CN102375066B - Creatinine content detecting reagent and kit, and manufacturing and using methods of kit - Google Patents
Creatinine content detecting reagent and kit, and manufacturing and using methods of kit Download PDFInfo
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- CN102375066B CN102375066B CN 201110279969 CN201110279969A CN102375066B CN 102375066 B CN102375066 B CN 102375066B CN 201110279969 CN201110279969 CN 201110279969 CN 201110279969 A CN201110279969 A CN 201110279969A CN 102375066 B CN102375066 B CN 102375066B
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Abstract
The invention relates to a creatinine content detecting reagent and kit and manufacturing and using methods of the kit. The creatinine content detecting reagent comprises creatininase, creatinase, creatine oxidase and horseradish peroxidase; and a fluorescent dye, i.e., 5(6)-carboxyl-2,7-dichlordihydro fluorescein ester dipivalate comprises a dye bottom layer, a protease fixing layer and a top layer protecting layer. The using method has high selectivity; a protease has high specificity to reactants; the creatininase can be only used for catalyzing the hydrolysis reaction of creatinine, and does not have catalysis activity on other reaction substrates; three enzymes with high specificity are related to the detection process, so that the selectivity of the method is increased exponentially; the response fed by the kit is fluorescent response; and compared with certain methods for detecting color variations, the invention has the advantages of high sensitivity and interference resistance under the action of the inherent characteristics of fluorescent response.
Description
Technical field
The invention belongs to the kreatinin detection field, especially a kind of kreatinin content detecting reagent and kit, and the making and use method of kit.
Background technology
Kreatinin is the product of phosphocreatine metabolism, and its chemical constitution is the lactams that creatine forms by dehydrating condensation.Kreatinin generally is present in the blood, arrives kidney and is filtered by blood circulation.For the crowd of normal renal function, the kreatinin concentration in the blood varies in different localities because of the people.Generally speaking, in the situation that without large physical load, the kreatinin concentration in women's the blood is at 45-60 μ M, the male sex's blood kreatinin concentration is floated in the scope of 60-110 μ M.When renal function goes wrong, kidney filters the ability of removing kreatinin will be reduced, cause the concentration abnormality of kreatinin in the blood to raise and kreatinin concentration reduction in the urine, therefore by measuring the concentration of kreatinin in blood and the urine, just can measure creatinine clearance even glomerulus rate filters, these two parameters have great importance for weighing renal function, therefore, can measure efficiently the kreatinin concentration in blood and the urine, for clinical and scientific research, all has very great application prospect.
The measurement of kreatinin is difficulty relatively.At present method commonly used is that mass spectroscopy (isotope dilution mass spectrometry), high performance liquid chromatography, liquid chromatography mass are used in conjunction method and antibody test (ELISA), these methods generally all need expensive instrument, such as mass spectrometer, liquid chromatograph or microplate reader etc., and the operation that was subjected to the analytical chemistry operating personnel of systematic training, greatly improve financial cost and time cost that kreatinin is measured, and kreatinin is measured in backwoodsman application.
Mass spectroscopy is by containing the kreatinin of certain isotope (such as 0-18) and this sample is carried out mass spectrophotometry artificial adding the in sample.The abundance of 0-18 and 0-18 can calculate kreatinin in the abundance of occurring in nature concentration among the comparison mass spectrum result.The artificial kreatinin that adds can strictly be controlled, and as a kind of internal standard (internal calibration), this method can effectively reduce error.According to current research, on the low side with the kreatinin concentration that mass spectroscopy obtains, this will cause series of problems clinically, and U.S. FDA also begins to consider new kreatinin detection means.
Liquid phase chromatography is to use high performance liquid chromatography that sample is carried out purifying on mass spectral:mass spectrographic basis and detect with mass spectrum.Mass spectroscopy and liquid phase chromatography can't robotizations, complicated operation; and need to analyze team instrument is carried out operation and maintenance; and the ELISA method need to be used expensive microplate reader; although its sensitivity is high; but selectivity is relatively poor; often can over-evaluate the concentration of kreatinin, cause the excessive or not enough of clinical application.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, provide a kind of selectivity kreatinin content detecting reagent and kit high, highly sensitive and easy to use, and the making and use method of kit.
The objective of the invention is to be achieved through the following technical solutions:
A kind of kreatinin content detecting reagent, comprise creatininase, kreatinase, sarcosine oxidase and horseradish peroxidase, fluorescent dye 5 (6)-carboxyls-2,7-dichloro-dihydro fluorescein two pivalates, kreatinin is hydrolyzed under the catalysis of creatininase and is creatine, creatine is hydrolyzed into methyl amimoacetic acid under the catalysis of kreatinase, methyl amimoacetic acid generates glycocoll and a small amount of hydrogen peroxide thereupon under the catalysis of sarcosine oxidase, under the catalysis of horseradish oxide enzyme, catalytic oxidation of hydrogen peroxide 5 (6)-carboxyls-2,7-dichloro-dihydro fluorescein two pivalates also generate the product with fluorescence response, speed and typical curve by the enhancing of monitoring fluorescence, obtain kreatinin concentration, wherein said creatininase: kreatinase: sarcosine oxidase: horseradish peroxidase: the weight ratio of fluorescent dye is: 1: 50: 60: 5.
And, comprise following three-decker:
(1) dyestuff bottom: cellulose and the mixing of D4 hydrogel and stirring with the fluorescent dye modified are coated with the film of last layer D4 hydrogel and make it natural air drying with scraper film coating machine on a clean plastic sheeting;
(2) proteinase fixed bed: in the aqueous solution of PVA-Sbq (POLYPROPYLENE GLYCOL phenylpyridine ethene complex compound), add 10% bovine serum albumin and react required creatininase, kreatinase, sarcosine oxidase and horseradish peroxidase, stir more than 25-40 minute, be coated with the PVA-Sbq solution of last layer enzyme with scraper film coating machine, uviol lamp is placed the top 30 minutes of rete, make phenylpyridine ethene crosslinked and rete is become dry
(3) top layer protective seam: in the above with scraper film coating machine be coated with D6 hydrogel that last layer contain 10% carbon black at last, and make the rete natural air drying.
And the concrete preparation process of three-decker is as follows:
(1) 5 (6)-carboxyls-2 that the 176mg cellulose is fixing, the 4%D4 hydrogel solution of 7-dichloro-dihydro fluorescein two pivalates and 2.6g mixes and stirs, and makes cellulose powder Uniform Dispersion in solution,
(2) evenly to make it to form a layer thickness be the film of 100 μ M and dry at plastic sheeting with solution prepared in the step (1);
(3) with 30mg kreatinase, 35mg horseradish peroxidase, 0.63mg creatininase, 2.6mg sarcosine oxidase, 340mg water and 1.33g concentration be 10% and the PVA-Sbq solution that contains 10% bovine serum albumin mix, and stir on ice:
(4) film of preparation is coated PVA-Sbq/ enzyme solutions prepared in the step (3) in step (2), and the thickness of rete is 100 μ m, and film was placed uviol lamp lower 30 minutes;
(5) prepared film is coated with solution and the natural air drying that last layer contains 6% carbon black and 1.8%D6 in step (4).
A kind of kreatinin reagent box for detecting content comprises above-mentioned detection reagent.
A kind of preparation method of kreatinin reagent box for detecting content is characterized in that: step is as follows:
(1) 5 (6)-carboxyls-2 that the 176mg cellulose is fixing, the 4%D4 hydrogel solution of 7-dichloro-dihydro fluorescein two pivalates and 2.6g mixes and stirs, and makes cellulose powder Uniform Dispersion in solution;
(2) evenly to make it to form a layer thickness be the film of 100 μ M and dry at plastic sheeting with solution prepared in the step (1);
(3) with 30mg kreatinase, 35mg horseradish peroxidase, 0.63mg creatininase, 2.6mg sarcosine oxidase, 340mg water and 1.33g concentration be 10% and the PVA-Sbq solution that contains 10% bovine serum albumin mix, and stir on ice;
(4) film of preparation is coated PVA-Sbq/ enzyme solutions prepared in the step (3) in step (2), and the thickness of rete is 100 μ m, and film was placed uviol lamp lower 30 minutes;
(5) prepared film is coated with solution and the natural air drying that last layer contains 6% carbon black and 1.8%D6 in step (4).
A kind of using method of kit, it is characterized in that: containing 100mMHEPES, 150mM sodium chloride, 5mM potassium chloride, 1.3mM in the damping fluid of lime chloride, pH7.3 adds the variable concentrations kreatinin, drives the sample solution of 150 μ L and assign on the kit and with luminoscope with kapillary two to begin to measure.
Advantage of the present invention and beneficial effect are:
1, method used in the present invention has the selectivity of height, as protease, it has the specificity of height to reactant, creatininase can only the catalysis kreatinin hydrolysis reaction, reaction substrate to other does not all have catalytic activity, owing to relating to the enzyme of three kinds of high degree of specificity in the testing process, the selectivity of this method is exponent increase especially, the response that kit provides at last is fluorescence response, compare with some method that detects change color, the inherent characteristic of fluorescence response is given the higher sensitivity of the present invention and jamproof ability.
2, the application provides a kind of novel kit for detection of kreatinin content in blood and the urine, this kit will have specific enzyme and the fluorescent dye of superoxide sensitivity will be fixed on the kit kreatinin, and enzymatic reaction is converted into fluorescence signal and detected, this kit can detect the above kreatinin of 10 μ M, meets request for utilization clinically.
3, enzyme used in the present invention has four kinds: creatininase, kreatinase, sarcosine oxidase and horseradish peroxidase, kreatinin in the sample is planted enzyme by first three and is degraded to glycocoll, generate simultaneously a small amount of hydrogen peroxide, under the catalysis of horseradish peroxidase, hydrogen peroxide can a kind of script of oxidation have the molecule of fluorescence, reaction product is the derivant of fluorescein, has stronger fluorescence response, by detecting the enhancing speed of fluorescence, can detect the generating rate of hydrogen peroxide, and indirect calculation goes out the concentration of kreatinin.
4, the present invention compares the kit that existing kreatinin detection technique and the application set forth, important parameter as renal function, the creatine that creatinine clearance and glomerulus rate are filtered need to repeatedly carry out the kreatinin of blood and urine and measure, therefore, the use that has of the mentioned kreatinin kit of the application characteristics simple, cheap and that have a high selectivity will have very great meaning.
The comparison of the existing kreatinin detection method of table 1
Description of drawings
Fig. 1 is the cross-linking reaction formula of phenyl vinylpyridine of the present invention;
Fig. 2 is the structure of the kit of creatinine sensor use of the present invention;
Fig. 3 is the fluorescence response in time of the present invention's sample of kit under different potassium concentrations; Fig. 3-1 and Fig. 3-2 represent that in two ways the time is corresponding.
Fig. 4 gain in strength for the fluorescence that calculates according to Fig. 4 and potassium concentration between correlativity.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
If the number percent that following examples are mentioned does not have special indicating all to be weight percentage, the mM of unit represents every liter of mM.
One, the principles of chemistry of kreatinin kit
Kreatinin kit set forth in the present invention comprises four kinds of enzymes and a kind of fluorescent dye, and they are respectively: creatininase, kreatinase, sarcosine oxidase and horseradish peroxidase.The chemical name of fluorescent dye is 5 (6)-carboxyls-2,7-dichloro-dihydro fluorescein two pivalates (the following A of structure), the fluorescent yield of this fluorescent dye is very low, therefore under general condition, its fluorescent emission efficient is very low, this fluorescent dye can be oxidized into 2,7-dichlorofluorescein (structural formula is seen B), 2, the 7-dichlorofluorescein has very high quantum yield, so these fluorescent dye 5 (6)-carboxyls-2, the oxidizing process of 7-dichloro-dihydro fluorescein two pivalates can be monitored by the mensuration to fluorescence.
5 (6)-carboxyls-2, the structure of 7-chlorine dihydrofluorescein two pivalates (A) and oxidation afterproduct (B) thereof
The present invention is described below the chemical process of the detection of kreatinin: kreatinin is hydrolyzed under the catalysis of creatininase and is creatine, creatine is hydrolyzed into methyl amimoacetic acid under the catalysis of kreatinase, methyl amimoacetic acid generates glycocoll and a small amount of hydrogen peroxide (reaction equation of above process is as follows) thereupon under the catalysis of sarcosine oxidase, under the catalysis of horseradish oxide enzyme, the catalytic oxidation of hydrogen peroxide that forms in the said process 5 (6)-carboxyls-2,7-dichloro-dihydro fluorescein two pivalates also generate the product with fluorescence response, by speed and the typical curve that monitoring fluorescence strengthens, can indirectly obtain the information of kreatinin concentration.
Two, protease and fluorescent dye is fixing
In kit, enzyme and fluorescent dye will be fixed in the solid phase substrate.The bottom is that (a kind of polyurethane polyureas ethylene glycol hydrogel, specifically see following patent: US 2008/0152864A1) supporting layer, principal ingredient are the linear polymers of polyglycol and polyesteramide to the D4 hydrogel.In view of the instability of fluorescent dye (structural formula is A), it is fixed on the cellulose by covalent bond, and the cellulosic particle of fluorescent dye is dispersed in the D4 hydrogel layer uniformly.Protease is fixed on the second layer, and its principal ingredient is the multipolymer of polyvinyl alcohol (PVA) and phenyl vinylpyridine.The hydrosol of polyvinyl alcohol (PVA) phenyl vinylpyridine with become collosol state after protease mixes.After potpourri was by ultraviolet irradiation, the phenyl vinylpyridine produced crosslinked, makes mixture solidified; And protease also will be fixed in the gel by physics, and its reaction equation is seen Fig. 1.
Because protease is fixed in the solid and the preservation that is dried, so its hydrolysis and degraded comparatively speaking can be difficult a lot: at first owing to lack of water, the possibility that hydrolysis occurs reduces greatly; Secondly owing to the existence of solid phase protective seam, protein decomposition enzyme contacted protein molecular proportion is difficulty, and the hydrolysis reaction that results in its catalysis can not occur, and therefore can expect, the protease that is fixed is also with more stable.
Three, the method for making of kit:
(1) dyestuff bottom: the cellulose of fluorescent dye modified and D4 hydrogel are mixed and stirred a hour, on a clean plastic sheeting, be coated with the film of last layer D4 hydrogel and make it natural air drying with scraper film coating machine immediately.
(2) proteinase fixed bed: in the aqueous solution of PVA-Sbq (POLYPROPYLENE GLYCOL phenylpyridine ethene complex compound), sneak into 10% bovine serum albumin and react required four kinds of enzymes (creatininase, kreatinase, sarcosine oxidase and horseradish peroxidase) and stir more than 30 minutes, this solution will be for the immobilization of enzyme, be coated with the PVA-Sbq solution of last layer enzyme with scraper film coating machine, uviol lamp is placed the top 30 minutes of rete, make phenylpyridine ethene crosslinked and rete is become dry.
US 2008/0152864A1) and make the rete natural air drying (3) top layer protective seam: last be coated with the D6 hydrogel that last layer contains 10% carbon black with scraper film coating machine in the above (a kind of polyurethane-polyglycol hydrogel is specifically seen following patent:.
(4) using cutting machine to extract diameter is about the disk of 5mm and is installed in the sky kit.The shape of last kit is seen Fig. 2.Blood sample enters kit and the valve by black by kapillary from the kit right part and contacts with sensor and produce signal and received by luminoscope.The position of having reserved at present other five other sensors on the kit has reached the purpose of a plurality of detection things of one-time detection.
Four, specifically prepare example:
1, kit preparation process:
(1) 5 (6)-carboxyls-2 that the 176mg cellulose is fixing, the 4%D4 hydrogel solution of 7-dichloro-dihydro fluorescein two pivalates and 2.6g mixes and stirs, and makes cellulose powder Uniform Dispersion in solution,
(2) evenly to make it to form a layer thickness be the film of 100 μ M and dry at plastic sheeting with solution prepared in the step (1);
(3) with 30mg kreatinase, 35mg horseradish peroxidase, 0.63mg creatininase, 2.6mg sarcosine oxidase, 340mg water and 1.33g concentration be 10% and the PVA-Sbq solution that contains 10% bovine serum albumin mix, and stir on ice:
(4) film of preparation is coated PVA-Sbq/ enzyme solutions prepared in the step (3) in step (2), and the thickness of rete is 100 μ m, and film was placed uviol lamp lower 30 minutes;
(5) prepared film is coated with solution and the natural air drying that 6% carbon black and 1.8%D6 are contained in the upper strata in step (4);
(6) extract film prepared in the step (5) and be installed on the kit and can measure.
In view of the instability of fluorescent dye (structural formula is A), it is fixed on the cellulose by covalent bond, and fixing means is as follows:
1g aminocellulose and 50mg fluorescent dye are mixed in solvent dimethylformamide and stir, slowly add approximately dicyclohexylcarbodiimide and the 100mg N-hydroxy-succinamide of 200mg, filtration and also dry with dimethyl imide flushing filter residue after stirring the mixture four hours.
2, sample measurement:
Containing 100mMHEPES, 150mM sodium chloride, 5mM potassium chloride, (pH7.3) adds the variable concentrations kreatinin in the damping fluid of 1.3mM lime chloride, drives the sample solution of about 150 μ L and assign on the kit and with Portable fluorescence instrument OPTTCCA-TS with kapillary two to begin measurement.
3, data analysis:
The computer that links to each other with luminoscope will record photo emissions intensity that reaction produces and the curve of time, can be in the hope of reaction rate according to this curve, this speed be exactly kit to the response of kreatinin, can be in the hope of the concentration of kreatinin according to typical curve.
4, experimental data: kit is to the response of kreatinin:
Response shows at Fig. 3, Fig. 4 the kit that above-mentioned steps obtains to kreatinin.Fig. 3 is the fluorescence response in time of the sample of kit under different kreatinin concentration.Can see, under different kreatinin concentration, the speed that fluorescence rises is different.When not having kreatinin in the solution, fluorescence intensity has risen approximately 40% in 60 seconds.And when containing the kreatinin of 0.5mM in the solution, fluorescence intensity has risen approximately 80% in 60 seconds.So the speed that fluorescence strengthens and the concentration of kreatinin are closely-related, also can calculate with the speed that fluorescence increases the concentration of kreatinin.
Fig. 4 represent according to the fluorescence that Fig. 3 calculates gain in strength and kreatinin concentration between correlativity.Can more obviously see the correlativity that kreatinin and fluorescence are advanced the speed.Kreatinin concentration and fluorescence are advanced the speed linear.This will greatly facilitate the calculating of typical curve.The sensitivity of calculating gained according to 3 σ methods is 10 μ M.
5, conclusion
Present patent application has been set forth and has been planted the novel kit for detection of kreatinin content in blood and the urine.This kit will have specific enzyme and the fluorescent dye of superoxide sensitivity will be fixed on the kit kreatinin, and enzymatic reaction is converted into fluorescence signal and detected.
This kit can detect the above kreatinin of 10 μ M at an easy rate, meets request for utilization clinically.
Claims (1)
1. kreatinin content detecting reagent, it is characterized in that: comprise creatininase, kreatinase, sarcosine oxidase and horseradish peroxidase, fluorescent dye 5(6)-carboxyl-2,7-dichloro-dihydro fluorescein two pivalates, kreatinin is hydrolyzed under the catalysis of creatininase and is creatine, creatine is hydrolyzed into methyl amimoacetic acid under the catalysis of kreatinase, methyl amimoacetic acid generates glycocoll and a small amount of hydrogen peroxide thereupon under the catalysis of sarcosine oxidase, under the catalysis of horseradish oxide enzyme, catalytic oxidation of hydrogen peroxide 5(6)-carboxyl-2,7-dichloro-dihydro fluorescein two pivalates also generate the product with fluorescence response, speed and typical curve by monitoring fluorescence strengthens obtain kreatinin concentration;
Comprise following three-decker:
⑴ dyestuff bottom: cellulose and the mixing of D4 hydrogel and stirring with the fluorescent dye modified are coated with the film of last layer D4 hydrogel and make it natural air drying with scraper film coating machine on a clean plastic sheeting;
⑵ proteinase fixed bed: in the aqueous solution of PVA-Sbq, add 10% bovine serum albumin and react required creatininase, kreatinase, sarcosine oxidase and horseradish peroxidase, stir more than 25-40 minute, be coated with the PVA-Sbq solution of last layer enzyme with scraper film coating machine, uviol lamp is placed the top 30 minutes of rete, make phenylpyridine ethene crosslinked and rete is become dry;
⑶ top layer protective seam: in the above with scraper film coating machine be coated with D6 hydrogel that last layer contain 10% carbon black at last, and make the rete natural air drying.
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| CN201310360937.5A CN103472041B (en) | 2011-09-20 | 2011-09-20 | A kind of using method of creatinine content detection kit |
| CN 201110279969 CN102375066B (en) | 2011-09-20 | 2011-09-20 | Creatinine content detecting reagent and kit, and manufacturing and using methods of kit |
| CN201310363461.0A CN103472239B (en) | 2011-09-20 | 2011-09-20 | A kind of preparation method of kreatinin content detecting reagent of three-decker |
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| CN201310360937.5A Division CN103472041B (en) | 2011-09-20 | 2011-09-20 | A kind of using method of creatinine content detection kit |
| CN201310363461.0A Division CN103472239B (en) | 2011-09-20 | 2011-09-20 | A kind of preparation method of kreatinin content detecting reagent of three-decker |
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| CN103278468B (en) * | 2013-05-24 | 2015-09-02 | 宁波美康生物科技股份有限公司 | A kind of creatinine detection reagent |
| CN104459164B (en) * | 2014-11-28 | 2016-03-23 | 山东博科生物产业有限公司 | A kind of serum creatinine detection reagent |
| EP3262182B1 (en) * | 2015-02-27 | 2019-12-11 | Radiometer Medical ApS | Modified creatinase |
| CN106645758B (en) * | 2017-01-03 | 2019-03-22 | 长沙中生众捷生物技术有限公司 | The Test paper of creatinine |
| CN107760759A (en) * | 2017-11-07 | 2018-03-06 | 上海纳米技术及应用国家工程研究中心有限公司 | The method for detecting prostate cancer target methyl amimoacetic acid |
| CN114774256B (en) * | 2022-04-14 | 2023-03-24 | 重庆云芯医联科技有限公司 | Optical differential signal processing blood creatinine detection card, preparation method and application |
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| EP0281000A2 (en) * | 1987-02-27 | 1988-09-07 | Konica Corporation | Multi-layer analytical element for creatinine analysis |
| EP1162451A1 (en) * | 1996-10-31 | 2001-12-12 | Kyoto Daiichi Kagaku Co., Ltd. | Dry measuring test device |
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| CN1766640A (en) * | 2004-10-28 | 2006-05-03 | 王尔中 | Creatinine content determination method and creatinine diagnosis kit |
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| CN103472239A (en) | 2013-12-25 |
| CN103472239B (en) | 2016-03-09 |
| CN103472041A (en) | 2013-12-25 |
| CN102375066A (en) | 2012-03-14 |
| CN103472041B (en) | 2016-01-20 |
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