 0.2, the color change is obvious, and the solid matrix containing the indicator can be used for quickly detecting different human metabolites.)

1. An indicator capable of rapidly responding to small pH value change, which comprises an acid-base indicator, and is characterized in that: also comprises a stable protein with isoelectric point of 3.0-10.0 and molecular weight of 10-200 kDa and a buffer solution.

2. The indicator that rapidly responds to a fine ph change according to claim 1, wherein: the protein is one of calmodulin, bovine serum albumin, myosin, collagen or lactoferrin.

3. The indicator that rapidly responds to a fine ph change according to claim 2, wherein: the protein is preferably one of myosin, lactoferrin or collagen.

4. The indicator that rapidly responds to a fine ph change according to claim 1, wherein: the acid-base indicator is bromocresol green, methyl orange, methyl red, nitroyellow, phenolphthalein, bromocresol green-methyl orange, bromocresol green-methyl red, bromocresol green-nitroyellow, bromocresol green-phenolphthalein, methyl orange-methyl red, methyl orange-nitroyellow, methyl orange-phenolphthalein, methyl red-nitroyellow, methyl red-phenolphthalein, nitroyellow-phenolphthalein, bromocresol green-methyl orange-methyl red, bromocresol green-methyl orange-nitroyellow, bromocresol green-methyl orange-phenolphthalein, bromocresol green-methyl red-nitroyellow, bromocresol green-methyl red-phenolphthalein, methyl orange-methyl red-nitroyellow, methyl orange-methyl red-phenolphthalein, methyl red-nitroyellow-phenolphthalein, nitroyellow, phenolphthalein, or mixtures thereof, One of bromocresol green-methyl orange-methyl red-nitro-yellow, bromocresol green-methyl orange-methyl red-phenolphthalein, bromocresol green-methyl red-nitro-yellow-phenolphthalein, methyl orange-methyl red-nitro-yellow-phenolphthalein, or bromocresol green-methyl orange-methyl red-nitro-yellow-phenolphthalein.

5. The indicator that rapidly responds to a fine ph change according to claim 4, wherein: the acid-base indicator is preferably one of bromocresol green-nitroyellow, methyl orange-methyl red, nitroyellow-phenolphthalein, bromocresol green-methyl red-nitroyellow, bromocresol green-methyl red-phenolphthalein, bromocresol green-methyl orange-methyl red-nitroyellow, methyl orange-methyl red-nitroyellow-phenolphthalein, or bromocresol green-methyl orange-methyl red-nitroyellow-phenolphthalein.

6. The indicator that rapidly responds to a fine ph change according to claim 1, wherein: the buffer solution comprises one of citric acid-sodium citrate, citric acid-sodium hydroxide, sodium citrate-hydrochloric acid, sodium dihydrogen phosphate-dipotassium hydrogen phosphate, sodium dihydrogen phosphate-sodium hydroxide, dipotassium hydrogen phosphate-hydrochloric acid, dipotassium hydrogen phosphate-phosphoric acid or acetic acid-sodium acetate or acetic acid-potassium acetate.

7. The indicator that rapidly responds to a fine ph change according to claim 6, wherein: the buffer solution is preferably one of citric acid-sodium citrate, sodium dihydrogen phosphate-dipotassium hydrogen phosphate or acetic acid-sodium acetate.

8. A method for preparing the indicator capable of rapidly responding to the small pH change according to any one of claims 1 to 7, wherein the method comprises the following steps: the method comprises the following steps:

1) weighing a certain amount of protein, dissolving the protein in glycerol, and preparing a protein mother solution with the mass fraction of 0.01-0.05%;

2) weighing and dissolving a certain amount of acid-base indicator to prepare the acid-base indicator with the mass fraction of 0.05-0.5%;

3) weighing a certain amount of each component of the buffer solution, and preparing 0.1M buffer solutions with different pH values, wherein the pH value is 2.0-14.0;

4) mixing the protein solution obtained in the step 1) with the acid-base indicator obtained in the step 2) in a volume ratio of 1: 5-1: 50;

5) the mixed solution of 4) is added with the buffer solution obtained in 3) to fix the volume.

9. Use of an indicator of rapid response to small ph changes according to claim 1 for the detection of metabolites in the human body.

10. Use of an indicator for rapid response to fine ph change in the detection of metabolites in the human body according to claim 9, wherein: the indicator is attached to a solid substrate for detecting the metabolites of the human body, and the solid substrate comprises an indicating swab, test paper and cellulose dressing.

Technical Field

The invention relates to the technical field of biochemistry, in particular to a color indicator capable of rapidly responding to slight pH value change and a preparation method and application thereof.

Background

The pH indicator is a kind of organic and inorganic compound and their combination capable of changing pH value via color change reaction in specific pH range. Currently commonly used pH indicators are: thymol blue indicator, color change range of pH1.2-2.8 (red → yellow); bromophenol blue indicator, color change range pH2.8-4.6 (yellow → blue green); methyl orange indicator, color change range pH3.2-4.4 (Red → yellow); congo red indicator, color change range pH3.5-5.2 (blue → red); bromcresol green indicator liquid, the color change range is pH3.6-5.2 (yellow → blue); alizarin sodium sulfonate indicator, color change range pH3.7-5.2 (yellow → purple); methyl red indicator, color change range pH4.2-6.3 (red → yellow); litmus indicator, color change range pH4.2-8.0 (red → blue); bromcresol purple indicator, color change range pH5.2-6.8 (yellow → purple); nitro-nitrogen yellow indicator, the color change range is pH6.0-7.0 (yellow → blue purple); bromothymol blue indicator, the color change range is pH6.0-7.6 (yellow → blue); a neutral red indicator, the color change range of which is pH6.8-8.0 (red → yellow); cresol red indicator, color change range ph7.2-8.8 (yellow → red); curcumin indicator, color change range ph7.8-9.2 (yellow → reddish brown); phenolphthalein indicator liquid, the color change range is pH8.3-10.0 (colorless → red); malachite green indicator, the color change range is pH11.0-13.5 (green → colorless), etc.

The pH detection is applied to various industries, for example, in the medical field, the pH range of human blood is in the range of 7.35-7.45, acidosis can occur when the pH is lower than 7.35, and severe acidosis coma and death can occur when the pH is lower than 7.0; the pH value of the blood is higher than 7.45, so that the alkalosis can occur, and the serious consequence of tetany and death caused by serious alkalosis can occur when the pH value is higher than 7.8; in agriculture, the pH of soil is related to the growth of crops, some crops such as sesame, rape, radish and the like can grow in a larger pH range, and some crops are very sensitive to the pH reaction of the soil, for example, tea trees are suitable for growing in the soil with the pH of about 4.0-5.5; in sewage treatment, the pH value is also an important index.

People are also constantly searching for novel fingersAnd the indicator component is used for adjusting and optimizing the detection range of the indicator, and most indicators are originally sourced from plants, such as litmus indicators, thymol blue indicators, curcumin indicators and the like. Simultaneously, the indicator is combined, the indication range is expanded, the mutation range is narrowed, for example, a neutral red ethanol solution and a thymol blue ethanol solution in a certain proportion are mixed, and the color change range is between pH7.0 and 7.4 (rose color → dark green); the bromocresol green indicator-methyl orange indicator in a certain proportion is mixed, and the color change range is pH3.5-4.3 (yellow → blue green). However, the detection in the subdivided field does not pursue a range for the pH indicator, and pays more attention to specific color change points, for example, the indication range of the acid-base indicator is expanded by mixing curcumin and alkannin according to a certain ratio by Jiangsu peacock-tolerant bioengineering technology limited (CN105784702A), and the mutation range is narrow; a benzothiazole azo compound acid-base indicator is disclosed and developed at Sichuan university (CN111896536A), the color mutation range is narrow, and the color change is visual and obvious and is convenient to observe; taiyuan Jiepong electronic technology Limited (CN108120796A) discloses a method for detecting CO of a railway storage battery₃ ^2-And (3) a method for measuring and calculating the pH value mutation point. However, the formula in the invention is based on simple indicator combination and changes the ionic environment of the solution, and the obtained color development approximate to mutation is obtained, for example, the color change interval of the pH indicator for vaginal secretion detection can only be limited to be 4.5-5.0, the range is wide, the color change interval can not be effectively indicated, and the formula has larger limitation for actual sample detection.

Disclosure of Invention

The invention aims to solve the technical problem of developing an indicator which can narrow the color change range and quickly respond to slight pH value change.

Based on the problems, the technical scheme provided by the invention is an indicator capable of rapidly responding to slight pH change, and the indicator comprises an acid-base indicator, a protein with an isoelectric point of 3.0-10.0 and a molecular weight of 10kDa-200kDa and a buffer solution.

When the pH of the buffer system in which the protein is located is not equal to pI, the protein belongs to a dissociation state and has net charges, the charge amount of the protein is changed by using the buffer solution, namely the net charges of the protein are adjusted by using the pH value of the buffer solution, and then the pH color development interval of the protein is adjusted by the synergistic effect of the pH value of the buffer solution and the acid-base indicator. Theoretically, the more the pH of the buffer solution of the protein deviates from the pI, the more the net charge is carried, the higher the solubility is, and conversely, the lower the solubility is, the solubility is the minimum when the pH is equal to the pI, but the larger the deviation is, the more the charge influences the ion balance system, so that the pH indicator deviates too much, the color change range is difficult to control, and the protein cannot respond to fine change, and therefore, the isoelectric point range of the selected protein is controlled to be between 3.0 and 10.0. The stability of the protein in the buffer solution is affected by the molecular weight of the protein being too large or too small, so that the molecular weight is limited to 10kDa-200kDa to ensure the stability.

The protein is one of calmodulin, bovine serum albumin, myosin, collagen or lactoferrin. Preferably bovine serum albumin, one of myosin, collagen and lactoferrin, and most preferably one of myosin, lactoferrin and collagen.

The acid-base indicator is bromocresol green, methyl orange, methyl red, nitroyellow, phenolphthalein, bromocresol green-methyl orange, bromocresol green-methyl red, bromocresol green-nitroyellow, bromocresol green-phenolphthalein, methyl orange-methyl red, methyl orange-nitroyellow, methyl orange-phenolphthalein, methyl red-nitroyellow, methyl red-phenolphthalein, nitroyellow-phenolphthalein, bromocresol green-methyl orange-methyl red, bromocresol green-methyl orange-nitroyellow, bromocresol green-methyl orange-phenolphthalein, bromocresol green-methyl red-nitroyellow, bromocresol green-methyl red-phenolphthalein, methyl orange-methyl red-nitroyellow, methyl orange-methyl red-phenolphthalein, methyl red-nitroyellow-phenolphthalein, nitroyellow, phenolphthalein, or mixtures thereof, One of bromocresol green-methyl orange-methyl red-nitro-yellow, bromocresol green-methyl orange-methyl red-phenolphthalein, bromocresol green-methyl red-nitro-yellow-phenolphthalein, methyl orange-methyl red-nitro-yellow-phenolphthalein, and bromocresol green-methyl orange-methyl red-nitro-yellow-phenolphthalein. Preferably one of bromocresol green, nitroflavin, bromocresol green-methyl orange, bromocresol green-nitroflavin, methyl orange-methyl red, methyl red-nitroflavin, nitroflavin-phenolphthalein, bromocresol green-methyl red-nitroflavin, methyl orange-methyl red-phenolphthalein, methyl red-nitroflavin-phenolphthalein, bromocresol green-methyl orange-methyl red-nitroflavin, bromocresol green-methyl red-nitroflavin-phenolphthalein, methyl orange-methyl red-nitroflavin-phenolphthalein or bromocresol green-methyl orange-methyl red-nitroflavin-phenolphthalein, most preferably bromocresol green-nitroflavin, methyl orange-methyl red, nitroflavin-phenolphthalein, One of bromocresol green-methyl red-nitro-yellow, bromocresol green-methyl red-phenolphthalein, bromocresol green-methyl orange-methyl red-nitro-yellow, methyl orange-methyl red-nitro-yellow-phenolphthalein, and bromocresol green-methyl orange-methyl red-nitro-yellow-phenolphthalein.

The buffer solution comprises citric acid-sodium citrate, citric acid-sodium hydroxide, sodium citrate-hydrochloric acid, sodium dihydrogen phosphate-dipotassium hydrogen phosphate, sodium dihydrogen phosphate-sodium hydroxide, dipotassium hydrogen phosphate-hydrochloric acid, dipotassium hydrogen phosphate-phosphoric acid, acetic acid-sodium acetate, and acetic acid-potassium acetate. Preferably citric acid-sodium citrate, citric acid-sodium hydroxide, sodium dihydrogen phosphate-dipotassium hydrogen phosphate, dipotassium hydrogen phosphate-hydrochloric acid, dipotassium hydrogen phosphate-phosphoric acid, acetic acid-sodium acetate, acetic acid-potassium acetate. Most preferably one of citric acid-sodium citrate, sodium dihydrogen phosphate-dipotassium hydrogen phosphate and acetic acid-sodium acetate.

A method of preparing the indicator of claim 1 that responds rapidly to small ph changes, characterized in that: the method comprises the following steps:

1) weighing a certain amount of protein, dissolving the protein in glycerol, and preparing a protein mother solution with the mass fraction of 0.01-0.05%;

2) weighing a certain amount of acid-base indicator and dissolving the acid-base indicator to prepare the acid-base indicator with the mass fraction of 0.05-0.5%, wherein the solvent can be ultrapure water, ethanol or methanol;

3) weighing a certain amount of each component of the buffer solution, and preparing 0.1M buffer solutions with different pH values, wherein the pH value ranges from 2.0 to 14.0;

4) mixing the protein solution obtained in the step 1) with the acid-base indicator obtained in the step 2) in a volume ratio of 1: 5-1: 50;

5) the mixed solution of 4) is added with the buffer solution obtained in 3) to fix the volume.

The indicator is attached to a solid substrate for detecting metabolites of a human body, such as saliva, blood, sweat, urine, semen, amniotic fluid, breast milk, vaginal mucus, cervical mucus and the like, wherein the solid substrate comprises an indicating swab, test paper, dust-free cloth and cellulose dressing.

The invention has the advantages and beneficial effects that:

the invention adds protein with different isoelectric points and molecular weights and matched buffer solution into the conventional indicator to obtain the super-sensitive color indicator capable of rapidly responding to the change of pH value, and the indicator additionally adds the protein with special isoelectric points and molecular weights, so that the color mutation interval of the indicator can be controlled between 0.1 and 0.2, the color change is obvious, and the solid matrix containing the indicator can be used for rapidly detecting different human metabolites.

Drawings

FIG. 1 is the development of a hypersensitive indicator with a mutation point of pH 6.2.

FIG. 2 is the development of a hypersensitive indicator with a mutation point of pH 3.8.

FIG. 3 is a super sensitive indicator swab visualization for detecting vaginal mucus.

FIG. 4 is a color development of a hypersensitive indicator strip for detecting urine.

Detailed Description

The following describes in detail embodiments of the present invention with reference to specific examples.

Example 1 preparation of a hypersensitive indicator having a mutated color development point of pH6.2

Preparing a 0.015% lactoferrin solution: weighing 1.5mg of lactoferrin, dissolving and fixing the volume in 10mL of glycerin; preparing a 0.2% nitro-nitrogen yellow indicator: weighing 0.2g of nitratine yellow to dissolve and fixing the volume in 100mL of absolute ethyl alcohol; preparing a 0.1mol/L citric acid solution: taking 21.01g of citric acid to dissolve and fix the volume in 1000mL of pure water, and preparing 0.1mol/L sodium citrate solution: dissolving 29.41g of sodium citrate and fixing the volume in 1000mL of pure water; mixing 19.0mL of citric acid solution and 81.0mL of sodium citrate solution to prepare SSC buffer solution with pH of 6.0; and uniformly mixing the lactoferrin solution of 100 mu L and the nitroflavin solution of 1.5mL, and fixing the volume to 10mL by SSC solution with the pH value of 6.0 to prepare the hypersensitive indicator with the mutation color development point of pH6.2.

As shown in figure 1, after the prepared hypersensitive indicator with the mutation point of pH6.2 is soaked in filter paper or dust-free cloth and dried, detection solutions with different pH values are dripped, and when the pH value is more than or equal to 6.2, the filter paper and the cloth are indicated to be mutated from yellow to green.

Example 2 preparation of a hypersensitive indicator with a mutated color point of pH 4.4

Preparation of 0.025% myosin solution: weighing 2.5mg of myosin, dissolving and fixing the volume in 100mL of glycerol; preparing 0.1% bromocresol green-nitro nitrogen yellow indicator: simultaneously weighing 0.02g of bromocresol green and 0.08g of nitratine yellow reagent, dissolving and fixing the volume in 100mL of absolute ethyl alcohol; preparing a 0.1mol/L citric acid solution: taking 21.01g of citric acid to dissolve and fix the volume in 1000mL of pure water, and preparing 0.1mol/L sodium citrate solution: dissolving 29.41g of sodium citrate and fixing the volume in 1000mL of pure water; mixing 65.5mL of citric acid solution and 34.5mL of sodium citrate solution to prepare SSC buffer solution with pH of 4.0; and (3) uniformly mixing the 50 mu L of myosin solution and 2mL of bromocresol green-nitroflavin indicator, and diluting to 10mL by using SSC solution with pH4.0 to prepare the super-sensitive indicator with the mutation color development point of pH 4.4.

Example 3 preparation of a hypersensitive indicator with a mutated color point of pH 3.8

Preparing a 0.02% bovine serum albumin solution: weighing 20mg of lactoferrin, dissolving and fixing the volume in 100mL of glycerin; preparing 0.47% bromocresol green-methyl orange-methyl red indicator: simultaneously weighing 0.25g of bromocresol green, 0.1g of methyl orange and 0.12g of methyl red reagent, dissolving and fixing the volume in 100mL of absolute ethyl alcohol; preparing a 0.1mol/L sodium acetate solution: dissolving 8.2g of sodium acetate and fixing the volume in 1000mL of pure water; preparing 0.1mol/L acetic acid solution: diluting 2.86mL of acetic acid solution to 500mL by using ultrapure water; mixing 15.0mL of acetic acid solution and 75.0mL of sodium acetate solution to prepare ABS buffer solution with pH of 3.5; and uniformly mixing 200 mu L of bovine serum albumin solution and 1.5mL of bromocresol green-methyl orange-methyl red indicator, and diluting to 10mL by using an ABS solution with pH of 3.5 to prepare the hypersensitive indicator with the mutation chromogenic point of pH 3.8.

As shown in FIG. 2, after the prepared hypersensitive indicator with the mutation point of pH 3.8 is soaked in filter paper or dust-free cloth and dried, detection solutions with different pH values are dripped, and when the pH value is more than or equal to 3.8, the filter paper and the cloth are indicated to be mutated from yellow to green.

Example 4 preparation of a hypersensitive indicator with a mutated color development point of ph8.0

Preparing a 0.04% bovine serum albumin solution: weighing 4.0mg of bovine serum albumin, dissolving and fixing the volume in 10mL of glycerol; preparing 0.48% bromocresol green-methyl red-nitro nitrogen yellow-phenolphthalein indicator: simultaneously weighing 0.1g of bromocresol green, 0.12g of methyl red, 0.16g of nitratine yellow and 0.1g of phenolphthalein, dissolving and fixing the volume in 100mL of methanol; preparing 0.1mol/L sodium dihydrogen phosphate solution: dissolving 12.0g of sodium dihydrogen phosphate and fixing the volume in 1000mL of pure water; preparing 0.1mol/L dipotassium hydrogen phosphate solution: dissolving 17.4g of dipotassium phosphate and fixing the volume in 1000mL of pure water; mixing 16.0mL of sodium dihydrogen phosphate solution and 84.0mL of dipotassium hydrogen phosphate solution to prepare PBS buffer solution with pH of 7.5; and (3) uniformly mixing the 30 mu L of bovine serum albumin solution and 1.0mL of bromocresol green-methyl red-nitro nitrogen yellow-phenolphthalein, and diluting to 10mL by using PBS (phosphate buffer solution) with pH 7.5 to prepare the super-sensitive indicator with the mutation chromogenic point of pH8.0, namely, when the pH of the detection substance is more than or equal to 8.0, the solution is mutated from gray to blue-purple.

Example 5 preparation of pH-mutant chromogenic Supersensitive indicating swab for detection of vaginal mucus

Preparation of 0.025% myosin solution: weighing 2.5mg of lactoferrin, dissolving and fixing the volume in 10mL of glycerin; preparing 0.26% bromocresol green-nitro nitrogen yellow indicator: simultaneously weighing 0.06g of bromocresol green and 0.2g of nitratine yellow reagent, dissolving and fixing the volume in 100mL of absolute ethyl alcohol; preparing a 0.1mol/L citric acid solution: taking 21.01g of citric acid to dissolve and fix the volume in 1000mL of pure water, and preparing 0.1mol/L sodium citrate solution: dissolving 29.41g of sodium citrate and fixing the volume in 1000mL of pure water; mixing 65.5mL of citric acid solution and 34.5mL of sodium citrate solution to prepare SSC buffer solution with pH of 4.0; and (3) uniformly mixing the 50 mu L of myosin solution and 2mL of bromocresol green-nitroflavin indicator, and diluting to 10mL by using SSC solution with pH4.0 to obtain the super-sensitive indicator with the mutation color development point of pH 4.6. And (3) dropwise adding the super-sensitive indicator on the sampling swab, completely soaking the sampling swab, putting the sampling swab in a vacuum drying oven, and drying the sampling swab for 1 hour at the temperature of 30 ℃ to prepare the super-sensitive indicating swab for super-sensitive detection of vaginal mucus.

The pH value of the vaginal mucus in normal production is 3.8-4.5, and when the pH value is higher than 4.5, the vaginal mucus is one of the manifestations of abnormal leucorrhea. Dipping a vaginal secretion sample by using an indicating swab for detecting vaginal mucus in an ultra-sensitive manner, wherein if the swab does not change color into yellow, the pH of the secretion is normal; if the swab turns green, an abnormal pH of the exudate is indicated. As shown in FIG. 3, different vaginal mucus samples are respectively dropped on a pH test paper and an ultra-sensitive indicating swab, and the actual pH value is judged according to the pH test paper, wherein the color of the swab with the pH value less than or equal to 4.6 is yellow, and the color of the swab with the pH value greater than or equal to 4.6 is green.

EXAMPLE 6 preparation of pH-mutation-developing hypersensitive test paper for detecting urine

Preparing a 0.015% lactoferrin solution: weighing 1.5mg of lactoferrin, dissolving and fixing the volume in 10mL of glycerin; preparing 0.2% nitro-nitrogen yellow-phenolphthalein indicator: weighing 0.12g of nitramine yellow indicator and 0.08g of phenolphthalein, dissolving and fixing the volume in 100mL of absolute ethyl alcohol; preparing a 0.1mol/L citric acid solution: taking 21.01g of citric acid to dissolve and fix the volume in 1000mL of pure water, and preparing 0.1mol/L sodium citrate solution: dissolving 29.41g of sodium citrate and fixing the volume in 1000mL of pure water; mixing 19.0mL of citric acid solution and 81.0mL of sodium citrate solution to prepare SSC buffer solution with pH of 6.0; and uniformly mixing the 280 mu L of lactoferrin solution and 1.5mL of nitrazexanthene-phenolphthalein indicator solution, and metering to 10mL by using SSC solution with pH6.0 to obtain the hypersensitive indicator with the mutation color developing point of pH 6.5.

And if the super-sensitive indicator is dripped on the filter paper to be completely soaked, the super-sensitive indicator is placed in a vacuum drying oven to be dried for 20 minutes at the temperature of 30 ℃, and the super-sensitive test paper for detecting the urine is prepared.

The normal pH value of the urine is 5.5-6.5, and when the pH value is higher than 6.5, the urine is one of the pre-warning manifestations of the body. Using super-sensitive urine to detect urine sample, if the test paper does not change color to yellow, indicating that the pH value of the urine is normal; if the test paper changes color to blue-green, the abnormal pH of the urine is indicated. As shown in FIG. 4, different urine samples are respectively dropped on the pH test paper and the hypersensitive test paper, and the actual pH value is judged according to the pH test paper, wherein the color of the swab with the pH less than or equal to 6.5 is yellow, and the color of the swab with the pH greater than 6.5 is blue-green.

EXAMPLE 7 preparation of pH-mutagenic chromogenic hypersensitive test paper for semen detection

Preparing a 0.04% bovine serum albumin solution: weighing 4.0mg of bovine serum albumin, dissolving and fixing the volume in 10mL of glycerol; preparing 0.48% bromocresol green-methyl red-nitro nitrogen yellow-phenolphthalein indicator: simultaneously weighing 0.1g of bromocresol green, 0.12g of methyl red, 0.16g of nitratine yellow and 0.1g of phenolphthalein, dissolving and fixing the volume in 100mL of methanol; preparing 0.1mol/L sodium dihydrogen phosphate solution: dissolving 12.0g of sodium dihydrogen phosphate and fixing the volume in 1000mL of pure water; preparing 0.1mol/L dipotassium hydrogen phosphate solution: dissolving 17.4g of dipotassium phosphate and fixing the volume in 1000mL of pure water; mixing 16.0mL of sodium dihydrogen phosphate solution and 84.0mL of dipotassium hydrogen phosphate solution to prepare PBS buffer solution with pH of 7.5; and (3) uniformly mixing the 30 mu L of bovine serum albumin solution and 1.0mL of bromocresol green-methyl red-nitro nitrogen yellow-phenolphthalein, and diluting to 10mL by using PBS (phosphate buffer solution) with pH 7.5 to prepare the super-sensitive indicator with the mutation color developing point of pH 8.0.

And (3) dropwise adding the hypersensitive indicator on the water absorption test paper, completely soaking the indicator, and then placing the indicator in a vacuum drying oven to dry for 20 minutes at the temperature of 30 ℃ to prepare the hypersensitive test paper for detecting the semen.

The pH value of the semen during production is 7.2-7.8, and when the pH value is higher than 8.0, inflammation can occur. Dipping the semen sample by using hypersensitive semen detection test paper, and if the test paper does not change color into gray, indicating that the pH value of the semen is normal; if the color of the test paper is bluish purple, the pH of the semen is higher.

Example 8 preparation of hypersensitive wound acidic dressing for pH indication on skin surface

The super-sensitive color indicator is mixed with the cellulose dressing to be used as a wound healing indicator, and the specific formula is as follows:

preparing a 0.025% collagen solution: weighing 2.5mg of collagen, dissolving and fixing the volume in 10mL of glycerol; preparing 0.13% nitro-nitrogen yellow-phenolphthalein indicator: simultaneously weighing 0.05g of nitratine yellow and 0.08g of phenolphthalein reagent, dissolving and fixing the volume in 100mL of sterile water; preparing a 0.1mol/L citric acid solution: dissolving 21.01g of citric acid and fixing the volume in 1000mL of pure water; mixing 27.5mL of citric acid solution and 72.5mL of sodium citrate solution to prepare SSC buffer solution with pH of 5.5; and (3) uniformly mixing the 50 mu L of collagen solution and 2mL of nitro-nitrogen yellow-phenolphthalein indicator, and diluting the mixture to 10mL by using SSC solution with pH 5.5 to obtain the hypersensitive indicator with the mutation color development point of pH 6.5.

Mixing 1mL of the hypersensitive indicator with a piece of cellulose dressing, and placing the mixture in a vacuum drying oven to dry for 10 minutes at the temperature of 25 ℃ after the mixture is uniformly permeated to prepare the hypersensitive wound acidic dressing.

The pH value range of normal skin is 4.0-6.0, and the pH value range of chronic wound surface is 6.5-8.5. When the acidic dressing is applied to the skin wound of a patient, the dressing is light blue; when the wound heals, the dressing may appear colorless or yellowish.

Although the present invention has been described in detail with reference to the preferred embodiments, the present invention is not limited to the embodiments, and the present invention is not limited to the embodiments. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.