CN108152282A - A kind of reagent for detecting urine creatinine and its application - Google Patents
A kind of reagent for detecting urine creatinine and its application Download PDFInfo
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- CN108152282A CN108152282A CN201711338598.5A CN201711338598A CN108152282A CN 108152282 A CN108152282 A CN 108152282A CN 201711338598 A CN201711338598 A CN 201711338598A CN 108152282 A CN108152282 A CN 108152282A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/775—Indicator and selective membrane
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
- G01N2021/7759—Dipstick; Test strip
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Abstract
The present invention relates to technical field of medical detection, disclose a kind of reagent for detecting urine creatinine and its application.Reagent of the present invention includes A liquid and B liquid, and the A liquid includes phytic acid, copper sulphate, EDTA and Iodophthaleine Sodigue using buffer solution as basic solution；The B liquid includes TMB, PVP and peroxide using methanol or ethyl alcohol as solvent.The present invention is by adjusting the component in optimization reagent, influence of each disturbing factor to detection in urine specimen is reduced, has reached higher accuracy and precision, and linear relationship is good in a wide range of, reliability is high, compensates for existing similar product insufficient in terms of accuracy and precision the defects of.
Description
Technical field
The present invention relates to technical field of medical detection, and in particular to a kind of reagent for detecting urine creatinine and its application.
Background technology
Creatinine is product of the muscle in human body metabolism, is mainly excreted by glomerular filtration.The creatine of human body 90%
It is present in muscle, and most of presence in the form of phosphocreatine.Phosphocreatine generates creatinine, Hou Zhesui after sloughing phosphoric acid
Urine excludes to be urine creatinine.The daily output quite stable of normal person's urine creatinine.When kidney is invaded by the various causes of disease
Afterwards, kidney generation fibrosis causes kidney injury, and the function of discharged waste decreases, and has resulted in the toxin such as creatinine in body
Interior aggregation, causes urine creatinine to decline.Renal function of concentration and dilution and glomerular filtration work(can be understood by measuring urine creatinine
Can, it is time saving, accurate, economical, provide a kind of conveniently approach for clinical diagnosis.
At present, the test paper of urine creatinine is detected in the market, is the detection method of sxemiquantitative mostly, can only be provided general content
Range, and accurate content cannot be provided.Further, since inappropriate reagent selection easily leads to the precision and standard of testing result
Exactness is poor.
Invention content
In view of this, the purpose of the present invention is to provide a kind of reagents for detecting urine creatinine so that the reagent is being examined
Has higher accuracy and precision when surveying urine creatinine；
Another object of the present invention is to provide the test strips based on mentioned reagent so that the test strips exist
Has higher accuracy and precision, while can be detected with quantitative and semi-quantitative when detecting urine creatinine；
Another object of the present invention is to provide the quantitatively or semi-quantitatively detection method based on mentioned reagent, makes
It obtains the detection method and has higher accuracy and precision when detecting urine creatinine.
To achieve these goals, the present invention provides following technical solution：
A kind of reagent for detecting urine creatinine, including A liquid and B liquid, the A liquid includes plant using buffer solution as basic solution
Acid, copper sulphate, EDTA and Iodophthaleine Sodigue；
The B liquid includes TMB, PVP and peroxide using methanol or ethyl alcohol as solvent.
For existing detection urine creatinine product insufficient in terms of accuracy and precision the defects of, the present invention passes through
The composition of detection reagent is adjusted, precision and accuracy when significantly improving detection.
Preferably, the A liquid includes buffer solution 0.2mol/L-1.0mol/L, phytic acid 0.1%-0.5% (w/v), sulfuric acid
Copper 0.1%-0.3% (w/v), EDTA 50mmol/L and Iodophthaleine Sodigue 0.1%-1% (w/v)；It is highly preferred that the A liquid includes pH
Value 6.0-7.0 buffer solutions 0.2-0.75mol/L, phytic acid 0.1%-0.5% (w/v), copper sulphate 0.1%-0.3% (w/v), EDTA
50mmol/L and Iodophthaleine Sodigue 0.1%-1% (w/v)；In the specific embodiment of the invention, the A liquid is buffered including pH value 6.5
Liquid 0.5mol/L, phytic acid 0.1%-0.5% (w/v), copper sulphate 0.1%-0.3% (w/v), EDTA 50mmol/L and Iodophthaleine Sodigue
0.1%-1% (w/v).
In the specific embodiment of the invention, the buffer solution is sodium citrate buffer solution and Tris buffer solutions；Specifically,
The buffer solution be sodium citrate 0.1mol/L-0.5mol/L, Tris buffer solution 0.1mol/L-0.25mol/L, pH value range
6.0-7.0；More specifically, the buffer solution is sodium citrate 0.25mol/L, Tris buffer solution 0.25mol/L, pH value 6.5.
Preferably, the B liquid using methanol or ethyl alcohol as solvent, includes TMB 0.3%-1% (w/v), PVP 1%-5%
(w/v) and peroxide 3%-5% (w/v)；It is highly preferred that the B liquid is using methanol as solvent, comprising TMB 0.5% (w/v),
PVP 3% (w/v) and peroxide 3%-5% (w/v)；In the present invention, the peroxide is bis- for 2,5- dimethyl -2,5-
One or more of (tert-butyl peroxy base) hexane, hydrogen peroxide cumene and urea peroxide；In a specific embodiment, institute
Peroxide is stated as bis- (tert-butyl peroxy base) hexanes of 2,5- dimethyl -2,5- of 5% (w/v) or the hydrogen peroxide of 5% (w/v)
Cumene is bis- (tert-butyl peroxy base) hexanes of 2,5- dimethyl -2,5- of 3% (w/v) and the hydrogen peroxide of person 2% (w/v)
The mixed peroxides of cumene.
Meanwhile the invention also provides the reagent prepare detect urine creatinine test strips in application or detection urinate
Application in liquid creatinine.
According to application the present invention provides a kind of test strips for detecting urine creatinine, by carrier filter paper successively through the reagent
A liquid and B liquid leaching it is even after drying prepare.More specifically preparation process is as follows：
Carrier filter paper is soaked in A liquid even, suck redundant solution, dried in drying box, 90 DEG C -105 DEG C of drying temperature is dried
Dry time 60-70min；
The filter paper for being soaked with A liquid of drying in B liquid is soaked even, suck redundant solution, dried in drying box, drying temperature 70
DEG C -90 DEG C, drying time 50-60min.
Operation and carrying for convenience, the test strips can cut into item, be pasted onto PVC on pieces, then cut into small
The shape of item, specification 5mm × 5mm, the specification and final products can be adjusted arbitrarily, this depends on practical demand.
Preferably, the carrier filter paper is 3MM or 3 grade of chromatography filter paper.
According to the test strips of preparation, the present invention also is able to carry out urine sample detection quantitatively or semi-quantitatively, wherein half
Quantitative detection method is：
The creatinine solution of normal gradients concentration is added drop-wise to respectively in the test strips, obtains standard color comparison card；
Urine sample is added drop-wise in the test strips, color and concentration on standard color comparison card are compared according to colour developing result
Correspondence, obtain semi-quantitative results.
More specifically, compound concentration is the creatinine standard solution of 320mg/L, and gradient dilution is into 320mg/dl, 160mg/
Dl, 80mg/dl, 40mg/dl, 20mg/dl, 10mg/dl, 0mg/dl8 concentration；The creatinine solution of above-mentioned standard gradient concentration
It adds in test strips, sample size 25ul is observed after 90 seconds, can be visually observed out from colourless to bottle-green apparent color range, be obtained
Obtain standard color comparison card；Sample-adding is originally in test strips, and sample size 25ul, compared with standard color comparison card, range estimation is closest with sample card
Standard color comparison card corresponding to concentration be sample to be tested concentration.
And the method for quantitatively detecting creatinine in urine includes：
The creatinine solution of normal gradients concentration is added drop-wise to respectively in the test strips, is drawn and marked according to Urine Analyzer
Directrix curve；
Urine sample is added drop-wise in the test strips, detected value is obtained by Urine Analyzer, according to standard curve
Obtain corresponding creatine concentration.
More specifically, the test strips are fitted into and special get stuck and in cartridge, be placed in dedicated full-automatic Urine Analyzer
Reagent card storehouse in, sample size 25ul, 90 seconds reaction time；
The creatinine solution of aforesaid standards gradient concentration is placed in the sample disk of full automatic urine analyzer, is detected one by one,
Draw standard curve；
Sample is placed in the sample disk of full automatic urine analyzer, after to be detected, instrument according to standard curve from
It is dynamic to calculate surveyed concentration of specimens.
According to actually detected result of the test, the present invention has higher accuracy, and relative deviation is less than 4%, far below mark
The 10% of alignment request；Precision result shows that standard rate is 2.7%, much smaller than the 10% of standard requirement, shows institute of the present invention
Method is stated with high precision；Linear relationship result of the test shows have in the range of 0.0mg/dl-320.0mg/dl good
The linearity, R values be 0.9999, close to 1.
In addition, the component in similar product is replaced on reagent of the present invention composition or deletes constituent part, composition control examination
Agent product is compared, the results show that bigger difference occur in the accuracy of reference product and precision, correlation R-value is only
It is more than the 10% of requirement for 0.8812, CV values.
By above technical scheme it is found that the present invention reduces each in urine specimen by adjusting the component in optimization reagent
Influence of the disturbing factor to detection, has reached higher accuracy and precision, and linear relationship is good in a wide range of, reliably
Property it is high, compensate for existing similar product insufficient in terms of accuracy and precision the defects of.
Description of the drawings
Fig. 1 show standard color comparison card；Wherein, it is its corresponding concentration below color lump, unit mg/dl.
Specific embodiment
The invention discloses a kind of reagent for detecting urine creatinine and its application, those skilled in the art can use for reference herein
Content is suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications are to people in the art
It is it will be apparent that they are considered as being included in the present invention for member.Reagent of the present invention and its related application have been led to
Preferred embodiment is crossed to be described, related personnel significantly can not depart from the content of present invention, in spirit and scope to this paper institutes
The reagent and its related application stated are modified or suitably changed with combining, to realize and using the technology of the present invention.
Just a kind of reagent for detecting urine creatinine provided by the present invention and its application are described further below.
Embodiment 1：Reagent of the present invention and test strips
1st, reagent forms
A liquid is：Sodium citrate 0.1mol/L, Tris 0.2mol/L, pH value range 7.0, phytic acid 0.3% (w/v), sulfuric acid
Copper 0.1% (w/v), EDTA 50mmol/L, Iodophthaleine Sodigue 1.0% (w/v)；
B liquid is：TMB 0.3% (w/v), PVP 5% (w/v), hydrogen peroxide cumene 5% (w/v), ethyl alcohol dissolving.
2nd, the preparation of test strips
Filter paper is soaked in A liquid even, suck redundant solution, dried in drying box, 90 DEG C -105 DEG C of drying temperature, during drying
Between 60-70min；
The filter paper for being soaked with A liquid of drying in B liquid is soaked even, suck redundant solution, dried in drying box, drying temperature 70
DEG C -90 DEG C, drying time 50-60min.
Dry filter paper cuts into item, is pasted onto PVC on pieces, then cuts into small item, test paper block specifications 5mm × 5mm.
Embodiment 2：Reagent of the present invention and test strips
1st, reagent forms
A liquid is：Sodium citrate 0.5mol/L, Tris 0.1mol/L, pH value range 6.0, phytic acid 0.1% (w/v), sulfuric acid
Copper 0.3% (w/v), EDTA 50mmol/L, Iodophthaleine Sodigue 0.1% (w/v)；
B liquid is：TMB 1% (w/v), PVP 1% (w/v), bis- (tert-butyl peroxy base) hexanes 5% of 2,5- dimethyl -2,5-
(w/v), ethyl alcohol dissolves.
2nd, the preparation of test strips
Filter paper is soaked in A liquid even, suck redundant solution, dried in drying box, 90 DEG C -105 DEG C of drying temperature, during drying
Between 60-70min；
The filter paper for being soaked with A liquid of drying in B liquid is soaked even, suck redundant solution, dried in drying box, drying temperature 70
DEG C -90 DEG C, drying time 50-60min.
Dry filter paper cuts into item, is pasted onto PVC on pieces, then cuts into small item, test paper block specifications 5mm × 5mm.
Embodiment 3：Reagent of the present invention and test strips
1st, reagent forms
A liquid is：Sodium citrate 0.25mol/L, Tris 0.25mol/L, pH value range 6.5, phytic acid 0.5% (w/v), sulphur
Sour copper 0.2% (w/v), EDTA 50mmol/L, Iodophthaleine Sodigue 0.5% (w/v)；
B liquid is：TMB 0.5% (w/v), PVP 3% (w/v), hydrogen peroxide cumene 2% (w/v), 2,5- dimethyl -2,
Bis- (tert-butyl peroxy base) hexanes 3% (w/v) of 5-, methanol dissolving.
2nd, the preparation of test strips
Filter paper is soaked in A liquid even, suck redundant solution, dried in drying box, 90 DEG C -105 DEG C of drying temperature, during drying
Between 60-70min；
The filter paper for being soaked with A liquid of drying in B liquid is soaked even, suck redundant solution, dried in drying box, drying temperature 70
DEG C -90 DEG C, drying time 50-60min.
Dry filter paper cuts into item, is pasted onto PVC on pieces, then cuts into small item, test paper block specifications 5mm × 5mm.
Embodiment 4：Semi-quantitative detection method
(1) compound concentration is the creatinine standard solution of 320mg/L, and gradient dilution is into 320mg/dl, 160mg/dl, 80mg/
Dl, 40mg/dl, 20mg/dl, 10mg/dl, 0mg/dl8 concentration.
(2) above-mentioned standard liquid is added in test strips, sample size 25ul is observed after 90 seconds, can be visually observed out from nothing
Color is to bottle-green apparent color range.
(3) sample-adding is originally in test strips, and sample size 25ul, compared with standard color comparison card, range estimation is immediate with sample card
Concentration corresponding to standard color comparison card is the concentration of sample to be tested.Standard color comparison card is as shown in Figure 1.
Embodiment 5：Quantitative detecting method
(1) test strips are fitted into the special reagent card for getting stuck and in cartridge, being placed in dedicated full-automatic Urine Analyzer
In storehouse, sample size 25ul, 90 seconds reaction time.
(2) titer in embodiment 4 is placed in the sample disk of full automatic urine analyzer, detected one by one, it is fixed to draw
Mark song line.
(3) in use, sample is placed in the sample disk of full automatic urine analyzer, after to be detected, instrument according to
Calibration curve calculates surveyed concentration of specimens automatically.
Embodiment 6：Accuracy analysis
Using the test strips of embodiment 1-3, according to the method for embodiment 5 be respectively to concentration 40mg/dl, 80mg/dl,
The creatinine standard items of 160mg/dl are detected, wherein, 1 ELISA test strip 40mg/dl standard items of embodiment, 2 test paper of embodiment
Item detects 80mg/dl standard items, and 3 ELISA test strip 160mg/dl standard items of embodiment the results are shown in Table 1.
Table 1
The results show that each ELISA test strip result relative deviation of the present invention is respectively less than the 10% of standard requirement, show this hair
Bright the method has high accuracy.
Embodiment 7：Precision Analyze
Using the test strips of embodiment 3 to any one urine sample, detection 10 times is repeated according to 5 method of embodiment, as a result
It is shown in Table 2.
Table 2
The results show that standard rate is 2.7%, much smaller than the 10% of standard requirement, show that the method for the invention has
High precision.In addition, the test strips according to Examples 1 and 2 are detected, standard deviation is respectively 2.9% and 2.8%, is not surpassed
Cross 3%.
Embodiment 8：Linear relationship is analyzed
Using the test strips of embodiment 3, using the high creatinine urine sample of 320mg/dl as detection sample, by creatinine urine sample
Product are diluted to 7 different concentration, be followed successively by 0mg/dl, 10mg/dl, 20mg/dl, 40mg/dl, 80mg/dl, 160mg/dl,
320mg/dl carries out 2 inspections using the test strips of embodiment 3 and the detection method of embodiment 5 to each concentration of above-mentioned sample
It surveys, calculates coefficient R value, the results are shown in Table 3.
Table 3
The results show that R values are 0.9999, close to 1, show reagent of the present invention and its test strips 0-320.0mg/dl's
In the range of have the splendid linearity.In addition, the test strips using embodiment 1-2 carry out linear analysis, R values are respectively 0.9999
And 0.9998.
Embodiment 9：With the contrast test of reference substance
1st, the contrast test of reference substance 1
On the basis of the test strips of embodiment 3, the methanol reagent in B liquid of the present invention is replaced using common chloroform solvent,
Reference substance 1 is made；
Using the test strips of embodiment 3 and reference substance 1, by the detection method of embodiment 5, same sample is examined
It surveys, each pattern detection 10 times, testing result is shown in Table 4
Table 4
The results show that the CV values of reference substance 1 have been over the 10% of requirement, show the preferred methanol of test strips of the present invention
Solvent precision is better than chloroform solvent, shows that the method for the invention precision is good.
2nd, the contrast test of reference substance 2
On the basis of the test strips of embodiment 3, remove phytic acid and edta reagent in A liquid of the present invention, and using citric acid
Potassium (0.3%w/v) and polyvinylpyrrolidone replace (0.1%w/v), and reference substance 2 is made；
To 20 samples of definite value, the test strips of embodiment 3 and reference substance 2 is respectively adopted, passes through the inspection of embodiment 5
Survey method detects, and each pattern detection 1 time, testing result is shown in Table 5.
Table 5
The results show that test strips of the present invention and definite value sample correlations are good, in accuracy or in precision
Upper better than reference substance 2, shows that the method for the invention accuracy and precision are all higher.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of reagent for detecting urine creatinine, which is characterized in that including A liquid and B liquid, the A liquid is molten based on buffer solution
Liquid includes phytic acid, copper sulphate, EDTA and Iodophthaleine Sodigue；
The B liquid includes TMB, PVP and peroxide using methanol or ethyl alcohol as solvent.
2. reagent according to claim 1, which is characterized in that the A liquid includes buffer solution 0.2mol/L-1.0mol/L, plants
Sour 0.1%-0.5% (w/v), copper sulphate 0.1%-0.3% (w/v), EDTA 50mmol/L and Iodophthaleine Sodigue 0.1%-1% (w/
v)。
3. reagent according to claim 1 or claim 2, which is characterized in that the buffer solution delays for sodium citrate buffer solution and Tris
Fliud flushing.
4. reagent according to claim 1, which is characterized in that the B liquid includes TMB using methanol or ethyl alcohol as solvent
0.3%-1% (w/v), PVP 1%-5% (w/v) and peroxide 3%-5% (w/v).
5. according to the reagent of claim 1 or 4, which is characterized in that the peroxide is the bis- (uncles of 2,5- dimethyl -2,5-
One or more of butyl peroxy base) hexane, hydrogen peroxide cumene and urea peroxide.
6. reagent described in claim 1-5 any one prepare detect urine creatinine test strips in application or detection urine
Application in creatinine.
7. a kind of test strips for detecting urine creatinine, which is characterized in that by carrier filter paper successively through claim 1-5 any one
The even rear drying of A liquid and B liquid leaching of the reagent prepares.
8. test strips according to claim 7, which is characterized in that the carrier filter paper is 3MM or 3 grade of chromatography filter paper.
9. a kind of method of creatinine in half-quantitative detection urine, which is characterized in that including：
The creatinine solution of normal gradients concentration is added drop-wise to respectively in the test strips described in claim 7 or 8, obtains standard colorimetric
Card；
Urine sample is added drop-wise in the test strips described in claim 7 or 8, face on standard color comparison card is compared according to colour developing result
The correspondence of color and concentration obtains semi-quantitative results.
A kind of 10. method for quantitatively detecting creatinine in urine, which is characterized in that including：
The creatinine solution of normal gradients concentration is added drop-wise to respectively in the test strips described in claim 7 or 8, according to urinalysis
Instrument draws standard curve；
Urine sample is added drop-wise in the test strips described in claim 7 or 8, detected value is obtained by Urine Analyzer, according to
Standard curve obtains corresponding creatine concentration.
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| CN108152282B (en) | 2020-12-25 |
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