CN111595839A - Saliva glucose detection test paper, preparation method and application thereof - Google Patents
Saliva glucose detection test paper, preparation method and application thereof Download PDFInfo
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- CN111595839A CN111595839A CN202010426719.7A CN202010426719A CN111595839A CN 111595839 A CN111595839 A CN 111595839A CN 202010426719 A CN202010426719 A CN 202010426719A CN 111595839 A CN111595839 A CN 111595839A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Abstract
The invention provides saliva glucose detection test paper which comprises an enzyme layer and a color development layer, wherein the enzyme layer comprises glucose oxidase, horseradish peroxidase and vitamin C oxidase. Also provides a preparation method and application thereof. The invention relates to a test paper for rapidly detecting the content of glucose in saliva, which is used for in vitro detection, has important significance for early prevention and preliminary judgment of oral diseases, and also has certain positive significance in the aspect of diabetes prevention.
Description
Technical Field
The invention belongs to the field of medical instruments, and particularly relates to a saliva glucose detection test paper and a preparation method and an application method thereof.
Background
At present, a great deal of research shows that the increase of the content of saliva glucose (called saliva sugar for short) has obvious negative effects on human health. On the one hand, an increase in the salivary sugar content can damage the oral environment, for example, increase the oral osmotic pressure, decrease the pH, slow the salivary flow rate and impair the self-cleaning effect; on the other hand, the increase of the content of the salivary sugar provides nutrient substances for the growth of the flora such as streptococcus mutans, candida, lactobacillus and the like, and causes the proliferation of oral microorganisms. The two factors together cause oral diseases such as xerostomia, periodontitis, gingivitis and dental caries. Meanwhile, some studies show that the saliva sugar concentration of a diabetic is positively correlated with the blood sugar level of the diabetic, so that the probability of suffering from oral diseases is higher than that of a normal person.
Generally, oral diseases occur slowly, often without obvious clinical symptoms at the early stage, and are not easily perceived by patients. The middle and late stages of the disease are reached when the uncomfortable symptoms such as pain appear, the treatment is complicated, and the patient suffers more pain and costs more. Therefore, the determination of the content of saliva sugar is of great significance for the prevention and treatment of oral diseases and diabetes.
Saliva components are complex (such as proteins: mucin, albumin, globulin; enzymes: salivary amylase, maltase, phospholipase; inorganic components: sodium, phosphorus, potassium, magnesium, oxides and sulfates, carbonates, etc.; in addition, urea, uric acid and trace amounts of ammonia, mucin, trace elements, leukocytes, epithelial cells, trace substances, microorganisms, etc.), and theoretically, the glucose concentration thereof is only 1/50-1/100(1.98-119.99mg/L) of the glucose concentration in blood, so that the detection of saliva sugar is currently mainly realized by means of high-precision instruments, such as gas chromatography, liquid chromatography, ion chromatography, gas/liquid-mass combination, etc. These instruments are expensive, complicated to operate, and difficult to perform real-time detection, and thus their applications are limited. Saliva glucose detection is also reported in the literature by electrochemical and optical methods, but both methods have difficulty in accurate detection of glucose in real saliva due to high non-specific interference. The quartz crystal microbalance sensor based on boric acid-based hydrogel in the prior art can realize the specific detection of the salivary sugar. The system converts a glucose concentration signal in saliva into a frequency signal of a quartz crystal oscillator sensor, so that the glucose concentration of the saliva is obtained. The quartz crystal microbalance saliva sugar sensor has the advantages of rapidness and precision, but the sample pretreatment process is complex, so that a professional can be required to complete the detection.
The test paper method for detecting the salivary sugar has the advantages of simple and convenient operation, low price and the likeAnd has better application prospect in the aspect of detecting the salivary sugar. At present, some prior art reports successfully synthesize a test strip for testing glucose, which utilizes glucose oxidase to oxidize glucose to generate hydrogen peroxide, and oxidizes colorless o-dianisidine into a colored product under the catalytic action of catalase so as to enable the test strip to develop color, and the quantitative detection of the glucose is carried out by detecting the color change of the test strip. Although the enzyme coated by the hollow polymer nanofiber has the advantages of small enzyme dosage, high storage stability and the like, the detection object is blood sugar, the enzyme can only respond to high-concentration glucose, and has obvious color change at 10mmol/L, so that the detection requirement of low-concentration saliva sugar cannot be met. In the prior art, glucose oxidase is used for oxidizing glucose to generate hydrogen peroxide, 10-acetyl-3, 7-dihydroxyphenazine is catalyzed into a colored product under the catalysis of catalase, and the change of color is observed by naked eyes to qualitatively judge the height of the glucose. However, only qualitative differences in the level of glucose in saliva are possible. In the prior art, blood is filtered by a membrane, and a chromogen reagent, a color-assisting reagent and H are utilized2O2The chromogenic reaction of (1) detects glucose in blood glucose, but it can respond only to high concentrations of glucose as well.
Disclosure of Invention
Therefore, the invention aims to overcome the defects in the prior art and provide the saliva glucose test paper and the preparation and application methods thereof.
In order to achieve the above object, a first aspect of the present invention provides a saliva glucose test strip, which includes an enzyme layer and a color-developing layer, wherein the enzyme layer includes glucose oxidase, horseradish peroxidase, and vitamin C oxidase;
preferably, the enzyme activity unit ratio of the glucose oxidase, the horseradish peroxidase and the vitamin C oxidase in the enzyme layer is as follows: 50-200: 50-200: 1 to 10, preferably 50 to 150: 100-200: 1-5, most preferably 100:150: 2; and/or
Preferably, the content of the glucose oxidase in the enzyme layer is 500-1500U, more preferably 800-1200U, and most preferably 1000U; the content of horseradish peroxidase is 1000-2000U, preferably 1200-1800U, and most preferably 1500U; the content of the vitamin C oxidase is 10-50U, preferably 10-30U, and most preferably 20U.
According to the saliva glucose test paper of the first aspect, the color development layer comprises 2, 4, 6-tribromo-3-hydroxybenzoic acid and 4-aminoantipyrine.
The saliva glucose test strip according to the first aspect of the invention, wherein the enzyme layer or the color development layer of the test strip further comprises an enzyme activator, a liquid diffusion controller and/or an enzyme stabilizer;
preferably, the enzyme activator is selected from one or more of the following: citral, glutathione, ethylenediamine tetraacetic acid; preferably citral;
preferably, the liquid diffusion controlling agent is selected from one or more of the following: polyethylene glycol 200, polyethylene glycol 600, polyvinyl alcohol; preferably polyethylene glycol 200; and/or
Preferably, the enzyme stabilizer is bovine serum albumin.
The second aspect of the present invention provides a method for preparing the saliva glucose test strip of the first aspect, wherein the method comprises preparing an enzyme layer and a color development layer of the test strip respectively by soaking and drying in an enzyme solution and a color development solution;
preferably, the soaking and drying comprises the following steps: placing the filter paper in the enzyme solution or the color development solution for soaking, and then refrigerating and drying the filter paper;
more preferably, the filter paper is soaked in the enzyme solution for 1 to 10 hours, preferably 2 to 6 hours, and most preferably 5 hours; the refrigeration temperature is-10 to-30 ℃, preferably-15 to-25 ℃, and most preferably-20 ℃; the refrigerating time is 6-24 hours, preferably 6-18 hours, and most preferably 12 hours; the drying temperature is 20-35 ℃, preferably 25-35 ℃, and most preferably 30 ℃; and/or the drying time is 10-60 minutes, preferably 20-50 minutes, and most preferably 30 minutes.
The preparation method according to the second aspect of the present invention, wherein the concentration of glucose oxidase in the enzyme solution is 50 to 200U/mL, preferably 50 to 150U/mL, and most preferably 100U/mL;
the concentration of the horseradish peroxidase is 50-200U/mL, preferably 100-200U/mL, and most preferably 150U/mL; and/or
The concentration of the vitamin C oxidase is 1-10U/mL, preferably 1-5U/mL, and most preferably 2U/mL.
The preparation method according to the second aspect of the invention, wherein in the developing solution, the concentration of 2, 4, 6-tribromo-3-hydroxybenzoic acid is 1-20 mg/mL, preferably 1-10 mg/mL, and most preferably 5 mg/mL; and/or
The concentration of the 4-aminoantipyrine is 10-50 mg/mL, preferably 10-30 mg/mL, and most preferably 20 mg/mL.
The preparation method according to the second aspect of the present invention, wherein the enzyme solution further comprises:
an enzyme activator, wherein the concentration of the enzyme activator is 0.5-2 mg/mL, preferably 1-1.5 mg/mL, and most preferably 1.22 mg/mL;
the addition amount of the liquid diffusion control agent is 10-500 mu L/mL, preferably 50-200 mu L/mL and most preferably 100 mu L/mL; and/or
The concentration of the enzyme stabilizer is 5-20 mg/mL, preferably 8-12 mg/mL, and most preferably 10 mg/mL.
The third aspect of the present invention provides a saliva glucose test device comprising the saliva glucose test strip according to the first aspect or the saliva glucose test strip prepared by the preparation method according to the second aspect.
The device according to the third aspect of the present invention, wherein the test paper further comprises a color chart, absorbent cotton, a water absorption pad and/or a packaging strip;
preferably, the package strip comprises an upper package strip and a lower package strip;
more preferably, the test paper is arranged on the absorbent pad, the absorbent cotton is arranged on the top of the lower packaging strip and above the test paper, and an enzyme layer of the test paper is close to the absorbent cotton.
A fourth aspect of the present invention provides a method for saliva glucose test, the method comprising performing saliva glucose test using the saliva glucose test device of the third aspect;
preferably, the detection method comprises: and placing the absorbent cotton end of the product in the oral cavity for 10 minutes, taking out, observing the color of the color development layer, and judging the content of the saliva glucose by comparing with a color chart.
The invention relates to test paper for rapidly detecting the content of glucose in saliva, which is used for in vitro detection, and the test result is only used for reference. Has important significance for early prevention and preliminary judgment of oral diseases and also has certain positive significance in the aspect of diabetes prevention.
The working principle of the product is as follows: reacting glucose in a saliva sample to be detected with oxygen under the catalytic action of glucose oxidase to generate gluconic acid and hydrogen peroxide; the hydrogen peroxide further reacts with 2, 4, 6-tribromo-3-hydroxybenzoic acid to generate 2, 4-dibromo-3, 6-dioxane-1, 4-diene formic acid under the catalysis of peroxidase, and the product reacts with 4-aminoantipyrine to generate red imine substances. The amount of red substance produced is directly related to the glucose content in saliva, so that the color of the reaction product can indirectly indicate the saliva sugar content.
The saliva glucose test paper can carry out semi-quantitative detection on the content of low-concentration glucose (0-200mg/L) in saliva.
The saliva glucose test strip of the present invention may have the following beneficial effects, but is not limited to:
the invention relates to a test paper for rapidly detecting the content of glucose in saliva, which is used for in vitro detection, has important significance for early prevention and preliminary judgment of oral diseases, and also has certain positive significance in the aspect of diabetes prevention.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows a schematic structural diagram of a saliva glucose detecting device according to the present invention.
FIG. 2 shows a plan view of a saliva glucose test device of the present invention.
FIG. 3 is a graph showing the test results of the saliva glucose test strip of the present invention.
FIG. 4 shows the linear relationship between the glucose concentration and the color intensity of the test strip.
Description of reference numerals:
1. a color comparison card; 2. mounting a packaging strip; 3. absorbent cotton; 4. test paper; 5. a water absorbent pad; 6. a lower packaging strip; 7. an enzyme layer; 8. and a color development layer.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
The reagents and materials used in the following examples are as follows:
reagents and materials:
qualitative filter paper, purchased from Whatman;
enzymatic reagents, purchased from seichen cheng clinical reagents ltd;
glucose oxidase, horseradish peroxidase, polyethylene glycol-200, 2, 4, 6-tribromo-3-hydroxybenzoic acid, citral, purchased from Macklin;
vitamin C oxidase, purchased from Sigma;
bovine serum albumin, purchased from beijing kel comet;
gelatin, phosphate buffer solution, purchased from beijing walford;
4-aminoantipyrine, available from Beijing YinoKay science and technology, Inc.
Example 1
This example is intended to illustrate the structure and preparation of a saliva glucose test device according to the invention.
As shown in FIG. 1, the saliva glucose detecting device of the present invention is composed of the following parts: 1, a color comparison card; an upper package strip 2; absorbent cotton 3; test paper 4; a water absorbent pad 5; a lower encapsulation strip 6.
The assembling process comprises the following steps: the test paper 4 is adhered to the water absorption pad 5, then placed on the lower packaging strip 6, the absorbent cotton 3 is placed in the circular area at the top of the lower packaging strip 6 and placed above the test paper 4, then the whole is assembled by the upper packaging strip 2, and the color card 1 is printed at the rear end of the upper packaging strip 2. The test paper 4 comprises an enzyme layer 7 and a color development layer 8, wherein the enzyme layer 7 is close to the absorbent cotton 3.
The preparation method of the test paper comprises the following steps: the test paper is the core component of the product, and the preparation method is explained below.
Cutting the filter paper into squares of 4cm x 4cm, soaking the filter paper in an enzyme solution prepared in the following step (1) at room temperature for 5 hours, taking the filter paper out of the enzyme solution, wiping off residues on the filter paper, refrigerating the filter paper in a refrigerator at-20 ℃ for 12 hours, and then keeping the filter paper in an oven at 30 ℃ for 30 minutes; then, the filter paper was placed upright (the immersion height was about 0.5cm) in the color developing solution described in the following (2) and soaked at room temperature for 1 to 2 hours, the filter paper was taken out of the color developing solution and the residue on the filter paper was wiped clean, and the filter paper was placed in a refrigerator and refrigerated at-20 ℃ for 12 hours and then kept in an oven at 30 ℃ for 30 minutes. And finally, cutting the filter paper into a proper size.
The first step is to completely immerse the filter paper in the enzyme solution, and the second step is to place the filter paper upright in a color developing solution (having a height of about 0.5cm), at which time the developer will be adsorbed to the lower end of the filter paper. Gelatin is also added into the color developing solution, so that the whole solution is uniform. In addition, due to capillary phenomenon, color developing agent is distributed at about 0.5-1cm position of the lower end of the filter paper. The color-developing agent is also mixed with the enzyme agent attached to the front, which is beneficial to increasing the reaction speed. And the 2-step soaking method ensures that the test paper shows colorless or light yellow, and the product-red can be effectively distinguished, thereby being convenient for observing the result.
(1) Preparation of enzyme solution: injecting 10mL of enzyme reagent into a 70 x 40 culture dish by using a pipette, sequentially adding 1000U of glucose oxidase, 1500U of horseradish peroxidase, 20U of vitamin C oxidase, 1mL of polyethylene glycol-200, 10uL of citral and 100mg of bovine serum albumin into the culture dish, uniformly mixing, and then adding 2mL of gelatin (adding 3g of gelatin into 10mL of distilled water, soaking overnight, and boiling before use);
(2) preparing a color developing solution: 10mL of Phosphate Buffered Saline (PBS) with pH 7.0 was injected into a 70 x 40 petri dish using a pipette, 50mg of 2, 4, 6-tribromo-3-hydroxybenzoic acid and 200mg of 4-aminoantipyrine were sequentially added to the petri dish, and then mixed well, and then 2mL of gelatin was added (10 mL of distilled water was added to 3g of gelatin, and the mixture was soaked overnight and boiled before use).
Test example 1
This example illustrates the use and effect of the saliva glucose test strip of the present invention.
The using method comprises the following steps: the round top end of the product is placed in the oral cavity for 10 minutes and then taken out, the color of the color development layer is directly observed by naked eyes, and the content of the saliva glucose is judged by comparing with a color comparison card.
The invention can realize the semi-quantitative determination of the glucose concentration in saliva, and the experimental verification result is as follows: PBS solutions with glucose concentrations of 0, 10, 20, 50, 100 and 200mg/L are prepared in sequence to test the effectiveness of the test paper. The developed portion of the filter paper was cut into 1cm by 1cm squares and a 30uL sample was applied to the filter paper by pipette. As can be seen from FIG. 3, when the glucose concentration is gradually increased, the red color generated by the reaction is increasingly heavier (the color development area of the test paper is white or light yellow). Thereafter, saliva (the cotton ball in the saliva collecting tube was chewed in the oral cavity for 3-5 minutes and then put back in the collecting tube, centrifuged at 4000r/min for 10 minutes to obtain saliva) was used instead of the PBS solution and glucose was added to the saliva in order at different contents to obtain artificial saliva solutions having concentrations of 0, 10, 20, 50, 100, and 200 in order, and the above experiment was repeated. As can be seen from FIG. 3, the red color generated by the reaction becomes heavier and heavier as the glucose concentration in saliva increases.
The test result in FIG. 3 is quantified by using the brightness of red in three primary colors (RGB) as an index (taking the average value of 100 pixels in the center of the test paper)The data listed in table 1 can be obtained. As can be seen from FIG. 4, the color development intensity of the saliva test paper monotonically decreased with the increase of the glucose concentration in the PBS buffer and saliva, and a better linear relationship (R) between the color development intensity of the test paper and the glucose concentration was found by linear fitting (R)20.9) from which the concentration of test saliva can be semi-quantitatively determined from the color of the test paper.
TABLE 1 salivary sugar concentration and test paper color brightness
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Claims (10)
1. The saliva glucose test paper is characterized by comprising an enzyme layer and a color development layer, wherein the enzyme layer comprises glucose oxidase, horseradish peroxidase and vitamin C oxidase;
preferably, the enzyme activity unit ratio of the glucose oxidase, the horseradish peroxidase and the vitamin C oxidase in the enzyme layer is as follows: 50-200: 50-200: 1 to 10, preferably 50 to 150: 100-200: 1-5, most preferably 100:150: 2; and/or
Preferably, the content of the glucose oxidase in the 10mL of enzyme solution is 500-1500U, more preferably 800-1200U, and most preferably 1000U; the content of horseradish peroxidase is 1000-2000U, preferably 1200-1800U, and most preferably 1500U; the content of the vitamin C oxidase is 10-50U, preferably 10-30U, and most preferably 20U.
2. The saliva glucose test strip of claim 1, wherein the color-developing layer comprises 2, 4, 6-tribromo-3-hydroxybenzoic acid and 4-aminoantipyrine.
3. A saliva glucose test strip according to claim 1 or 2, wherein the enzyme layer of the test strip further comprises an enzyme activator, a liquid diffusion controller and/or an enzyme stabilizer;
preferably, the enzyme activator is selected from one or more of the following: citral, glutathione, ethylenediamine tetraacetic acid; preferably citral; and/or
Preferably, the liquid diffusion controlling agent is selected from one or more of the following: polyethylene glycol 200, polyethylene glycol 600, polyvinyl alcohol; preferably polyethylene glycol 200; and/or
Preferably, the enzyme stabilizer is bovine serum albumin.
4. The method of producing a saliva glucose test strip according to any one of claims 1 to 3, characterized in that the method comprises preparing an enzyme layer and a color-developing layer of the test strip by immersion-drying in an enzyme solution and a color-developing solution, respectively;
preferably, the soaking and drying comprises the following steps: placing the filter paper in the enzyme solution or the color development solution for soaking, and then refrigerating and drying the filter paper;
more preferably, the soaking time of the filter paper is 1-10 hours, preferably 2-6 hours, and most preferably 5 hours; the refrigeration temperature is-10 to-30 ℃, preferably-15 to-25 ℃, and most preferably-20 ℃; the refrigerating time is 6-24 hours, preferably 6-18 hours, and most preferably 12 hours; the drying temperature is 20-35 ℃, preferably 25-35 ℃, and most preferably 30 ℃; and/or the drying time is 10-60 minutes, preferably 20-50 minutes, and most preferably 30 minutes.
5. The preparation method according to claim 4, wherein the concentration of glucose oxidase in the enzyme solution is 50 to 200U/mL, preferably 50 to 150U/mL, and most preferably 100U/mL;
the concentration of the horseradish peroxidase is 50-200U/mL, preferably 100-200U/mL, and most preferably 150U/mL; and/or
The concentration of the vitamin C oxidase is 1-10U/mL, preferably 1-5U/mL, and most preferably 2U/mL.
6. The preparation method according to claim 4 or 5, wherein the concentration of 2, 4, 6-tribromo-3-hydroxybenzoic acid in the color developing solution is 1-20 mg/mL, preferably 1-10 mg/mL, most preferably 5 mg/mL; and/or
The concentration of the 4-aminoantipyrine is 10-50 mg/mL, preferably 10-30 mg/mL, and most preferably 20 mg/mL.
7. The method according to any one of claims 4 to 6, wherein the enzyme solution further comprises:
an enzyme activator, wherein the concentration of the enzyme activator is 0.5-2 mg/mL, preferably 1-1.5 mg/mL, and most preferably 1.22 mg/mL;
the addition amount of the liquid diffusion control agent is 10-500 mu L/mL, preferably 50-200 mu L/mL and most preferably 100 mu L/mL; and/or
The concentration of the enzyme stabilizer is 5-20 mg/mL, preferably 8-12 mg/mL, and most preferably 10 mg/mL.
8. A saliva glucose test device comprising the saliva glucose test strip according to any one of claims 1 to 3 or the saliva glucose test strip produced by the production method according to any one of claims 4 to 7.
9. The saliva glucose detecting device of claim 8, wherein the test strip further comprises a color chart, absorbent cotton, a water absorption pad and/or a packaging strip;
preferably, the package strip comprises an upper package strip and a lower package strip;
more preferably, the test paper is arranged on the absorbent pad, the absorbent cotton is arranged on the top of the lower packaging strip and above the test paper, and an enzyme layer of the test paper is close to the absorbent cotton.
10. A method for saliva glucose testing, comprising using the saliva glucose testing device of claim 9 to perform saliva glucose testing;
preferably, the detection method comprises: and placing the absorbent cotton end of the product in the oral cavity for 10 minutes, taking out, observing the color of the color development layer, and judging the content of the saliva glucose by comparing with a color chart.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114107436A (en) * | 2021-11-26 | 2022-03-01 | 华南理工大学 | Nano-cellulose/wood fiber composite paper-based saliva glucose detection chip |
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