Preparation reagent of bilirubin dry chemical test paper, test paper and preparation method of test paper

Abstract

The invention discloses a preparation reagent of bilirubin dry chemical test paper, the test paper and a preparation method of the test paper. The reagent comprises a main reactant, an acid, an accelerator, an accelerator stabilizer and a solvent, wherein the accelerator is selected from one or more of carrageenan, pectin, gelatin and agar. When the reagent is used for preparing the dry chemical test paper, the preparation process is safe, the acceleration effect is good, and the detection sensitivity and accuracy of the bilirubin dry chemical test paper are improved.

Description

Technical Field

[0001] The present application relates to the field of in vitro diagnosis, and in particular to a dry chemical test paper for detecting bilirubin and a preparation method thereof. Background Art

[0002] Urine testing is one of the important items in routine clinical testing. Positive urine bilirubin may indicate liver cell damage or bile duct obstruction.

[0003] At present, dry chemical test strips can be used to semi-quantitatively detect bilirubin in urine. This type of test is usually performed by adsorbing a reaction reagent that can react with bilirubin to develop color on the test strip, such as a diazonium salt. When the test strip is in contact with urine, the diazonium salt reacts with the bilirubin in the urine to present a purple-red color, and displays different shades of color based on the concentration of bilirubin, thereby presenting a semi-quantitative result for the content of bilirubin in urine. Bilirubin can be divided into indirect bilirubin (or unconjugated bilirubin) that has not been processed by the liver and direct bilirubin (or conjugated bilirubin) formed by the liver and glucuronic acid. Direct bilirubin can react directly with diazonium salts. Indirect bilirubin is insoluble in water due to the mutual binding of intermolecular hydrogen bonds, and is bound to proteins in the blood. It cannot react directly with diazonium salts, and the intermolecular hydrogen bonds need to be broken. When no intramolecular hydrogen bonds are formed, the reaction occurs more fully, and the sensitivity is improved. Insufficient participation of indirect bilirubin in the detection reaction will lead to unreliable test results.

[0004] Therefore, dry chemical test strips usually also contain accelerators, which are used to destroy the hydrogen bonds of bilirubin and maintain the stability of the bilirubin structure without forming intramolecular hydrogen bonds, so that bilirubin can fully react with diazonium salts. The commonly used accelerator is caffeine, and the dosage is large. However, caffeine is a psychotropic drug under state control, its use is restricted, and it is difficult to obtain qualifications. In addition, nitrites are also used as accelerators, but nitrites are highly toxic substances, and the acceleration effect is not ideal.

[0005] Therefore, it is necessary to provide a urine bilirubin dry chemical test paper with good safety and improved test result accuracy. Summary of the invention

[0006] The purpose of the present application is to provide a reagent for preparing dry chemical test paper for detecting bilirubin, especially urinary bilirubin, a method for preparing dry chemical test paper using the reagent, and the dry chemical test paper obtained thereby. The dry chemical test paper uses one or more of carrageenan, pectin, gelatin, and agar as an accelerator, the preparation process is safe, and the acceleration effect is good, thereby improving the sensitivity and accuracy of the test paper detection.

[0007] To this end, in a first aspect, the present invention provides a reagent for preparing a dry chemical test paper for detecting bilirubin, the reagent comprising a main reactant, an acid, an accelerator, an accelerator stabilizer and a solvent, wherein the accelerator is selected from one or more of carrageenan, pectin, gelatin and agar.

[0008] According to some embodiments, the content of the accelerator is 1-5 g / L.

[0009] According to some embodiments, the pH value of the reagent is 1.5-3.0, preferably 1.5-2.5.

[0010] The acid is used to adjust the pH value of the reagent within the above range. According to some embodiments, the acid is selected from one or more of 5-sulfosalicylic acid, metaphosphoric acid, maleic acid, and citric acid, preferably, the acid includes 5-sulfosalicylic acid, and more preferably, the acid includes 5-sulfosalicylic acid and maleic acid.

[0011] According to some embodiments, the acid is present in an amount of 10-30 g / L.

[0012] According to some embodiments, the accelerator stabilizer is D-(+) trehalose. Preferably, the content of the D(+) trehalose is 10-30 g / L.

[0013] According to some embodiments, the main reactant is a diazonium salt. Preferably, the main reactant is 2,4-dichlorobenzene diazonium salt. More preferably, the main reactant is 2,4-dichlorobenzene diazonium tetrafluoroborate.

[0014] According to some embodiments, the content of the main reactant is 0.2-1.0 g / L.

[0015] According to some embodiments, the solvent is water.

[0016] According to some embodiments, the reagent further comprises 0.05-0.1 g / L of yellow dye.

[0017] According to some embodiments, the reagent is provided in the form of a plurality of sub-reagents. Preferably, the plurality of sub-reagents include a first reagent and a second reagent, wherein the first reagent includes the main reactant and the acid dissolved in the solvent; and the second reagent includes the accelerator and the accelerator stabilizer dissolved in the solvent. In some embodiments, the second reagent also includes 0.05-0.1 g / L of yellow dye. According to a preferred embodiment, the reagent is used for the preparation of dry chemical test paper for detecting urinary bilirubin.

[0018] In a second aspect, the present invention provides a method for preparing a dry chemical test paper for detecting bilirubin, comprising the steps of impregnating filter paper with the aforementioned reagent and drying.

[0019] According to some embodiments, the reagent is provided in the form of a plurality of sub-reagents, and the method comprises the steps of treating the filter paper with each of the sub-reagents in sequence and drying.

[0020] Preferably, in the embodiment, the method comprises: treating the filter paper with a first reagent and drying it, the first reagent comprising a main reactant and an acid dissolved in a solvent; and treating the filter paper obtained by being treated and dried by the first reagent with a second reagent and drying it, the second reagent comprising an accelerator, an accelerator stabilizer and an optional yellow dye dissolved in the solvent. In a third aspect, the present invention provides a dry chemical test paper for detecting bilirubin prepared by the above method.

[0021] Preferably, the dry chemical test paper is used to check urine bilirubin.

[0022] The present invention uses water-soluble protein or polysaccharide as an accelerator, which can effectively combine with free bilirubin that breaks the intermolecular hydrogen bond, prevent the free bilirubin from forming intramolecular hydrogen bonds, so that the bilirubin in the sample can fully react with the main reactant, thereby improving the reaction efficiency and the detection sensitivity and accuracy of the test paper. Moreover, since the bilirubin in the sample fully participates in the reaction, the amount of the required main reactant, that is, the diazonium salt, can also be further reduced, thereby reducing the cost.

[0023] The present invention further controls the pH of the reagent for preparing the dry chemical test paper to be relatively acidic at 1.5-2.5, thereby creating an external environment for indirect bilirubin to break the intermolecular hydrogen bonds, which is more conducive to the accelerator to play a role.

[0024] The accelerator stabilizer in the reagent can stabilize the accelerator, thereby further improving the detection sensitivity of the test paper.

[0025] In addition, the reagent has good anti-interference properties to reducing substances such as vitamin C, so that the detection is not interfered by reducing substances in samples, especially urine samples. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the implementation methods of the present invention, not all of the implementation methods. Based on the implementation methods of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0028] As mentioned above, bilirubin can be divided into indirect bilirubin and direct bilirubin, wherein indirect bilirubin cannot directly react with diazonium salts because bilirubin is mutually bound by intermolecular hydrogen bonds. In addition, the free bilirubin formed after the intermolecular hydrogen bonds of indirect bilirubin are disconnected is also very easy to form intramolecular hydrogen bonds, which curls all the hydrophilic groups in the bilirubin molecules inside the molecules, and makes the bilirubin molecules as a whole hydrophobic and insoluble in water. Therefore, when the dry chemical test paper contacts with a sample (such as urine), whether the intermolecular hydrogen bonds of indirect bilirubin can be disconnected to form free bilirubin that does not form intramolecular hydrogen bonds is the key to improving the detection sensitivity and accuracy of the chemical dry test paper for detecting bilirubin.

[0029] Reagents for preparing test strips

[0030] To this end, the present invention provides a reagent for preparing a dry chemical test paper for detecting bilirubin, the reagent comprising a main reactant, an acid, an accelerator, an accelerator stabilizer and a solvent, wherein the accelerator is selected from one or more of carrageenan, pectin, gelatin and agar.

[0031] In the above reagents, the accelerator uses a water-soluble macromolecular protein, such as carrageenan, gelatin or agar; or a water-soluble polysaccharide, such as pectin. Free bilirubin is easily adsorbed on such macromolecules, and the hydrophilic groups (such as hydroxyl / keto groups, imino groups and carboxyl groups) in the bilirubin molecules can combine with a large number of hydrophilic groups in these water-soluble proteins or polysaccharides, thereby preventing the free bilirubin molecules from forming intramolecular hydrogen bonds. These substances used as accelerators have large molecular weights, but can be dissolved in polar solvents such as water, so that they can be evenly attached to the test paper when preparing dry chemical test paper. In addition, the accelerators are all of biological origin, highly safe, non-toxic, and friendly to production and the environment.

[0032] The accelerator may be one of the above substances, or a combination of two or more thereof. The more preferred accelerators are gelatin and agar, and the most preferred is gelatin.

[0033] Because a large number of hydrophilic groups in these macromolecules can fully bind to free bilirubin, the amount of accelerator is reduced. In the reagent, the content of the accelerator is 1-5g / L. For example, the content of the accelerator can be 1g / L, 2g / L, 3g / L, 4g / L, and 5g / L. If the content of the accelerator is too low, it cannot fully bind to bilirubin, especially for samples with high bilirubin content, which may cause part of the bilirubin in the sample to be unable to participate in the reaction. If the content of the accelerator is too high, the viscosity of the reagent may increase, which increases the difficulty of the dipping and finished product preparation steps when preparing the chemical dry test paper, and is not convenient for operation and mass production of products.

[0034] The acid in the reagent is used to adjust the pH of the reagent to 1.5-3.0, preferably 1.5-2.5, so as to provide a strong acidic environment during detection and destroy the intermolecular hydrogen bonds of indirect bilirubin. The acid in the reagent and the accelerator cooperate with each other, so that the accelerator is easy to combine with free bilirubin that has not yet formed intramolecular hydrogen bonds, so as to play a better role.

[0035] Acids that can adjust the pH of the reagent to be within the above range can be used as long as they do not affect the detection. From the perspective of convenience and controllability of preparing test paper, it is preferred to use an acid that is solid at room temperature and soluble in water. According to one embodiment, the acid is selected from one or more of 5-sulfosalicylic acid, metaphosphoric acid, maleic acid, and citric acid. According to a preferred embodiment, the acid includes 5-sulfosalicylic acid, for example, the acid is 5-sulfosalicylic acid; 5-sulfosalicylic acid and maleic acid; 5-sulfosalicylic acid and metaphosphoric acid; 5-sulfosalicylic acid and citric acid, etc. More preferably, the acid includes or is 5-sulfosalicylic acid and maleic acid. According to other embodiments, the acid may also be a mixture of three or all of the above acids.

[0036] The content of the acid in the reagent is 10-30 g / L. When a mixture of acids is used, the amount ratio of each acid is not particularly limited. According to a preferred embodiment, when the acid includes 5-sulfosalicylic acid, the content of 5-sulfosalicylic acid is at least 10 g / L. Those skilled in the art can adjust the amount of the acid according to the type of acid selected and the above pH value.

[0037] The main reactant in the reagent is diazonium salt. Bilirubin can react with diazonium salt to generate purple-red substance. Different amounts of the product result in different shades of color, thus reflecting the amount of bilirubin in the sample.

[0038] The present invention has no particular restrictions on the type of diazonium salt, as long as it can react with bilirubin and generate a color that can qualitatively reflect whether the sample contains bilirubin and the approximate concentration range of the bilirubin contained. Preferably, the diazonium salt is 2,4-dichlorobenzene diazonium salt. More preferably, the diazonium salt is 2,4-dichlorobenzene diazonium tetrafluoroborate. The tetrafluoroboric acid in 2,4-dichlorobenzene diazonium tetrafluoroborate is also an acidic substance, which also plays a certain role in destroying the intermolecular hydrogen bonds of bilirubin, and is therefore more preferred, but the 2,4-dichlorobenzene diazonium salt that can be used in the present invention is not limited thereto.

[0039] Since the reagent adopts the above-mentioned accelerator, the indirect bilirubin can be immediately combined with the hydrophilic groups in the biomacromolecules of the accelerator after breaking the intermolecular hydrogen bonds, and can no longer form intramolecular hydrogen bonds. Therefore, the chemical dry test paper prepared by the reagent is more fully reacted, the main reactant utilization rate is high, and the side reactions are less. As a result, the content of the main reactant in the reagent can be as low as 0.2g / L, and the effect of a detection limit of 8μmol / L can still be obtained. Since diazonium salts are expensive, the cost of chemical dry test paper prepared using the reagent of the present invention can be significantly reduced. For example, when nitrite is used as an accelerator, the amount of 2,4-dichlorobenzene diazonium salt can be 2-6g / L, for example, up to 5g / L. The content of diazonium salt in the reagent of the present invention may not be higher than 1.0g / L. For example, the dosage of the diazonium salt may be 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1.0 g / L, and is more preferably 0.4 to 0.6 g / L, for example 0.5 g / L.

[0040] The function of the accelerator stabilizer in the reagent is to stabilize the accelerator so that it can function better. According to one embodiment, the accelerator stabilizer is D-(+) trehalose. As shown in the following examples, when the reagent does not contain D-(+) trehalose, the detection limit of the prepared test paper does not reach the detection limit of 8 μmol / L determined in most clinical tests, resulting in false negative results or low bilirubin content.

[0041] The content of D(+) trehalose in the reagent is at least 10 g / L, preferably not more than 30 g / L. A higher content of D(+) trehalose (such as 35 g / L or even higher) is also possible, but further increasing its content will not significantly improve the detection effect, but increase the cost. For example, the content of D(+) trehalose can be 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L.

[0042] The solvent of the reagent is not particularly limited and may be a polar solvent. For example, the solvent is any one of water and methanol or a mixed solvent of the two, and the preferred solvent is water. Methanol is often used in the preparation of existing test papers. Due to the toxicity of methanol, it is not preferred from the perspective of production. Moreover, as an organic solvent, the danger of methanol storage and operation is significantly higher than that of water. The components in the reagent are all water-soluble, so water can be used completely as a solvent, further improving the safety of production.

[0043] According to a preferred embodiment, the reagent also includes a yellow dye, preferably a water-soluble yellow dye. The purpose of adding the yellow dye is to make the purple-red substance generated by the reaction of bilirubin and diazonium salt appear orange together with the yellow dye, so that the color development is more stable and it is convenient to carry out instrumental determination. The present invention has no particular restrictions on the specific type of the yellow dye, and one or more of the dyes such as orange yellow G and hydrazine yellow can be cited. The present invention has no particular restrictions on the content of the yellow dye, and can be adjusted to make the test paper present the desired color after color development. For example, the content of the yellow dye can be 0.05-0.1g / L.

[0044] The reagent is provided in the form of a single reagent, or in the form of multiple sub-reagents. Preferably, the reagent is provided in the form of multiple sub-reagents. In other words, each component in the reagent can be provided as a sub-reagent individually or in a mixture of two or more. In each sub-reagent, the corresponding component is provided in the above-mentioned content, and the sub-reagent containing the acid provides the required pH value.

[0045] According to a specific embodiment, the plurality of sub-reagents include a first reagent and a second reagent. The first reagent contains the above-mentioned acid and a main reactant; the second reagent contains the above-mentioned accelerator and an accelerator stabilizer. In the case of a yellow dye, the second reagent further contains a yellow dye.

[0046] In the case of a single reagent, the preparation method of the chemical dry test paper as described in detail below is to treat the filter paper with the single reagent (such as impregnation) and dry it. In the case of multiple sub-reagents, the preparation method of the chemical dry test paper as described in detail below is to treat the filter paper with each sub-reagent in turn (such as impregnation) and dry it. For the latter case, the sensitivity of the prepared test paper can be further improved. For example, for the same reagent formula, when detecting the same sample, the color presented by the test paper prepared by a single reagent will be lighter than the test paper prepared by the sub-reagent. It is explained that the method of using sub-reagents, especially the method of using the first and second reagents and treating the filter paper in this order and drying it, can make the reaction more sufficient.

[0047] However, in either case, substantially the same detection result can be obtained by adjusting the comparison basis during detection (eg, the color of the color card or the threshold of the device).

[0048] Preparation method of test paper

[0049] The present application also provides a method for preparing a chemical dry test paper for detecting bilirubin using the above reagent, wherein the method comprises the steps of treating the filter paper with the reagent and drying it.

[0050] Treating the filter paper with the reagent of any of the above embodiments may be done by immersing the filter paper in the reagent or by wetting the filter paper with the reagent, and the present invention has no particular restrictions on this. The purpose of drying is to volatilize the solvent, such as water. Drying may be performed in an oven at a temperature of 60° C. to 80° C. Other drying methods may also be used, such as air drying or vacuum drying. The present invention has no particular restrictions on the form of drying.

[0051] The method further comprises the step of cutting the dried test paper into a suitable size and shape. The chemical dry test paper finally obtained for detecting bilirubin can be a test paper strip or a test paper block, and the shape can be rectangular, circular, or other polygonal. The present invention has no particular restrictions on the size and shape of the test paper.

[0052] For the single reagent form, the chemical dry test paper can be obtained by performing one treatment and drying.

[0053] For the form of the above sub-reagents, multiple treatments and drying are performed with the sub-reagents in sequence. According to a preferred embodiment, the filter paper is treated and dried with the above first reagent, and then the filter paper treated and dried with the above second reagent is treated and dried.

[0054] Test strips

[0055] The present application also provides a chemical dry test paper for detecting bilirubin prepared by the above method using the above reagent.

[0056] The test paper of the present invention is used for detecting bilirubin in a sample, especially a urine sample, and the bilirubin is urine bilirubin.

[0057] The filter paper prepared by the above reagent has excellent anti-interference ability. It is well known that the components in urine are complex, and the reducing substances therein have obvious interference effects on the detection of bilirubin. Ascorbic acid is the most common reducing substance in urine. As confirmed by the following examples, the filter paper prepared by the above reagent can also resist the interference of, for example, 1.4mmol / L ascorbic acid (vitamin C), and can be applied to urine samples of different subjects.

[0058] Detection Methods

[0059] The chemical dry test paper of the present invention can perform qualitative or semi-quantitative detection on samples, especially urine samples. The detection result can be obtained by comparing with a standard color plate, or by detecting the reflectivity of the test paper after color development by an instrument, and the detection result is given according to a threshold value set in the instrument.

[0060] According to the most widely used index in clinical application, the bilirubin content in the sample is 0, which is a negative result; the bilirubin content in the sample is in the range of 8 mol / L to less than 33 μmol / L, which is one plus sign (1+); the bilirubin content in the sample is in the range of 33 μmol / L to less than 100 μmol / L, which is two plus signs (2+); the bilirubin content in the sample is above 100 μmol / L, which is three plus signs (3+). The following examples confirm that the detection limit of the test paper prepared with the reagent of the present invention is stable at 8 μmol / L, and the test results are accurate.

[0061] The advantages of the present invention are further described below through specific examples.

[0062] Example

[0063] The reagents and bilirubin used below were all commercially available.

[0064] The standard bilirubin stock solution (0.2 g / L) used was diluted as shown in the following table to obtain a gradient solution, wherein the bilirubin content was 0 μmol / L, 8 μmol / L, 33 μmol / L and 100 mol / L, respectively. The following examples and comparative examples were all tested using the bilirubin gradient solution.

[0065] Gradient ratio Mother liquor (mL) Pure water (mL) 0μmol / L 0 1000 8μmol / L 50 950 33μmol / L 150 850 100μmol / L 300 700

[0066] The following examples and comparative examples were tested using Mindray UA-5600 after color development.

[0067] The obtained qualitative test results are determined according to the reflectivity value measured by the instrument. Specifically, when the reflectivity is ≥0.69, the test result is "-", that is, a negative result, which means that the bilirubin level in the sample is normal. When 0.46≤reflectivity<0.69, the test result is "1+", that is, one plus sign; when 0.3≤reflectivity<0.46, the test result is "2+", that is, two plus signs; when the reflectivity is <0.3, the test result is "3+", that is, three plus signs, all of which represent that the bilirubin level in the sample is higher than the normal level, and the more plus signs represent the higher the bilirubin level in the tested sample.

[0068] Example 1

[0069] Measure about 500mL of pure water and add it to the first reagent beaker. Weigh 10g of 5-sulfosalicylic acid and add it to the beaker. After it is fully dissolved, add 20g of maleic acid and stir until it is completely dissolved. Then add 0.5g of 2,4-dichloroaniline diazonium salt tetrafluoroborate and stir until it is completely dissolved. Transfer the dissolved liquid to a 1L volumetric flask and add pure water to make up to 1L to obtain the first reagent, pH = 1.8.

[0070] Measure about 500mL of pure water and add it to another beaker of the second reagent. Weigh 5g of gelatin and add it to the beaker, place the beaker in a heated magnetic stirrer and heat and stir at 60℃ to dissolve. After the gelatin is fully dissolved, cool the solution to room temperature. Weigh 30g of D-(+) trehalose and dissolve it fully, then add 0.1g of hydrazine yellow and dissolve it fully. Transfer the dissolved liquid to a 1L volumetric flask, add pure water to make up to 1L to prepare the second reagent.

[0071] Transfer the first reagent into a glass container, fully soak the filter paper with a specification of 70mm*265mm in the first reagent, scrape off the excess impregnation liquid on the surface of the filter paper and dry it. Transfer the second reagent into another glass container, immerse the dried filter paper in the second reagent, scrape off the excess impregnation liquid on the surface of the filter paper and dry it to obtain the bilirubin dry chemical test paper. Cut the prepared test paper into small pieces of 5*5mm and stick them on the PET bottom plate with double-sided tape. Use the test paper to perform color development reaction on the standard bilirubin gradient solution, and use Mindray UA-5600 for determination after color development. Each gradient solution is tested in parallel with three test papers. The test results are shown in Table 1 below.

[0072] Table 1:

[0073] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 2+ 2+ 3+(100μmol / L) 3+ 3+ 3+

[0074] Comparative Example 1

[0075] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0076] First reagent:

[0077] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.8.

[0078] Second reagent:

[0079] 6g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0080] The test results are shown in Table 2 below:

[0081] Table 2:

[0082] gradient Test 1 Test 2 Test 3 -(0μmol / L) - 1+ - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 3+ 3+ 3+(100μmol / L) 3+ 3+ 3+

[0083] When the amount of accelerator used is too much, false positive results may appear in the test and the accuracy may be reduced.

[0084] Example 2

[0085] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0086] First reagent:

[0087] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.8.

[0088] Second reagent:

[0089] 1g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0090] The test results are shown in Table 3 below:

[0091] Table 3:

[0092] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 1+ 2+ 3+(100μmol / L) 2+ 3+ 3+

[0093] Example 3

[0094] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0095] First reagent:

[0096] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.9.

[0097] Second reagent:

[0098] 5g pectin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0099] The test results are shown in Table 4 below:

[0100] Table 4:

[0101] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 3+ 2+ 3+(100μmol / L) 3+ 3+ 3+

[0102] Example 4

[0103] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0104] First reagent:

[0105] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.8.

[0106] Second Reagent

[0107] 3g pectin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is adjusted to 1L.

[0108] The test results are shown in Table 5 below:

[0109] Table 5:

[0110] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 1+ 2+ 3+(100μmol / L) 3+ 2+ 3+

[0111] Example 5

[0112] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0113] First reagent:

[0114] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.9.

[0115] Second reagent:

[0116] 5g gelatin, 35g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0117] The test results are shown in Table 6 below:

[0118] Table 6:

[0119] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 2+ 2+ 3+(100μmol / L) 3+ 3+ 3+

[0120] Example 6

[0121] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0122] First reagent:

[0123] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.8.

[0124] Second reagent:

[0125] 5g gelatin, 20g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0126] The test results are shown in Table 7 below:

[0127] Table 7:

[0128] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 2+ 2+ 3+(100μmol / L) 3+ 3+ 3+

[0129] Example 7

[0130] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0131] First reagent:

[0132] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is diluted to 1L, pH = 1.8.

[0133] Second reagent:

[0134] 5g gelatin, 10g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0135] The test results are shown in Table 8 below:

[0136] Table 8:

[0137] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 1+ 2+ 3+(100μmol / L) 3+ 3+ 3+

[0138] Comparative Example 2

[0139] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation, wherein the second reagent did not contain D-(+) trehalose as an accelerator stabilizer.

[0140] First reagent:

[0141] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.9.

[0142] Second reagent:

[0143] 5g gelatin, 0.1g hydrazine yellow, and dilute the solution to 1L.

[0144] The test results are shown in Table 9 below:

[0145] Table 9:

[0146] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) - 1+ - 2+(33μmol / L) 2+ 1+ 2+ 3+(100μmol / L) 2+ 3+ 3+

[0147] When no accelerator or stabilizer was used, 5 g of gelatin was used, but the sensitivity of the test was still reduced, and the lower limit of detection did not reach 8 μmol / L, resulting in a false negative result.

[0148] Comparative Example 3

[0149] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation, wherein the second reagent did not contain D-(+) trehalose as an accelerator stabilizer.

[0150] First reagent:

[0151] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.8.

[0152] Second reagent:

[0153] 5g pectin, 0.1g hydrazine yellow, and dilute the solution to 1L.

[0154] The test results are shown in Table 10 below:

[0155] Table 10:

[0156] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) - - - 2+(33μmol / L) 1+ 1+ 1+ 3+(100μmol / L) 2+ 2+ 2+

[0157] When no accelerator or stabilizer was used, the detection sensitivity was reduced more significantly when 5 g of pectin was used, and the detection limit could only reach 33 μmol / L.

[0158] Example 8

[0159] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0160] First reagent:

[0161] 10g 5-sulfosalicylic acid, 20g maleic acid, 1.0g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.8.

[0162] Second reagent:

[0163] 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0164] The test results are shown in Table 11 below:

[0165] Table 11:

[0166] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 2+ 2+(33μmol / L) 2+ 2+ 2+ 3+(100μmol / L) 3+ 3+ 3+

[0167] Example 9

[0168] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0169] First reagent:

[0170] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.2g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.9.

[0171] Second reagent:

[0172] 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0173] The test results are shown in Table 12 below:

[0174] Table 12:

[0175] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 1+ 2+ 2+ 3+(100μmol / L) 3+ 3+ 2+

[0176] Example 10

[0177] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0178] First reagent:

[0179] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.15g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is diluted to 1L, pH = 1.8.

[0180] Second reagent:

[0181] 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0182] The test results are shown in Table 13 below:

[0183] Table 13:

[0184] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) - 1+ 1+ 2+(33μmol / L) 1+ 1+ 1+ 3+(100μmol / L) 2+ 1+ 2+

[0185] Embodiment 11

[0186] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0187] First reagent:

[0188] 10g 5-sulfosalicylic acid, 20g metaphosphoric acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 2.5.

[0189] Second reagent:

[0190] 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0191] The test results are shown in Table 14 below:

[0192] Table 14:

[0193] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 2+ 1+ 3+(100μmol / L) 2+ 3+ 3+

[0194] Example 12

[0195] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0196] First reagent:

[0197] 10g 5-sulfosalicylic acid, 20g citric acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.7.

[0198] Second reagent:

[0199] 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0200] The test results are shown in Table 15 below:

[0201] Table 15:

[0202] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 1+ 2+ 1+ 3+(100μmol / L) 2+ 2+ 3+

[0203] Example 13

[0204] The first reagent and the second reagent were prepared in the same manner as in Example 1, and the standard bilirubin gradient solution was detected in the same manner, except that the following formula was used for preparation.

[0205] First reagent:

[0206] 30g 5-sulfosalicylic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is diluted to 1L, pH = 2.8.

[0207] Second reagent:

[0208] 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0209] The test results are shown in Table 16 below:

[0210] Table 16:

[0211] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 2+ 1+ 3+(100μmol / L) 2+ 2+ 2+

[0212] Embodiment 14

[0213] Measure about 500mL of pure water and add it to the first reagent beaker. Weigh 10g of 5-sulfosalicylic acid and add it to the beaker. After it is fully dissolved, add 20g of maleic acid, 0.5g of 2,4-dichloroaniline diazonium salt tetrafluoroborate, 30g of D-(+) trehalose and 0.1g of hydrazine yellow in sequence. Stir each substance until it is completely dissolved to obtain a mixed solution. Weigh 5g of gelatin and mix it with an appropriate amount of pure water in another beaker. Place the beaker in a heated magnetic stirrer and heat and stir at 60°C to dissolve. After the gelatin is fully dissolved, combine the dissolved gelatin liquid with the aforementioned mixed solution, transfer it to a 1L volumetric flask, add pure water to make up to 1L, and obtain the reagent, pH = 1.8.

[0214] Transfer the prepared reagent into a glass container, fully soak the filter paper with a specification of 70mm*265mm in the reagent, scrape off the excess impregnation liquid on the surface of the filter paper and dry it to obtain the bilirubin dry chemical test paper. Cut the prepared test paper into small pieces of 5*5mm and stick them on the PET bottom plate with double-sided tape. Use the test paper to perform color development reaction on the standard bilirubin gradient solution, and then use Mindray UA-5600 to measure it after color development. Use three test papers for each gradient solution for parallel testing.

[0215] The test results are shown in Table 17 below.

[0216] Table 17:

[0217]

[0218]

[0219] Embodiment 15

[0220] The reagent was prepared in the same manner as in Example 15, and the standard bilirubin gradient solution was tested in the same manner, except that the following formula was used for preparation:

[0221] 10g 5-sulfosalicylic acid, 20g maleic acid, 2g 2,4-dichloroaniline diazonium tetrafluoroborate, 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, the solution is diluted to 1L, pH = 1.8.

[0222] The test results are shown in Table 18 below.

[0223] Table 18:

[0224] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 2+ 1+ 3+(100μmol / L) 3+ 2+ 2+

[0225] Example 16

[0226] The reagent was prepared in the same manner as in Example 15, and the standard bilirubin gradient solution was tested in the same manner, except that the following formula was used for preparation:

[0227] 10g 5-sulfosalicylic acid, 20g maleic acid, 4g 2,4-dichloroaniline diazonium tetrafluoroborate, 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, the solution is diluted to 1L, pH = 1.8.

[0228] The test results are shown in Table 19 below.

[0229] Table 19:

[0230] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 2+ 2+ 3+(100μmol / L) 3+ 3+ 2+

[0231] Embodiment 17

[0232] The first reagent and the second reagent were prepared in the same manner as in Example 1, except that the following formulas were used for the preparation.

[0233] First reagent:

[0234] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.8.

[0235] Second reagent:

[0236] 5g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0237] The standard bilirubin gradient solution was tested in the same manner as in Example 1, except that 1.4 mmol / L of vitamin C was added to each standard bilirubin gradient solution.

[0238] The test results are shown in Table 20 below.

[0239] Table 20:

[0240] gradient Test 1 Test 2 Test 3 -(0μmol / L) - - - 1+(8μmol / L) 1+ 1+ 1+ 2+(33μmol / L) 2+ 2+ 2+ 3+(100μmol / L) 3+ 3+ 3+

[0241] Embodiment 18

[0242] The first reagent and the second reagent were prepared in the same manner as in Example 1, except that the following formulas were used for the preparation.

[0243] First reagent:

[0244] 10g 5-sulfosalicylic acid, 20g maleic acid, 0.5g 2,4-dichloroaniline diazonium tetrafluoroborate, the solution is adjusted to 1L, pH = 1.8.

[0245] Second reagent:

[0246] 1g gelatin, 30g D-(+) trehalose, 0.1g hydrazine yellow, and the solution is diluted to 1L.

[0247] The standard bilirubin gradient solution was tested in the same manner as in Example 1, except that 1.4 mmol / L of vitamin C was added to each standard bilirubin gradient solution.

[0248] The test results are shown in Table 21 below.

[0249] Table 21:

[0250]

[0251]

[0252] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A reagent for preparing a dry chemical test paper for detecting bilirubin, the reagent comprising a main reactant, an acid, an accelerator, an accelerator stabilizer and a solvent, wherein: The accelerator is one or more selected from carrageenan, pectin, gelatin and agar.

2. The reagent according to claim 1, wherein the content of the accelerator is 1-5 g / L.

3. The reagent according to claim 1, wherein the pH value of the reagent is 1.5-3.0, preferably 1.5-2.

5.

4. The reagent according to claim 3, wherein the acid is selected from one or more of 5-sulfosalicylic acid, metaphosphoric acid, maleic acid, and citric acid, preferably, the acid comprises 5-sulfosalicylic acid, more preferably, the acid comprises 5-sulfosalicylic acid and maleic acid. The reagent according to claim 4 , wherein the content of the acid is 10-30 g / L.

6. The reagent according to claim 1, wherein the accelerator stabilizer is D-(+) trehalose, preferably, the content of the D(+) trehalose is 10-30 g / L.

7. The reagent according to claim 1, wherein the main reactant is a diazonium salt, preferably, the main reactant is 2,4-dichlorobenzene diazonium salt, more preferably, the main reactant is 2,4-dichlorobenzene diazonium tetrafluoroborate.

8. The reagent according to claim 7, wherein the content of the main reactant is 0.2-1.0 g / L.

9. The reagent according to claim 1, wherein the solvent is water.

10. The reagent according to any one of claims 1 to 9, wherein the reagent further comprises 0.05-0.1 g / L of a yellow dye.

11. The reagent according to any one of claims 1 to 9, wherein the reagent is provided in the form of a plurality of sub-reagents, preferably, the plurality of sub-reagents include a first reagent and a second reagent, wherein the first reagent includes the main reactant and the acid dissolved in the solvent; and the second reagent includes the accelerator and the accelerator stabilizer dissolved in the solvent, wherein preferably, the second reagent also includes 0.05-0.1 g / L of a yellow dye.

12. A method for preparing a dry chemical test paper for detecting bilirubin, comprising the steps of treating filter paper with a reagent according to any one of claims 1 to 11 and drying.

13. The method according to claim 12, wherein: The reagent is provided in the form of a plurality of sub-reagents, and the method comprises the steps of treating the filter paper with each of the sub-reagents in sequence and drying. Preferably, the method comprises: Treating the filter paper with a first reagent and drying the first reagent, wherein the first reagent includes a main reactant and an acid dissolved in a solvent; The filter paper obtained by being treated with the first reagent and dried is treated with a second reagent comprising an accelerator, an accelerator stabilizer and optionally a yellow dye dissolved in the solvent and dried.

14. A dry chemical test paper for detecting bilirubin, prepared by the method according to claim 12 or 13.

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