CN108489869A - A kind of kit and its application changing detection glucose in urine exception based on contact angle - Google Patents
A kind of kit and its application changing detection glucose in urine exception based on contact angle Download PDFInfo
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- CN108489869A CN108489869A CN201810317337.3A CN201810317337A CN108489869A CN 108489869 A CN108489869 A CN 108489869A CN 201810317337 A CN201810317337 A CN 201810317337A CN 108489869 A CN108489869 A CN 108489869A
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- glucose
- urine
- silicon chip
- sensitive polymer
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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 present invention relates to a kind of kit and its applications changing detection glucose in urine exception based on contact angle, belong to medical science.The kit includes：It is coated with the silicon chip of glucose sensitive polymer, urine pretreatment fluid and resuscitation fluid.Described to be coated in the silicon chip of glucose sensitive polymer, glucose sensitive polymer refers to the polymer for sensitive primitive with glucose oxidase, concanavalin A or phenyl boric acid.The urine pretreatment fluid is the ethanol solution containing 0.3 1wt% Sodium azides, and the resuscitation fluid is the aqueous solution containing 0.1wt% citric acids and 0.9%NaCl.The present invention has further related to the preparation method and application of the kit.
Description
Technical field
The present invention relates to a kind of kit and its applications changing detection glucose in urine exception based on contact angle, belong to medicine detection
Field.
Background technology
Diabetes are a kind of diseases occurred frequently, and it is often difficult to find be in chronic disease process, suffer from early days or without the classical symptom phase
Person often generates complication (such as in 15-20：Cardiovascular and cerebrovascular diseases, hypopsia, hypercoagulable blood etc.) see a doctor after just begin know once
Suffer from diabetes for many years.In order to control diabetic complication on clinical treatment, the glucose being periodically required in detection blood of human body
Concentration.This is not only necessary for type-1 diabetes mellitus patient, for type 2 diabetes patient and so.Since blood glucose is resistance to
Amount detection, blood sugar test refer to end slightly trace detection, are required to carry out multiple periodical dynamic detection own insulin point on the one
The regulation and control for secreting function are horizontal, it is necessary to which patient overcomes the mental handicape for stabbing skin before each measurement.
It for Most patients, does not need to measure blood glucose, as long as and controlling the concentration of glucose in urine.
If the stimulation by metabolism has been more than the threshold value of kidney, the outer glucose of physiological requirements is resulted in pass through as glucose in urine
Kidney is discharged, and the glucose in urine is just presented as the concentration that can be detected.Therefore, although blood sugar test is quite general
And but a kind of non-invasive detection methods easy to operate of glucose in urine detection conduct, still welcome deeply by diabetic.
Currently, clinically glucose in urine Test paper or Urine Analyzer is generally used to carry out glucose in urine detection.Glucose in urine detects
Test paper majority is the color reaction that glucose compliance is generated on the basis of enzymology.The disadvantage is that accuracy is poor, sensitivity
It is relatively low, it is only used for the half-quantitative detection of glucose in urine.Although and increase compared with Test paper in Urine Analyzer precision, but still
It is simply possible to use in half-quantitative detection, can not accomplish accurate quantification.
In addition, either using colorimetric bounce technique or electrochemica biological sensor method, various impurity examine glucose in urine in urine
Survey causes severe jamming.There are the amounts of chaff interferent bigger difference in different specific weight urine, contains more urine in baruria
The reproducibilities such as acid chaff interferent, a large amount of electrolyte ions and metal ion etc. can influence the specificity of glucose in urine detection, cause to test
As a result inaccuracy.
With the development of the times, the synthesis and application of functional polymer are more and more paid close attention to by scientific circles.Stimulation is rung
The type polymer of answering belongs to one kind in functional polymer, mostly amphiphilic structure, produces the aggregation to come in every shape in water,
Such aggregation is under extraneous environmental stimulus, such as the variation of pH value, temperature, light, molecule, electrolyte, voltage or other factors, meeting
Generate the response variation of specificity.Wherein, glucose responding type polymer is generated using the interaction between sugar and receptor
A kind of polymer of respective change.In general, the interaction between sugar and receptor mainly has covalent and non-covalent two kinds of forms.
For example, some artificial synthesized macromolecules can be responded by hydrogen bond and covalent bond cooperative achievement sugar.It is this at the same using covalent bond with
Non-covalent bond synergistic effect realizes the strategy of sugar response, to develop the biomolecule response body of new a variety of binding modes driving
System provides good thinking.
Invention content
The first aspect of the present invention is to provide a kind of kit changing detection glucose in urine based on contact angle, including：It is coated with
The silicon chip of glucose sensitive polymer, urine pretreatment fluid and resuscitation fluid.
In one embodiment, described to be coated in the silicon chip of glucose sensitive polymer, glucose sensitive polymer refers to Portugal
Grape carbohydrate oxidase, concanavalin A or phenyl boric acid are the polymer of sensitive primitive；It is preferably the poly- of sensitive primitive with phenyl boric acid
Close object.Response can be identified to glucose with specific in above-mentioned polymer, and then the property of polymer is caused to change, and
This change is concentration-dependant, so as to accurately measure the variation of sugared content in liquid.
In further carrying out scheme, the glucose sensitive polymer refers to N-isopropylacrylamide-acryl thiourea base
Amino phenyl boric acid copolymer.The polymer passes through hydrogen bond action between thiourea group and glycan molecule and phenyl boric acid and glycan molecule
Between the covalent bond synergistic effect that is formed, realize to the identification of the high specific of glycan molecule, when glycan molecule and glucose sensitive polymer knot
After conjunction, the hydrophobicity of glucose sensitive polymer can change, and then the contact angle by measuring between solution and material changes in fact
The measurement of sugared content in existing solution.
In another embodiment, the urine pretreatment fluid is that the absolute ethyl alcohol containing 0.3-1wt% Sodium azides is molten
Liquid；Ethanol solution preferably containing 0.5wt% Sodium azides.
In yet another embodiment, the resuscitation fluid is the aqueous solution containing 0.1wt% citric acids and 0.9%NaCl.
In the present invention, the silicon chip for being coated with glucose sensitive polymer may be reused, and after measuring sample, silicon chip is placed in multiple
15min in Soviet Union's liquid, can be measured again after being dried up with nitrogen.
Second aspect of the present invention is to provide the preparation method of the kit comprising：
1) it is coated with the preparation of the silicon chip of glucose sensitive polymer：By N-isopropylacrylamide, acryl thiourea base aminobenzene
Boric acid, dithio benzyl benzoate and initiator azodiisobutyronitrile in molar ratio 180:20:2:1 is added in schlenk pipes,
It is 4 that volume ratio, which is added,:1 Isosorbide-5-Nitrae-dioxane/methanol solvate is dissolved, using freeze-thaw pump circulation method degassing at least 3
It is secondary, it is reacted 20 hours under 70 DEG C of oil baths, chloroform is added and terminates polymerisation, product is precipitated with a large amount of n-hexane, will be precipitated
It is dissolved in chloroform, then is precipitated with n-hexane, repeats 3~4 times, product vacuum is dried overnight, light orange powder is obtained；It then will be shallow
Orange powder is spare after being dissolved with chloroform；It takes the NaOH of 5 × 5cm silicon chips 0.1M to impregnate 10min, the HNO of 0.1M is used after drying3
10min is cleaned, is then washed with water repeatedly 3 times, the polymer solution of chloroform dissolving before is coated on silicon chip after cleaning,
Naturally dry, to form the glucose sensitive polymer film of 20-50nm on silicon chip to obtain the final product.
2) urine pretreatment fluid and resuscitation fluid are conventionally prepared.
In the present invention, the assay method of the kit is as follows：
1) by urine specimen according to urine and urine pretreatment fluid volume than 5：1 mixing, centrifuging and taking supernatant；
2) supernatant is added dropwise in the silicon chip for being coated with glucose sensitive polymer, contact angle is measured with contact angle measurement；Meter
Count the difference of clear liquid contact angle and water contact angle in；
3) sugared content in conversion acquisition urine specimen is compared by standard items.
The present invention using glucose sensitive polymer combined with glycan molecule after hydrophobicity variation, and then by contact angle variation come
Measure content sugared in sample.It is contemplated that the complexity of urinary fractions, needs to pre-process urine specimen, shadow is removed
Ring the Related Component that contact angle changes in urine.Furthermore, it is contemplated that the repeatable usability of kit of the present invention, additionally provides sugar
The resuscitation fluid of sensitive polymer material dramatically saves cost to realize the reuse of kit.
Specific implementation mode
Can also the present invention further be understood by embodiment, wherein the embodiment illustrates some preparations or user
Method.It is to be appreciated, however, that these embodiments do not limit the present invention.The change of the present invention of currently known or further exploitation
Change is considered within the scope of the invention described herein and claimed below.
The preparation for the glucose in urine detection kit that embodiment 1 is changed based on contact angle
1) it is coated with the preparation of glucose sensitive polymer silicon chip：By N-isopropylacrylamide, acryl thiourea base aminobenzene boron
Acid, dithio benzyl benzoate and initiator azodiisobutyronitrile in molar ratio 180:20:2:1 is added in schlenk pipes, adds
It is 4 to enter volume ratio:1 Isosorbide-5-Nitrae-dioxane/methanol solvate is dissolved, and is deaerated at least 3 times using freeze-thaw pump circulation method,
It is reacted 20 hours under 70 DEG C of oil baths, chloroform is added and terminates polymerisation, product is precipitated with a large amount of n-hexane, precipitation is dissolved in
Chloroform, then precipitated with n-hexane, it repeats 3~4 times, product vacuum is dried overnight, light orange powder is obtained；Then by light orange
Powder is spare after being dissolved with chloroform；It takes the NaOH of 5 × 5cm silicon chips 0.1M to impregnate 10min, the HNO of 0.1M is used after drying3Cleaning
Then 10min is washed with water 3 times repeatedly, the polymer solution of chloroform dissolving before is coated on silicon chip after cleaning, natural
It dries, to form the glucose sensitive polymer film of 20-50nm on silicon chip to obtain the final product.
2) urine pretreatment fluid and resuscitation fluid are conventionally prepared.
Urine pretreatment fluid：Ethanol solution containing 0.5wt% Sodium azides.
Resuscitation fluid：Aqueous solution containing 0.1wt% citric acids and 0.9%NaCl.
The detection for the glucose in urine detection kit that embodiment 2 is changed based on contact angle
1) configuration 0.05,0.1,0.5,1,5,10,20,50, the glucose solution of 100mmol/L are as standard items；
2) various concentration standard solution is measured to be added dropwise respectively in the silicon chip (system of embodiment 1 for being coated with glucose sensitive polymer
It is standby, similarly hereinafter) on, contact angle of the bioassay standard product solution in silicon chip after 5min.
3) contact angle of the water on above-mentioned silicon chip is measured, the difference (Δ CA) of standard items contact angle and water contact angle is calculated.
4) using Δ CA as ordinate, standard curve is established using standard concentration as abscissa, specially Y (Δ CA)=
1.569X+0.21 R2Value>0.99, illustrate that kit of the present invention is linear good within the scope of 0.05-100mmol/L.
3 kit performance test of embodiment
Reproducibility：Known high concentration (50mmol/L) and low concentration (0.5mmol/L) standard items are taken, is made using embodiment 1
Standby kit replication 10 times, handles silicon chip using resuscitation fluid between measuring every time, calculates CV.The result shows that being
The CV of 50mmol/L standard items is 0.67%；The CV of 0.5mmol/L standard items is 0.79%.
4 kit of embodiment detects urine specimen
Human urine sample, wherein 15, normal person's sample, 23, diabetic's sample, using embodiment 1 are taken from hospital
The kit of preparation is detected.It is as follows：
1) by urine specimen according to urine and pretreatment fluid volume than 5：1 mixing, centrifuging and taking supernatant；
2) supernatant is added dropwise in the silicon chip for being coated with glucose sensitive polymer, contact angle is measured with contact angle measurement；Meter
Count the difference of clear liquid contact angle and water contact angle in；
3) standard curve obtained by embodiment 2 calculates the sugared content in each urine specimen.
The result shows that：15 testing results of normal person's sample are feminine gender, and glucose in urine is without detection.In diabetic's sample,
3, the sample of 0.1-5.5mmol/L, 14, the sample of 5.5-27.8mmol/L,>6, the sample of 27.8mmol/L.
Concrete outcome is as follows：
Table 1：The urine sugar concentrations of diabetic's sample
| Sample number | Urine sugar concentrations | Sample number | Urine sugar concentrations |
| 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 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 present invention uses the kit of embodiment 1, normal person's urine specimen is detected, but in detection process
Urine specimen is not pre-processed, the results showed that：In 15 normal samples, there are 5 false positive results occur, illustrate to use
When above-mentioned glucose sensitive polymer is detected glucose in urine, the pre-treatment of urine is necessary.
The replication of 5 kit of embodiment
Diabetic's sample in Example 4, detection show a concentration of 56.9mmol/L, use single coating repeatedly
There is the silicon chip (preparation of embodiment 1) of glucose sensitive polymer to test it, with resuscitation fluid to silicon chip processing between measuring every time
5min, the results showed that：After being repeatedly measured 200 times, as a result with first measurement result error within 1.5%, this illustrates the present invention
Kit performance during reuse is stablized.
The content of present invention merely illustrates some claimed specific embodiments, wherein one or more technologies
Recorded technical characteristic can be combined with arbitrary one or more technical solutions in scheme, obtained from these are combined
Technical solution also in the application protection domain, just as these it is combined obtained from technical solution in the present invention discloses
It is specifically recorded in appearance the same.
Claims (8)
1. a kind of kit changing detection glucose in urine based on contact angle comprising：It is coated with the silicon chip of glucose sensitive polymer, urine
Pretreatment fluid and resuscitation fluid.
2. kit according to claim 1, which is characterized in that described to be coated in the silicon chip of glucose sensitive polymer, sugar
Sensitive polymer refers to the polymer for sensitive primitive with glucose oxidase, concanavalin A or phenyl boric acid.
3. kit according to claim 2, which is characterized in that described to be coated in the silicon chip of glucose sensitive polymer, sugar
Sensitive polymer refers to the polymer for sensitive primitive with phenyl boric acid.
4. kit according to claim 3, which is characterized in that the glucose sensitive polymer refers to isopropyl acrylamide
Amine-acryl thiourea base amino phenyl boric acid copolymer.
5. kit according to claim 1, which is characterized in that the urine pretreatment fluid is folded containing 0.3-1wt%
The ethanol solution of nitrogen sodium.
6. kit according to claim 5, which is characterized in that the urine pretreatment fluid is to contain 0.5wt% nitrine
The ethanol solution of sodium.
7. kit according to claim 1, which is characterized in that the resuscitation fluid be containing 0.1wt% citric acids and
The aqueous solution of 0.9%NaCl.
8. the preparation method of any one of the claim 1-7 kits comprising：
1) it is coated with the preparation of the silicon chip of glucose sensitive polymer：By N-isopropylacrylamide, acryl thiourea base amino phenyl boric acid,
Dithio benzyl benzoate and initiator azodiisobutyronitrile in molar ratio 180:20:2:1 is added in schlenk pipes, is added
Volume ratio is 4:1 Isosorbide-5-Nitrae-dioxane/methanol solvate is dissolved, and is deaerated at least 3 times using freeze-thaw pump circulation method,
It is reacted 20 hours under 70 DEG C of oil baths, chloroform is added and terminates polymerisation, product is precipitated with a large amount of n-hexane, and precipitation is dissolved in chlorine
It is imitative, then precipitated with n-hexane, it repeats 3~4 times, product vacuum is dried overnight, light orange powder is obtained；Then by light orange powder
End is spare after being dissolved with chloroform；It takes the NaOH of 5 × 5cm silicon chips 0.1M to impregnate 10min, the HNO of 0.1M is used after drying3Cleaning
Then 10min is washed with water 3 times repeatedly, the polymer solution of chloroform dissolving before is coated on silicon chip after cleaning, natural
It dries, to form the glucose sensitive polymer film of 20-50nm on silicon chip to obtain the final product；
2) urine pretreatment fluid and resuscitation fluid are conventionally prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201810317337.3A CN108489869B (en) | 2018-04-10 | 2018-04-10 | Kit for detecting urine glucose abnormality based on contact angle change and application thereof |
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| CN201810317337.3A CN108489869B (en) | 2018-04-10 | 2018-04-10 | Kit for detecting urine glucose abnormality based on contact angle change and application thereof |
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| CN108489869B CN108489869B (en) | 2021-07-27 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110967281A (en) * | 2019-12-25 | 2020-04-07 | 吉林工程技术师范学院 | Super-hydrophobic aluminum foil for detecting blood sugar and detection method thereof |
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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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| CN110967281A (en) * | 2019-12-25 | 2020-04-07 | 吉林工程技术师范学院 | Super-hydrophobic aluminum foil for detecting blood sugar and detection method thereof |
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| CN108489869B (en) | 2021-07-27 |
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