CN108489869B - Kit for detecting urine glucose abnormality based on contact angle change and application thereof - Google Patents
Kit for detecting urine glucose abnormality based on contact angle change and application thereof Download PDFInfo
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- CN108489869B CN108489869B CN201810317337.3A CN201810317337A CN108489869B CN 108489869 B CN108489869 B CN 108489869B CN 201810317337 A CN201810317337 A CN 201810317337A CN 108489869 B CN108489869 B CN 108489869B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N13/00—Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects
- G01N13/02—Investigating surface tension of liquids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N13/00—Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects
- G01N13/02—Investigating surface tension of liquids
- G01N2013/0208—Investigating surface tension of liquids by measuring contact angle
Abstract
The invention relates to a kit for detecting abnormal urine glucose based on contact angle change and application thereof, belonging to the field of medical detection. The kit comprises: a silicon chip coated with a sugar-sensitive polymer, a urine pretreatment solution and a resuscitation solution. In the silicon chip coated with the sugar-sensitive polymer, the sugar-sensitive polymer is a polymer taking glucose oxidase, concanavalin A or phenylboronic acid as a sensitive element. The urine pretreatment solution is an absolute ethyl alcohol solution containing 0.3-1 wt% of sodium azide, and the resuscitation solution is an aqueous solution containing 0.1 wt% of citric acid and 0.9% of NaCl. The invention further provides a preparation method and application of the kit.
Description
Technical Field
The invention relates to a kit for detecting abnormal urine glucose based on contact angle change and application thereof, belonging to the field of medical detection.
Background
Diabetes is a high-incidence disease, is difficult to find in the early stage or the atypical symptom stage, is a chronic disease process, and is known to have diabetes for many years after complications (such as cardiovascular and cerebrovascular diseases, hypopsia, hyperviscosity and the like) are developed in 15 to 20 years. In clinical treatment, in order to control diabetic complications, it is necessary to periodically measure the glucose concentration in the blood of a human body. This is not only necessary for type I diabetics, but also for type II diabetics. Because the blood sugar tolerance test, the blood sugar test and the slight amount test of the finger tip are required to periodically and dynamically test the regulation level of the insulin secretion function of a patient for a plurality of times a day, the psychological barrier of pricking the skin of the patient is required to be overcome before each measurement.
For most patients, it is not necessary to measure blood glucose, but only to control the glucose concentration in the urine. If the threshold value of the kidney is exceeded by metabolic stimulation, this leads to the physiologically undesirable excretion of glucose as urine glucose via the kidney, the glucose in the urine being present in detectable concentrations. Therefore, although blood glucose detection has become quite popular, urine glucose detection is still popular among diabetic patients as a noninvasive detection method with easy operation.
Currently, urine glucose test paper or a urine analyzer is generally used clinically for urine glucose test. Most urine glucose test strips produce a glucose-dependent color reaction based on enzyme chemistry. The method has the defects of poor accuracy and low sensitivity, and can only be used for semi-quantitative detection of the urine glucose. Although the precision of the urine analyzer is improved compared with that of the test paper, the urine analyzer can only be used for semi-quantitative detection and cannot realize precise quantification.
In addition, whether colorimetric reflectance or electrochemical biosensor methods are used, various impurities in urine cause serious interference in urine glucose detection. The amount of interferents in urine with different specific gravity is greatly different, and high-specific gravity urine contains more reductive interferents such as uric acid and the like, and a large amount of electrolyte ions, metal ions and the like, which all affect the specificity of urine glucose detection, so that the test result is inaccurate.
With the development of the times, the synthesis and application of functional polymers are receiving more and more attention from the scientific community. The stimulus-responsive polymer belongs to one of functional polymers, is mostly in an amphiphilic structure, can generate aggregates with different forms in water, and the aggregates can generate specific response changes under external environment stimulus, such as changes of pH value, temperature, light, molecules, electrolyte, voltage or other factors. Among them, glucose-responsive polymers are a class of polymers that utilize the interaction between a sugar and a receptor to produce a corresponding change. Generally, the interaction between the sugar and the receptor is mainly in both covalent and non-covalent forms. For example, some synthetic macromolecules can achieve sugar responses synergistically through hydrogen bonds and covalent bonds. The strategy for realizing the sugar response by utilizing the synergistic effect of the covalent bond and the non-covalent bond simultaneously provides a good idea for developing a novel biomolecule response system driven by various action modes.
Disclosure of Invention
The first aspect of the present invention provides a kit for detecting urine glucose based on contact angle change, comprising: a silicon chip coated with a sugar-sensitive polymer, a urine pretreatment solution and a resuscitation solution.
In one embodiment, in the silicon wafer coated with the sugar-sensitive polymer, the sugar-sensitive polymer refers to a polymer taking glucose oxidase, concanavalin A or phenylboronic acid as a sensitive element; preferably, the polymer takes phenylboronic acid as a sensitive element. The polymers are specifically responsive to glucose recognition, resulting in changes in the properties of the polymers, and the changes are concentration dependent, allowing accurate determination of changes in the sugar content of the fluid.
In a further embodiment, the saccharide-sensitive polymer is an isopropylacrylamide-acrylothioureido-aminobenzeneboronic acid copolymer. The polymer realizes high-specificity recognition of sugar molecules through the hydrogen bond action between thiourea groups and the sugar molecules and the covalent bond synergistic action formed between phenylboronic acid and the sugar molecules, and after the sugar molecules are combined with the sugar-sensitive polymer, the hydrophobicity of the sugar-sensitive polymer can be changed, so that the sugar content in the solution can be measured by measuring the contact angle change between the solution and the material.
In another embodiment, the urine pretreatment solution is an absolute ethanol solution containing 0.3-1 wt% sodium azide; preferably an absolute ethanol solution containing 0.5 wt% sodium azide.
In yet another embodiment, the resuscitation fluid is an aqueous solution containing 0.1 wt% citric acid and 0.9% NaCl. In the invention, the silicon wafer coated with the sugar-sensitive polymer can be reused, after a sample is measured, the silicon wafer is placed in the resuscitation solution for 15min, and the measurement can be carried out again after the silicon wafer is dried by nitrogen.
The second aspect of the present invention provides a method for preparing the kit, which comprises:
1) preparation of silicon wafers coated with sugar-sensitive polymers: adding isopropyl acrylamide, acrylyl thioureido aminobenzene boric acid, benzyl dithiobenzoate and an initiator azobisisobutyronitrile into a schlenk tube according to a molar ratio of 180:20:2:1, adding a 1, 4-dioxane/methanol solvent with a volume ratio of 4:1 for dissolving, degassing for at least 3 times by adopting a freezing-thawing pump circulation method, reacting for 20 hours in an oil bath at 70 ℃, adding chloroform for terminating polymerization reaction, precipitating a product by using a large amount of n-hexane, dissolving the precipitate in chloroform, precipitating by using the n-hexane, repeating for 3-4 times, and drying the product in vacuum overnight to obtain light orange powder; then mixing light orange powderDissolving with chloroform for later use; soaking 5 × 5cm silicon wafer in 0.1M NaOH solution for 10min, sun drying, and adding 0.1M HNO3Cleaning for 10min, repeatedly cleaning with water for 3 times, coating the polymer solution dissolved with chloroform on the cleaned silicon wafer, and naturally drying to form 20-50nm sugar-sensitive polymer film on the silicon wafer.
2) Preparing a urine pretreatment solution and a resuscitation solution according to a conventional method.
In the present invention, the assay method of the kit is as follows:
1) and (3) mixing the urine sample according to the volume ratio of the urine to the urine pretreatment liquid of 5: 1, mixing, centrifuging and taking supernatant;
2) dripping the supernatant into a silicon wafer coated with a sugar sensitive polymer, and measuring a contact angle by using a contact angle measuring instrument; calculating the difference between the supernatant contact angle and the water contact angle;
3) and (5) obtaining the sugar content in the urine sample through comparison and conversion of the standard substance.
The method utilizes the hydrophobicity change after the sugar sensitive polymer is combined with the sugar molecules, and further determines the content of the sugar in the sample through the contact angle change. However, considering the complexity of urine components, the urine sample needs to be pretreated to remove the relevant components affecting the contact angle change in urine. In addition, in consideration of the reusability of the kit, the invention also provides the resuscitation solution of the sugar-sensitive polymer material, thereby realizing the reuse of the kit and greatly saving the cost.
Detailed Description
The invention may be further understood by reference to the following examples, which illustrate some methods of making or using. However, it is to be understood that these examples do not limit the present invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.
Example 1 preparation of urine glucose assay kit based on contact Angle Change
1) Preparation of silicon wafer coated with sugar-sensitive polymer: reacting isopropyl acrylamide and acrylylthioureido ammoniaAdding phenylboronic acid, benzyl dithiobenzoate and an initiator azobisisobutyronitrile into a schlenk tube according to a molar ratio of 180:20:2:1, adding a 1, 4-dioxane/methanol solvent with a volume ratio of 4:1 for dissolving, degassing for at least 3 times by adopting a freezing-thawing pump circulation method, reacting for 20 hours under an oil bath at 70 ℃, adding chloroform to terminate a polymerization reaction, precipitating a product by using a large amount of n-hexane, dissolving the precipitate in chloroform, precipitating by using the n-hexane, repeating for 3-4 times, and drying the product in vacuum overnight to obtain light orange powder; dissolving the light orange powder with chloroform for later use; soaking 5 × 5cm silicon wafer in 0.1M NaOH solution for 10min, sun drying, and adding 0.1M HNO3Cleaning for 10min, repeatedly cleaning with water for 3 times, coating the polymer solution dissolved with chloroform on the cleaned silicon wafer, and naturally drying to form 20-50nm sugar-sensitive polymer film on the silicon wafer.
2) Preparing a urine pretreatment solution and a resuscitation solution according to a conventional method.
Urine pretreatment liquid: an absolute ethanol solution containing 0.5 wt% sodium azide.
And (3) resuscitation solution: an aqueous solution containing 0.1% by weight of citric acid and 0.9% of NaCl.
Example 2 detection of urine glucose assay kit based on contact Angle Change
1) Preparing 0.05, 0.1, 0.5, 1, 5, 10, 20, 50 and 100mmol/L glucose aqueous solution as a standard substance;
2) the standard solutions with different concentrations were dropped on a silicon wafer coated with a sugar-sensitive polymer (prepared in example 1, the same applies below), and the contact angle of the standard solution on the silicon wafer was measured after 5 min.
3) The contact angle of water on the silicon wafer was measured, and the difference (. DELTA.CA) between the contact angle of the standard and the contact angle of water was calculated.
4) Establishing a standard curve by taking the delta CA as an ordinate and the standard substance concentration as an abscissa, wherein Y (delta CA) is 1.569X +0.21, and R is2Value of>0.99, which shows that the kit of the invention has good linearity in the range of 0.05-100 mmol/L.
Example 3 kit Performance test
Reproducibility: the determination was repeated 10 times using the kit prepared in example 1, using known standards of high concentration (50mmol/L) and low concentration (0.5mmol/L), and the CV was calculated by treating the silicon wafer with the resuscitating solution between each determination. The result showed a CV of 0.67% for the 50mmol/L standard; the CV of 0.5mmol/L standard was 0.79%.
Example 4 detection of urine sample by the kit
Samples of human urine, 15 samples of normal persons and 23 samples of diabetic patients, were taken from hospitals and tested using the kit prepared in example 1. The method comprises the following specific steps:
1) and (3) mixing the urine sample according to the volume ratio of the urine to the pretreatment liquid of 5: 1, mixing, centrifuging and taking supernatant;
2) dripping the supernatant into a silicon wafer coated with a sugar sensitive polymer, and measuring a contact angle by using a contact angle measuring instrument; calculating the difference between the supernatant contact angle and the water contact angle;
3) the sugar content in each urine sample was calculated from the standard curve obtained in example 2.
The results show that: the detection results of 15 normal human samples are negative, and urine glucose is not detected. Of the diabetic samples, 3 samples of 0.1 to 5.5mmol/L, 14 samples of 5.5 to 27.8mmol/L, and 6 samples of >27.8 mmol/L.
The specific results are as follows:
table 1: urine glucose concentration in diabetic samples
| Sample numbering | Urine glucose concentration | Sample numbering | Urine glucose concentration |
| Sample 1 | 0.7mmol/L | Sample 13 | 41.7mmol/L |
| Sample 2 | 23.6mmol/L | Sample 14 | 20.1mmol/L |
| Sample 3 | 10.3mmol/L | Sample 15 | 26.9mmol/L |
| Sample 4 | 26.5mmol/L | Sample 16 | 18.6mmol/L |
| Sample 5 | 33.7mmol/L | Sample 17 | 30.4mmol/L |
| Sample 6 | 1.2mmol/L | Sample 18 | 21.7mmol/L |
| Sample 7 | 11.8mmol/L | Sample 19 | 65.8mmol/L |
| Sample(s)8 | 5.2mmol/L | Sample 20 | 23.6mmol/L |
| Sample 9 | 22.4mmol/L | Sample 21 | 45.3mmol/L |
| Sample 10 | 56.9mmol/L | Sample 22 | 18.9mmol/L |
| Sample 11 | 8.5mmol/L | Sample 23 | 13.3mmol/L |
| Sample 12 | 13.2mmol/L |
In addition, the kit of embodiment 1 is adopted to detect the urine sample of a normal person, but the urine sample is not preprocessed in the detection process, and the result shows that: in 15 normal samples, 5 of the samples showed false positive results, indicating that pretreatment of urine is necessary for the detection of urine glucose using the above-mentioned sugar-sensitive polymer.
Example 5 repeated assay of the kit
A sample of the diabetic patient of example 4 was taken and tested to a concentration of 56.9mmol/L, and the test was repeated using a single silicon wafer coated with a sugar-sensitive polymer (prepared in example 1) and treated with a resuscitating fluid for 5min between each measurement, indicating that: after 200 times of repeated measurement, the error of the result and the result of the primary measurement is within 1.5 percent, which indicates that the kit has stable performance in the repeated use process.
This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.
Claims (1)
1. A kit for detecting urine glucose abnormality based on contact angle change is characterized by comprising a silicon chip coated with an isopropyl acrylamide-acrylylthioureidoaminobenzeneboronic acid copolymer, a urine pretreatment liquid and a resuscitation liquid;
the preparation method of the silicon chip coated with the sugar-sensitive polymer comprises the following steps: adding isopropyl acrylamide, acrylyl thioureido aminobenzene boric acid, benzyl dithiobenzoate and an initiator azobisisobutyronitrile into a schlenk tube according to a molar ratio of 180:20:2:1, adding a 1, 4-dioxane/methanol solvent with a volume ratio of 4:1 for dissolving, degassing for at least 3 times by adopting a freezing-thawing pump circulation method, reacting for 20 hours in an oil bath at 70 ℃, adding chloroform for terminating polymerization reaction, precipitating a product by using a large amount of n-hexane, dissolving the precipitate in chloroform, precipitating by using the n-hexane, repeating for 3-4 times, and drying the product in vacuum overnight to obtain light orange powder; dissolving the light orange powder with chloroform for later use; soaking silicon wafer in 0.1M NaOH for 10min, sun drying, and adding 0.1M HNO3Cleaning for 10min, repeatedly cleaning with water for 3 times, coating the polymer solution dissolved with chloroform on the cleaned silicon wafer, and naturally drying to form 20-50nm sugar-sensitive polymer film on the silicon wafer;
the urine pretreatment solution is an absolute ethyl alcohol solution containing 0.5 wt% of sodium azide,
the resuscitation solution is an aqueous solution containing 0.1 wt% of citric acid and 0.9% of NaCl;
the use method of the kit comprises the following steps:
1) and (3) mixing the urine sample according to the volume ratio of the urine to the urine pretreatment liquid of 5: 1, mixing, centrifuging and taking supernatant;
2) dripping the supernatant into a silicon wafer coated with a sugar sensitive polymer, and measuring a contact angle by using a contact angle measuring instrument; calculating the difference between the supernatant contact angle and the water contact angle;
3) obtaining the sugar content in the urine sample through comparison and conversion of standard substances, wherein the standard substances are glucose aqueous solutions with different molar concentrations;
the silicon wafers were treated with a resuscitating solution for 5min between each measurement.
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| US5801060A (en) * | 1990-10-10 | 1998-09-01 | Chimera Research & Chemical, Inc. | Method of using automated analyzer testing of urine for presence of a pH abnormality with single reagent indicator |
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