CN109115760B - A kind of rapid detection kit and detection method of urine sugar content based on chloroauric acid color reaction - Google Patents

Abstract

The invention discloses a rapid detection kit and a detection method for urine glucose content based on chloroauric acid color reaction, wherein the kit comprises a chloroauric acid reagent bottle and a urine glucose concentration colorimetric card, and can also comprise a urine glucose concentration accurate quantitative card.

Description

Rapid detection kit and detection method for urine sugar content based on chloroauric acid color reaction

Technical Field

The invention belongs to the technical field of detection of urine sugar, and particularly relates to a urine sugar rapid detection kit based on color development of gold ions in an oxidation-reduction reaction process, and a method for rapidly and qualitatively detecting the content of urine sugar by using the kit.

Background

Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. Diabetes mellitus is a long-standing hyperglycemia, causes chronic damage and dysfunction of various tissues, particularly eyes, kidneys, heart, blood vessels and nerves, and is one of the most known complications at present.

At present, the diagnosis and monitoring of diabetes mainly comprises the detection of blood sugar and urine sugar concentration, wherein the blood sugar detection is generally carried out by a hospital for blood drawing test or a household blood glucose meter for self-test, the two methods both need the patients to suffer from the pain of puncturing, and particularly, the patients with confirmed diabetes need to monitor urine sugar every day, which brings extra burden to the bodies and the minds of the patients; the current commonly used method for detecting urine sugar comprises a qualitative detection method of the urine sugar of the speckles, a urine sugar test paper method and the like, and the method is used for carrying out semi-quantitative analysis on the content of glucose in urine by colorimetry. Due to the difference of the color sensitivity of different individuals, the detection method by taking color comparison as a means has strong subjectivity, and particularly has low judgment reliability on the color near the critical value, so that patients with urine glucose values near the critical value need to be rechecked at home or in hospitals, and the time and economic cost of the patients and even medical systems are increased.

Nano-gold particles, also known as colloidal gold. At present, various gold nanoparticles with different sizes (1.0-50nm) are prepared by a chemical agent synthesis method, solutions of the gold nanoparticles sequentially show blue, purple red, peach red and wine red with different degrees according to different sizes of the gold nanoparticles, corresponding peak absorbance can be detected near 520nm by an ultraviolet visible spectrophotometer, and the wavelength corresponding to the peak absorbance is in a linear relation with the particle size of the gold nanoparticles.

Glucose (glucose) is an organic compound of formula C₆H₁₂O₆The monosaccharide is a monosaccharide which is most widely distributed and important in the nature, is polyhydroxy aldehyde, has reducibility, and can be used as a reducing agent for synthesizing nano gold particles. The glucose solution reacts with gold ions to generate gold nanoparticles with different particle sizes, the glucose concentration and the size of the gold nanoparticles have a linear relation, and the solution shows different colors according to the size of the particles.

Based on the characteristics, most of the existing urine sugar detection methods use a color reaction and a colorimetric card for reading, for example, CN102749327A uses a liquid chemical reagent to react with urine sugar to color to judge the approximate urine sugar concentration, and the sensitivity is 1 mM; CN104535753A was developed by reaction with urine glucose using a test paper and the concentration of urine glucose was judged by combining with a colorimetric plate, and the sensitivity was 0.5 mM. There is no correlation research result for accurately detecting the concentration of urine sugar by using a urine sugar reducing gold ion color reaction and colorimetric qualitative determination of the concentration of urine sugar, or using a curve fitting formula for correlation between the concentration of urine sugar and the size of nano-gold particles.

Disclosure of Invention

The invention aims to provide a rapid urine glucose detection kit based on a chloroauric acid color reaction and a method for qualitatively and quantitatively detecting the urine glucose content by adopting the kit. By adopting the kit and the detection method, the application field of the color reaction for preparing the nano-gold by the chloroauric acid is expanded; meanwhile, the efficiency of qualitative and quantitative detection of the urine glucose is effectively improved.

Aiming at the purposes, the urine glucose rapid detection kit adopted by the invention comprises a chloroauric acid reagent bottle and a urine glucose concentration colorimetric card; wherein the chloroauric acid reagent bottle is a disposable covered sealed glass bottle filled with 2-5 mL of chloroauric acid color developing solution, and the chloroauric acid color developing solution is prepared by adjusting the pH value of 0.5mM chloroauric acid aqueous solution to 7-9 by 0.2M sodium hydroxide aqueous solution; the urine glucose concentration colorimetric card consists of a color block A, a color block B, a color block C, a color block D, a color block E, a color block F, a color block G, a color block H and a color block I, wherein subscripts of the color block A and the color block B are more than or equal to 12mM, and the corresponding urine glucose concentration is more than or equal to 12 mM; 11-10mM is marked below the color blocks B and C, and the corresponding urine glucose concentration is 10-11 mM; 9-8mM is marked below the color blocks D and E, and the corresponding urine glucose concentration is 8-9 mM; 7-6mM is marked below the color blocks F and G, and the corresponding urine glucose concentration is 6-7 mM; 5-4mM is marked below the color blocks G and H, and the concentration of corresponding urine sugar is 4-5 mM; the color block H and the color block I are marked with less than or equal to 3mM below and the corresponding urine glucose concentration is less than or equal to 3 mM.

The manufacturing method of the urine glucose concentration colorimetric card comprises the following steps:

1. dissolving glucose dry powder with normal human urine to prepare 100mM glucose stock solution for diabetes, then diluting the glucose stock solution with normal human urine to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12mM respectively, and then sequentially diluting the glucose stock solution with deionized water by 20 times to prepare the glucose standard solution for urine.

2. According to the volume ratio of the urine sugar standard solution to the chloroauric acid color developing solution of 1:15, preheating the urine sugar standard solution through a boiling water bath, adding the preheated urine sugar standard solution into a chloroauric acid reagent bottle preheated through the boiling water bath, rapidly stirring for 5-10 seconds, and continuing to keep the boiling water bath for heating reaction for 5-6 minutes to obtain a nano gold solution; and manufacturing the urine glucose concentration colorimetric card according to the colors of the nano-gold solutions corresponding to the urine glucose standard solutions with different concentrations.

The capacity of the disposable capped and sealed glass bottle is 8mL, the height of the glass bottle is 70-80 mm, the diameter of the glass bottle is 10-15 mm, the glass bottle is made of a screw quartz glass material, and a heat-resistant leakage-proof gasket is additionally arranged inside the heat-resistant plastic material of the bottle cap.

In order to further accurately detect the content of the urine sugar, the kit can further comprise a urine sugar concentration accurate quantitative card, and the accurate quantitative card is written with a formula: Y-3E +44X^-15.95(in the formula, E is a scientific notation, E + n represents the power of 10 to the power of n, and the formula expression can also be:

) The formula is obtained by the following method: dripping 1.5-2 mL of the nanogold solution reacted in thestep 2 into a quartz cuvette, detecting the wavelength corresponding to the peak absorbance of the nanogold solution within the range of 500-600 nm by using an ultraviolet-visible spectrophotometer, and drawing a correlation curve of the wavelength corresponding to the peak absorbance and the glucose concentration to obtain the formula Y-3E +44X^-15.95Wherein X represents the wavelength corresponding to the peak absorbance of the nano-gold solution within the range of 500-600 nm, and Y represents the concentration of glucose in the urine to be detected.

The method for rapidly detecting the content of the urine sugar by adopting the kit comprises the following steps: diluting the urine to be detected by 20 times by using deionized water to obtain a diluted solution of the urine to be detected; preheating the urine diluent to be detected by using a boiling water bath, adding the preheated urine diluent to be detected into a chloroauric acid reagent bottle preheated by the boiling water bath according to the volume ratio of the urine diluent to be detected to the chloroauric acid developing solution of 1:15, rapidly stirring for 5-10 seconds, then continuously keeping the boiling water bath for heating and reacting for 5-6 minutes to enable glucose in the urine and the chloroauric acid to generate oxidation-reduction developing reaction, comparing the color of the obtained nanogold solution with a urine glucose concentration colorimetric card after the reaction is finished, and judging the concentration range of the glucose.

After the concentration range of glucose is judged, 1.5-2 mL of nano-gold solution is dripped into a quartz cuvette, an ultraviolet-visible spectrophotometer is adopted to detect the wavelength corresponding to the peak absorbance within the range of 500-600 nm, and the formula Y on the accurate urine glucose concentration quantification card is 3E +44X^-15.95To be detectedThe glucose concentration in the urine was quantitatively calculated.

The invention has the following beneficial effects:

1. according to the method, glucose in urine is used for reducing the tetrachloroauric acid, the content of urine glucose can be rapidly and qualitatively determined by comparing a colorimetric card according to different colors developed by the nano-gold solution, the steps are simple, and the reaction speed is high. The chloroauric acid reagent bottle is stored in a shade dark place and can be stored for half a year.

2. When the urine glucose content needs to be accurately detected, an ultraviolet-visible spectrophotometer is adopted, the glucose content in urine is accurately calculated through a correlation fitting formula of the wavelength of the peak absorbance of the nanogold solution and the glucose concentration in the urine, and the sensitivity is 0.01 mM.

3. The invention completes qualitative and quantitative detection of two kinds of urine sugar contents through one-time chemical color reaction, and effectively improves the urine sugar detection efficiency.

Drawings

FIG. 1 is a photograph of a urine glucose concentration colorimetric card in example 1.

FIG. 2 is a graph showing the results of absorbance detection of the nano-gold solution in the range of 450 to 700nm by an ultraviolet-visible spectrophotometer corresponding to the standard solutions of glucose in urine of different concentrations in example 1.

FIG. 3 is a graph of the correlation between the peak absorbance of the nanogold solution and the wavelength of the nanogold solution corresponding to the standard solution of urine glucose at different concentrations in example 1, and the fitting curve.

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

The kit for rapidly detecting the urine glucose content of the embodiment consists of a chloroauric acid reagent bottle, a urine glucose concentration colorimetric card and a urine glucose concentration accurate quantification card.

The chloroauric acid reagent bottle is a disposable capped sealed glass bottle filled with 3mL of chloroauric acid color developing solution, the chloroauric acid color developing solution is prepared by adjusting the pH value of 0.5mM aqueous solution of chloroauric acid to 8 with 0.2M aqueous solution of sodium hydroxide, the disposable capped sealed glass bottle has the capacity of 8mL, the height of 75mM and the diameter of 12mM, is made of a screw quartz glass material, and the bottle cap is made of a heat-resistant plastic material and is internally provided with a heat-resistant leakage-proof gasket.

The urine glucose concentration colorimetric card is shown in figure 1 and comprises a color block A, a color block B, a color block C, a color block D, a color block E, a color block F, a color block G, a color block H and a color block I, wherein the subscripts of the color block A and the color block B are more than or equal to 12mM, and the corresponding urine glucose concentration is more than or equal to 12 mM; 11-10mM is marked below the color blocks B and C, and the corresponding urine glucose concentration is 10-11 mM; 9-8mM is marked below the color blocks D and E, and the corresponding urine glucose concentration is 8-9 mM; 7-6mM is marked below the color blocks F and G, and the corresponding urine glucose concentration is 6-7 mM; 5-4mM is marked below the color blocks G and H, and the concentration of corresponding urine sugar is 4-5 mM; the color block H and the color block I are marked with less than or equal to 3mM below and the corresponding urine glucose concentration is less than or equal to 3 mM. The urine glucose concentration colorimetric card is manufactured by the following method:

1. weighing 0.18g of glucose dry powder, dissolving the glucose dry powder by using 10mL of normal human urine to prepare 100mM of grape diabetes stock solution, then respectively diluting the grape diabetes stock solution to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12mM by using the normal human urine, and sequentially diluting the grape diabetes stock solution by using deionized water for 20 times to prepare the urine glucose standard solution.

2. Preheating the standard urine sugar solution in a boiling water bath for 5 minutes, adding 200 mu L of the standard urine sugar solution into a chloroauric acid reagent bottle preheated in the boiling water bath for 5 minutes, rapidly stirring for 5 seconds, continuing to keep the boiling water bath for heating and reacting for 6 minutes until the color of the solution is stable, and ending the reaction, wherein the color of the mixture is changed from yellow to colorless, blue or red, which indicates that the nano gold solution is prepared; according to the color of the nano-gold solution corresponding to the standard urine glucose solution with different concentrations, a urine glucose concentration colorimetric card is manufactured, as shown in fig. 1.

The accurate quantitative card for the concentration of urine glucose is written with the formula: Y-3E +44X^-15.95The formula is obtained according to the following method:

taking 1.5mL of each of the nanogold solutions reacted in thestep 2, respectively dripping the nanogold solutions into a clean quartz cuvette, sequentially detecting the absorbance of the nanogold solutions within the wavelength range of 450-700 nm by using an ultraviolet-visible spectrophotometer (see fig. 2), and drawing a correlation curve (see fig. 3) of the wavelength corresponding to the peak absorbance within the wavelength range of 500-600 nm and the glucose concentration to obtain the formulaY & lt 3E & gt +44X^-15.95，R²0.9822, wherein X represents the wavelength corresponding to the peak absorbance of the nano-gold solution in the range of 500-600 nm, and Y represents the glucose concentration in the urine to be detected. As can be seen from fig. 3, the fitting formula has a higher goodness of fit with the correlation curve.

Example 2

The method for rapidly detecting the content of urine sugar by using the kit of theembodiment 1 comprises the following steps:

diluting the urine to be detected by 20 times by using deionized water to obtain a diluted solution of the urine to be detected; preheating the urine diluent to be detected by using a boiling water bath, adding 200 mu L of the urine diluent to a chloroauric acid reagent bottle preheated by the boiling water bath for 5 minutes, rapidly stirring for 5 seconds, continuously keeping the boiling water bath for heating and reacting for 6 minutes, enabling glucose in the urine and chloroauric acid to generate an oxidation-reduction color reaction, comparing the color of the obtained nanogold solution with a urine glucose concentration colorimetric card after the reaction is finished, and judging the glucose concentration range.

After the concentration range of glucose is judged, 1.5mL of the nanogold solution obtained after the reaction is finished is dripped into a quartz cuvette, the absorbance of the nanogold solution in the wavelength range of 450-700 nm is detected by adopting an ultraviolet-visible spectrophotometer, the wavelength corresponding to the peak absorbance in the wavelength range of 500-600 nm is obtained, and the formula Y on the urine glucose concentration accurate quantitative card is 3E +44X^-15.95And quantitatively calculating the concentration of glucose in the urine to be detected.

Claims (3)

Translated fromChinese

1.一种基于氯金酸显色反应的尿糖含量快速检测试剂盒，其特征在于：该试剂盒包括氯金酸试剂瓶和尿糖浓度比色卡；其中，所述的氯金酸试剂瓶是装有2～5 mL氯金酸显色液的一次性加盖密封玻璃瓶，氯金酸显色液是将0.5 mM氯金酸水溶液用0.2 M氢氧化钠水溶液调节pH值7～9配制而成；所述的尿糖浓度比色卡由色块A、色块B、色块C、色块D、色块E、色块F、色块G、色块H、色块I组成，其中色块A和色块B下标注有≥12 mM，对应尿糖浓度≥12mM；色块B和色块C下方标注有11-10 mM，对应尿糖浓度为10～11 mM；色块D和色块E下方标注有9-8 mM，对应尿糖浓度8～9 mM；色块F和色块G下方标注有7-6 mM，对应尿糖浓度6～7mM；色块G和色块H下方标注有5-4 mM，对应尿糖浓度4～5 mM；色块H和色块I下方标注有≤3mM，对应尿糖浓度≤3 mM；1. a kind of urine sugar content rapid detection kit based on chloroauric acid color reaction, it is characterized in that: this test kit comprises chloroauric acid reagent bottle and urine sugar concentration colorimetric card; Wherein, described chloroauric acid reagent The bottle is a one-time capped and sealed glass bottle filled with 2-5 mL of chloroauric acid chromogenic solution. The chloroauric acid chromogenic solution is a 0.5 mM chloroauric acid aqueous solution adjusted with a 0.2 M sodium hydroxide aqueous solution to pH 7 to 9 Formulated; the urine sugar concentration color chart is composed of color block A, color block B, color block C, color block D, color block E, color block F, color block G, color block H, color block I , in which the color block A and color block B are marked with ≥12 mM, and the corresponding urine sugar concentration is ≥12 mM; the color block B and color block C are marked with 11-10 mM, and the corresponding urine sugar concentration is 10-11 mM; color blocks D and color block E are marked with 9-8 mM, corresponding to the urine sugar concentration of 8-9 mM; color block F and color block G are marked under 7-6 mM, corresponding to the urine sugar concentration of 6-7 mM; color block G and color Block H is marked with 5-4 mM, corresponding to the urine sugar concentration of 4-5 mM; color blocks H and I are marked with ≤3 mM, corresponding to the urine sugar concentration of ≤3 mM;所述的尿糖浓度比色卡的制作方法如下：The preparation method of described urine sugar concentration colorimetric card is as follows:（1）将葡萄糖干粉用正常人尿液溶解，制备成100 mM葡萄糖尿液原液，然后将其用正常人尿液分别稀释至1、2、3、4、5、6、7、8、9、10、11、12mM，再依次用去离子水稀释20倍，制成尿糖标准液；(1) Dissolve the glucose dry powder in normal human urine to prepare a 100 mM glucose urine stock solution, and then dilute it with normal human urine to 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12mM, and then diluted 20 times with deionized water in turn to make urine sugar standard solution;（2）按照尿糖标准液与氯金酸显色液的体积比为1:15，将尿糖标准液经过沸水浴预热后加入沸水浴预热的氯金酸试剂瓶中，并迅速搅拌5～10秒钟后继续保持沸水浴加热反应5～6分钟，得到纳米金溶液；根据不同浓度尿糖标准液对应的纳米金溶液的颜色，制作成尿糖浓度比色卡；(2) According to the volume ratio of urine sugar standard solution and chloroauric acid chromogenic solution of 1:15, add urine sugar standard solution to the preheated chloroauric acid reagent bottle in a boiling water bath after preheating in a boiling water bath, and stir quickly. After 5 to 10 seconds, continue to keep the heating reaction in a boiling water bath for 5 to 6 minutes to obtain a nano-gold solution; according to the color of the nano-gold solution corresponding to the urine sugar standard solution of different concentrations, make a urine sugar concentration colorimetric card;所述试剂盒还包括尿糖浓度精确定量卡，该精确定量卡上写有公式：Y=3×10⁴⁴X^-15.95；所述尿糖浓度精确定量卡上的公式由下述方法得到：取上述步骤（2）中反应后的纳米金溶液1.5～2 mL滴入石英比色皿中，采用紫外可见分光光度计检测其在500～600 nm范围内的峰值吸光度所对应的波长，绘制峰值吸光度所对应的波长与葡萄糖浓度的相关性曲线，即得公式Y=3×10⁴⁴X^-15.95，其中X代表纳米金溶液在500～600 nm范围内的峰值吸光度所对应的波长，Y代表待检测尿液中的葡萄糖浓度。The kit also includes an accurate quantification card for urine sugar concentration, on which the formula is written: Y=3×10⁴⁴ X^-15.95 ; the formula on the precise quantification card for urine sugar concentration is obtained by the following method: 1.5-2 mL of the reacted nano-gold solution in the above step (2) was dropped into a quartz cuvette, and the wavelength corresponding to its peak absorbance in the range of 500-600 nm was detected by an ultraviolet-visible spectrophotometer, and the peak absorbance was drawn. The correlation curve between the corresponding wavelength and the glucose concentration, that is, the formula Y=3×10⁴⁴ X^-15.95 , where X represents the wavelength corresponding to the peak absorbance of the nano-gold solution in the range of 500-600 nm, and Y represents the wavelength to be detected Glucose concentration in urine.2.根据权利要求1所述的尿糖含量快速检测试剂盒，其特征在于：所述一次性加盖密封玻璃瓶的容量为8 mL、高70～80 mm、直径10～15 mm，为螺口石英玻璃材质，瓶盖为耐热塑料材质内部加装耐热防漏垫片。2. Urine sugar content rapid detection kit according to claim 1 is characterized in that: the capacity of described disposable capped and sealed glass bottle is 8 mL, height 70～80 mm, diameter 10～15 mm, is a screw. The mouth is made of quartz glass, and the bottle cap is made of heat-resistant plastic.3.一种采用权利要求1或2所述的试剂盒进行尿糖含量的非疾病诊断目的的检测的方法，其特征在于：将待检测尿液用去离子水稀释20倍，得到待检测尿液稀释液；将待检测尿液稀释液用沸水浴预热后，按照待检测尿液稀释液与氯金酸显色液的体积比为1:15，将预热后的待检测尿液稀释液加入沸水浴预热的氯金酸试剂瓶中，并迅速搅拌5～10秒钟后继续保持沸水浴加热反应5～6分钟，使尿液中的葡萄糖与氯金酸发生氧化还原显色反应，反应完成后将所得纳米金溶液的颜色与尿糖浓度比色卡进行对比，判断葡萄糖浓度范围；判断葡萄糖浓度范围后，取纳米金溶液1.5～2 mL滴入石英比色皿中，采用紫外可见分光光度计检测其在500～600 nm范围内的峰值吸光度所对应的波长，根据尿糖浓度精确定量卡上的公式Y=3×10⁴⁴X^-15.95，对待检测尿液中的葡萄糖浓度进行定量计算。3. a method that adopts the test kit described in claim 1 or 2 to carry out the detection of the non-disease diagnosis purpose of urine sugar content, it is characterized in that: urine to be detected is diluted 20 times with deionized water, to obtain urine to be detected After preheating the urine diluent to be tested with a boiling water bath, dilute the preheated urine to be tested according to the volume ratio of the urine diluent to be tested and the chloroauric acid color developing solution of 1:15 Add the liquid to the chloroauric acid reagent bottle preheated in the boiling water bath, and stir rapidly for 5 to 10 seconds, then continue to keep the boiling water bath for 5 to 6 minutes, so that the redox color reaction between glucose and chloroauric acid in the urine occurs. After the reaction is completed, the color of the obtained nano-gold solution is compared with the urine sugar concentration color chart to determine the glucose concentration range; after judging the glucose concentration range, take 1.5-2 mL of the nano-gold solution and drop it into a quartz cuvette, and use ultraviolet Visible spectrophotometer detects the wavelength corresponding to its peak absorbance in the range of 500-600 nm. According to the formula Y=3×10⁴⁴ X^-15.95 on the accurate quantification card of urine glucose concentration, the glucose concentration in urine to be detected is carried out. Quantitative calculations.

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