CN109916894A - A kind of dry chemistry reagent piece and preparation method thereof quantitative determining concentration of glucose - Google Patents
A kind of dry chemistry reagent piece and preparation method thereof quantitative determining concentration of glucose Download PDFInfo
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Abstract
The present invention relates to clinical in vitro diagnosis in vitro reagent technique field, in particular to a kind of dry chemistry reagent piece and preparation method thereof for quantitative determining concentration of glucose.The dry chemistry reagent piece includes four laminations, is followed successively by support layer, color layer, reagent layer and diffusion layer；Reagent in reagent layer includes buffer system, hydrophilic colloid, glucose oxidase, peroxidase, activator, chelating agent and stabilizer；Reagent in color layer includes chromogen, hydrophilic colloid and surfactant.Compared with prior art, the invention has the following advantages that operating process is simple, pattern detection is directly added dropwise without preparing in reagent；Analoids dry no moisture, and analoids have good stability；Diffusion layer has filtering function, and haemolysis, bilirubin and ascorbic acid sample do not significantly interfere with measurement result；Testing result is reproducible, accuracy is high, can carry out quantitative detection, and the range of linearity is wide；Preparation process is easy, and easy to operate, harmfulness, pollution are small.
Description
Technical field
The present invention relates to clinical in vitro diagnosis in vitro reagent technique fields, in particular to a kind of to quantitative determine the dry of concentration of glucose
Chemical reagent piece and preparation method thereof.
Background technique
Glucose is the important composition ingredient of human body and the important sources of energy.Glucose is new old generation in organism
Thank to indispensable nutriment.The heat that its oxidation reaction is released is the important sources of energy needed for human life activity.
Normal human needs many sugar to provide energy daily, and the normal operation for various tissues, internal organs provides power.Portugal in blood
Grape sugar is known as blood glucose, so blood glucose must keep certain horizontal needs that could maintain internal each organ and tissue.Hyperglycemia
The harm generated to human health is further serious, such as generates obesity, diabetes disease.Currently, diabetes have become it is only secondary
In cancer and cardiovascular disease, the third-largest disease of human health is threatened, the investigation knot according to related international organization in 2010
Fruit shows that global diabetic has been up to 2.85 hundred million, and the number of annual diabetic is still constantly soaring.Meanwhile sugar
Urine disease can also generate multiple complications, such as cerebral infarction, cerebral hemorrhage, hypertension, life peace that is indirect or directly affecting individual
Entirely, the state of an illness of diabetic is carried out real time monitoring and treated to be an effective way for guaranteeing diabetic's life security
Diameter, and observe Diabetes Mellitus with the most effective means of detection in real time is grape in measurement diabetic's blood
The concentration of sugar.Therefore, have with detection to the diagnosis, treatment and control of the health and disease of human body to the analysis of concentration of glucose
Significance.
The method of measurement glucose can be divided into two methods of liquid reagent and dry chemistry reagent by types of agents at present.Liquid
The detection method of reagent mainly has spectrophotometry, Optical Rotation, gas chromatography, high performance liquid chromatography etc..These methods
Detection is carried out to concentration of glucose and needs specific equipment, equipment itself costly, does not have economy；Profession is needed simultaneously
Technician operates instrument, does not have due universality.The method measurement period is long, and analytic process is complex, cannot achieve
The real time measure of glucose.
Dry chemistry reagent is mainly used in emergency treatment, on-site test.With easy, quickly, flexibly and be not necessarily to professional
The advantages that operation.Domestic at present to be essentially qualitative or semiquantitative determination, this dry chemistry reagent piece is by filter paper, cellulose
The one kind such as piece, porous glue film multilayer material whole reagents fixed in advance, two layers and three layers by the processing such as stacking, bonding, punching press
In conjunction with.It is little to measure the range of linearity, encountering has using meeting interference experiment result under drug condition.There are many examinations in analysis test
Agent cannot be pre-mixed, and complicated test fluid needs to pre-process, and which limits dry chemistry reagent piece application fields.Quantitative inspection
The dry chemistry reagent card of survey is all external production.Using Johson & Johnson and company, Fuji as the multilayer dry slides method of representative, sense is utilized
The reagents such as reagent layer, auxiliary reagent layer, optical diffusion layer and distribution layer are coated in transparent support layer by the coating technology of ray film,
From the variation of the reverse side of support layer measurement optical density.Though above-mentioned dry chemistry reagent piece can quantitative detection, distribution layer or diffusion layer
Organic reagent need to be used, requires material installation high, production technology complexity, and very harmful to preparation personnel health, pollution
Environment.
Summary of the invention
In view of this, the present invention provides a kind of dry chemistry reagent piece for quantitative determining concentration of glucose and its preparation sides
Method.The dry chemistry reagent piece testing result accuracy is high, and precision is good, and stability is good, and long shelf-life is easy to operate, is applicable in model
It encloses wide.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of dry chemistry reagent pieces for quantitative determining concentration of glucose, include four laminations, are followed successively by
Support layer, color layer, reagent layer and diffusion layer；
Reagent in reagent layer include buffer system, hydrophilic colloid, glucose oxidase, peroxidase, activator,
Chelating agent and stabilizer；Dosage of each component are as follows:
Reagent in color layer includes chromogen, hydrophilic colloid and surfactant.
Testing principle are as follows: liquid sample is added dropwise in analoids diffusion layer, reagent layer is uniformly distributed and is transported to, in reagent layer
By the catalysis of glucose oxidase, the glucose in sample, which is oxidized, generates hydrogen peroxide and gluconate, subsequent peroxide
Hydrogen peroxide is decomposited the free radical of oxygen by compound enzyme, makes the 4-AA in color layer and 3,5- dichloro hydroxy benzenes sulphur
Sour sodium is oxidized to red quinonoid compound, and under 510nm wavelength, the variation through reflective spectrophotometry measurement reflectivity passes through
The concentration of standard curve calculating glucose.By reaction temperature control under the conditions of 37 DEG C ± 1 DEG C, measurement in 5 minutes can be complete
At.Sample is added dropwise in diffusion layer, is measured in opposite one side, colored intensity is high, reduces the bubble that the measurement of sample-adding end generates, excessive
Liquid, turbidity and caused by interfere.Simultaneously because color layer is hydrophilic colloid, there is very high colour developing evenness.
The dry chemistry reagent piece of above structure is mainly used for measuring serum, urine, whole blood equal samples, mixes the sample with before measurement
Suitable isotonic solution is diluted, then is measured.
Preferably, buffer system is MOPS or phosphate buffer, and pH value is 7.0~7.5 in reagent layer；Activator
For sodium chloride；Chelating agent is EDTA；Stabilizer is polyethylene glycol-6000.
Preferably, the pH value of buffer system is 7.1~7.3.
Preferably, dosage of each component in reagent layer are as follows:
Preferably, hydrophilic colloid is selected from polyvinyl alcohol, amylopectin, cellulose esters, agarose, polyvinylpyrrolidine
One of ketone, polyacrylamide, hydroxypropyl methyl cellulose, Copolymer of Methyl Vinyl Ether/Maleic Anhydride are a variety of.
Preferably, hydrophilic colloid is the mixture of hydroxypropyl methyl cellulose and polyvinyl alcohol.
Preferably, chromogen is made of A and B in color layer, the A is 4-AA, and B is selected from phenol, to chlorine
One of phenol, P-hydroxybenzoic acid, 3,5- dichloro sodium hydroxybenzenesulfonate or a variety of, dosage of each component in color layer are as follows:
0.6~30mmol/m of chromogen2；
10~100g/m of hydrophilic colloid2；
0.1~5g/m of surfactant2。
Preferably, chromogen is made of 4-AA and 3,5- dichloro sodium hydroxybenzenesulfonate in color layer, color layer
Middle surfactant is polyethylene glycol to isooctyl phenyl ether, dosage of each component in color layer are as follows:
It is highly preferred that dosage of each component in color layer are as follows:
Preferably, diffusion layer is made of hydrophilic high molecular polymer, surfactant and particulate matter；In diffusion layer
Dosage of each component are as follows:
15~25g/m of hydrophilic high molecular polymer2；
0.1~5g/m of surfactant2；
200~300g/m of particulate matter2。
Preferably, in diffusion layer, hydrophilic high molecular polymer be selected from polyurethane, polyamide, sodium carboxymethylcellulose,
One of polyvinyl alcohol, amylopectin, xanthan gum, sodium alginate, gum arabic, peach gum, chitosan, polyvinylacetate
Or it is a variety of；Hydrophilic high molecular polymer has porous structure, can filter out the interfering substance in sample；
Preferably, hydrophilic high molecular polymer is polyurethane.
Preferably, surfactant is polyethylene glycol to isooctyl phenyl ether；
Preferably, particulate matter is selected from pigment, crystallite colloid, silica, resin bead, bead, diatomite, fiber
One of plain ester is a variety of.
Preferably, particulate matter is titanium dioxide and cellulose esters.
Preferably, support layer is the polymer of polyethylene terephthalate, with a thickness of 50~300 microns.
Preferably, support layer is with a thickness of 100~200 microns.
Preferably, the surface of support layer is by ultraviolet irradiation, corona or coating bottom layer treatment.
Preferably, support layer is handled by ultraviolet irradiation.
The present invention also provides the preparation methods of the dry chemistry reagent piece, include the following steps:
Developing solution reagent, reaction solution reagent are sequentially coated on support layer, diffusion liquid reagent is then sprayed on support
It is dry on layer, obtain successively include support layer, color layer, reagent layer and diffusion layer dry chemistry reagent piece；
Reagent in reagent layer include buffer system, hydrophilic colloid, glucose oxidase, peroxidase, activator,
Chelating agent and stabilizer；Dosage of each component are as follows:
Reagent in color layer includes chromogen, hydrophilic colloid and surfactant.
Preferably, the surface of support layer is by ultraviolet irradiation, corona or coating bottom layer treatment.
Preferably, the coating method of color layer and reagent layer includes prepared by the modes such as levelling, extension stream, the viscous, coating of rolling, work
To be preferred, preparation method is coating.
The present invention provides a kind of dry chemistry reagent pieces and preparation method thereof for quantitative determining concentration of glucose.The dry chemical
Analoids include four laminations, are followed successively by support layer, color layer, reagent layer and diffusion layer；Reagent in reagent layer includes buffering
System, hydrophilic colloid, glucose oxidase, peroxidase, activator, chelating agent and stabilizer；Reagent in color layer
Including chromogen, hydrophilic colloid and surfactant.Compared with prior art, the invention has the following advantages that
(1) operating process is simple, and pattern detection is directly added dropwise without preparing in reagent.
(2) the dry no moisture of reagent layer, analoids have good stability.
(3) diffusion layer has filtering function, and haemolysis, bilirubin and ascorbic acid sample obviously do not do measurement result
It disturbs.
(4) testing result is reproducible, accuracy is high, can carry out quantitative detection, and the range of linearity is wide.
(5) preparation process is easy, and easy to operate, harmfulness, pollution are small.
Experiment shows the dry chemistry reagent piece of quantitatively determining human blood glucose concentration of the present invention, testing result
Accuracy is high, and precision is good, and specificity is good, and stability is good, and long shelf-life is easy to operate, applied widely, is not necessarily to professional
The advantages that supplement the shortcoming of wet chemistry method well, accuracy of measurement and precision are already close to wet chemical determination side
Method is provided a great convenience in clinical field of fast detection.
Specific embodiment
The invention discloses a kind of dry chemistry reagent piece and preparation method thereof for quantitative determining concentration of glucose, this field skills
Art personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replacements and
Change apparent to those skilled in the art, they are considered as being included in the present invention.Method of the invention and
Using being described by preferred embodiment, related personnel can obviously not depart from the content of present invention, spirit and scope
It is interior that method described herein and application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
In dry chemistry reagent piece and preparation method thereof of quantitative determination concentration of glucose provided by the invention agents useful for same or
Instrument is available on the market.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1: the preparation method of glucose dry chemistry reagent piece of the present invention
Transparent support layer material uses polyethylene terephthalate material, and with a thickness of 0.20mm, length 30cm is wide
Degree is 20cm.Surface is handled by ultraviolet irradiation, irradiation distance 10cm, and irradiation time 60 minutes.
The formula of developing solution reagent is as follows:
The formula of reaction solution reagent is as follows:
Color layer is coated on the polyethylene terephthalate support layer Jing Guo ultraviolet processing, and reagent layer is continuously applied
It overlays on color layer, is put into drying box, 30 DEG C dry 20 minutes.
The formula of diffusion liquid reagent is as follows:
Diffusion liquid is uniformly sprayed on reagent layer, is put into drying box and dries.12mm is cut to strip cutting machine
The test-paper of × 12mm size, is sealed with aluminium foil bag.
Detection method: 10 μ L test samples are added dropwise and are incubated for 5 minutes under analoids, 37 DEG C of constant temperatures, use is reflective
Photometer is measured, and the concentration of glucose in sample is calculated according to standard curve.
Embodiment 2: the accuracy analysis of the method for the invention
Test sample: 20 examinee's blood samples；
Contrasting detection method: with commercial glucose detection kit (hexokinase method) single liquid reagent in Hitachi 7080
It is detected on automatic clinical chemistry analyzer, 2.5 μ L samples is added by automatic clinical chemistry analyzer, 250 μ L reagents, 37 DEG C are reacted 10 points
Clock measures the absorbance value of 340nm, and the concentration of glucose in sample is calculated according to standard curve.
20 samples are measured respectively with 1 detection method of embodiment and contrasting detection method, and testing result is shown in Table 1.
Table 1 analyzes result
The results show that calculated R according to testing result2Value is 0.999, is greater than 0.95, shows the method for the invention
Testing result and contrast agent box testing result no significant difference have high accuracy (degree of conformity).
Embodiment 3: the Precision Analyze of the method for the invention
Test sample: any one blood serum sample；
Detection 10 times is repeated to same sample to be tested with 1 detection method of embodiment, testing result is shown in Table 2.
2 test sample concentration of glucose of table (10 times) and standard rate (coefficient of variation)
The results show that standard rate is 0.62%, less than the 10% of standard requirements, show that the method for the invention has height
Precision.
Embodiment 4: the linear analysis of the method for the invention
Test sample: high concentration glucose sample (30mmol/L)；
Glucose sample is diluted to 7 different concentration, be followed successively by 0mmol/L, 2.5mmol/L, 5.0mmol/L,
7.5mmol/L, 10mmol/L, 20mmol/L, 30mmol/L are dense to each of above-mentioned sample using the detection method of embodiment 1
Degree carries out 2 detections, calculates coefficient R value, and testing result is shown in Table 3.
3 linear test result of table
The results show that be 0.9994 according to the calculated R value of testing result that 1 detection method of embodiment obtains, close to
1, show that the method for the invention has the good linearity in the range of 0.0mmol/L-30.0mmol/L.
Embodiment 5: the preparation method of glucose dry chemistry reagent piece of the present invention
Transparent support layer material uses ethylene glycol terephthalate material, with a thickness of 0.20mm, length 30cm, width
For 20cm.Surface pass through sided corona treatment, corona intensity 900W, the corona time 5 seconds.
The formula of developing solution reagent is as follows:
The formula of reaction solution reagent is as follows:
Color layer is coated on the polyethylene terephthalate support layer Jing Guo sided corona treatment, and reagent layer is continuously applied
It overlays on color layer, is put into drying box and dries.
The formula of diffusion liquid reagent is as follows:
Diffusion liquid is uniformly sprayed on reagent layer, is put into drying box and dries.10mm is cut to strip cutting machine
The test-paper of × 10mm size, is sealed with aluminium foil bag.
Detection method: 10 μ L test samples are added dropwise and are incubated for 5 minutes under analoids, 37 DEG C of constant temperatures, use is reflective
Photometer is measured, and the concentration of glucose in sample is calculated according to standard curve.
Embodiment 6: the stability analysis of the method for the invention
Condition of storage: sealing is placed in 2-8 DEG C of refrigerator.
Round of visits: when 2-8 DEG C 0, March, September, 15 months, 18 months, 21 months.
It is detected with the detection method of embodiment 5 in above-mentioned round of visits, accuracy in computation, precision, linear model
It encloses, testing result is shown in Table 4.
4 stability test result of table
The results show that the calculated R of testing result obtained according to 5 detection method of embodiment2Value is all larger than 0.95, standard
Rate is respectively less than 10%, the R value of standard requirements all close to 1, shows that the method for the invention stores 21 under the conditions of 2-8 DEG C
Month, analoids still have good accuracy, precision, the range of linearity.
Embodiment 7: the anti-Interference Analysis of the method for the invention
Test sample: bilirubin (20mg/dL) is added in check sample serum, check sample serum in check sample serum
Middle addition ascorbic acid (3mg/dL)；
3 detections are carried out to above-mentioned sample using the detection method of embodiment 5, experiment with computing sample is relative to check sample
Bias produced by measurement result, testing result are shown in Table 5.
5 anti-interference test result of table
The results show that the calculated D of testing result obtained according to 5 detection method of embodimentobsRespectively less than Dc, do not observe
It is influenced to clinical significance, shows the method for the invention in the bilirubin of 20mg/dL concentration and the Vitamin C of 3mg/dL concentration
Acid does not significantly interfere with measurement result.
Comparative example 1: the preparation method of glucose dry chemistry reagent piece described in other methods
Transparent support layer material uses polyethylene terephthalate material, with a thickness of 0.20mm, color layer and reaction
Layer uses PES film, and diffusion layer uses glass fibre.Color layer, conversion zone and diffusion layer are separately immersed in developing solution, reaction solution
In diffusion liquid, takes out, strike off after soaking completely.It is put into 30-50 DEG C of drying box, is taken out after dry, color layer is pasted
In on support layer, then successively it is compacted conversion zone and diffusion layer.
The formula of developing solution reagent is as follows:
Polyethylene glycol is to isooctyl phenyl ether 2g/L；
3,5- dichloro sodium hydroxybenzenesulfonate 20mmol/L；
4-AA 4.0mmol/L；
The formula of reaction solution reagent is as follows:
The formula of diffusion liquid reagent is as follows:
Polyethylene glycol is to isooctyl phenyl ether 1.5g/L；
It is cut to the test-paper of 12mm × 12mm size with strip cutting machine, is sealed with aluminium foil bag.
Detection method: 10 μ L test samples are added dropwise and are incubated for 5 minutes under analoids, 37 DEG C of constant temperatures, use is reflective
Photometer is measured, and the concentration of glucose in sample is calculated according to standard curve.
The Precision Analyze of 2 comparative example of comparative example, 1 method the method
Test sample: any one blood serum sample；
Detection 10 times is repeated to same sample to be tested with 1 detection method of comparative example, testing result is shown in Table 6.
6 test sample concentration of glucose of table (10 times) and standard rate (coefficient of variation)
The results show that standard rate is 5.48%, hence it is evident that be greater than embodiment standard rate 0.62%, show of the present invention
Method has high precision.
The linear analysis of 3 comparative example of comparative example, 1 method the method
Test sample: high concentration glucose sample (30mmol/L)；
Glucose sample is diluted to 7 different concentration, be followed successively by 0mmol/L, 2.5mmol/L, 5.0mmol/L,
7.5mmol/L, 10mmol/L, 20mmol/L, 30mmol/L are dense to each of above-mentioned sample using the detection method of comparative example 1
Degree carries out 2 detections, calculates coefficient R value, and testing result is shown in Table 7.
7 linear test result of table
The results show that being 0.9957 according to the calculated R value of testing result that 1 detection method of comparative example obtains, and implement
It is good to show that the method for the invention has in the range of 0.0mmol/L-30.0mmol/L for example R value 0.9994 closer to 1
The linearity.
The above is only a 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 are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of dry chemistry reagent piece for quantitative determining concentration of glucose, which is characterized in that include four laminations, be followed successively by support
Layer, color layer, reagent layer and diffusion layer；
Reagent in the reagent layer include buffer system, hydrophilic colloid, glucose oxidase, peroxidase, activator,
Chelating agent and stabilizer；Dosage of each component are as follows:
Reagent in the color layer includes chromogen, hydrophilic colloid and surfactant.
2. dry chemistry reagent piece according to claim 1, which is characterized in that in the reagent layer, buffer system MOPS
Or phosphate buffer, pH value is 7.0~7.5；Activator is sodium chloride；Chelating agent is EDTA；Stabilizer is polyethylene glycol-
6000。
3. dry chemistry reagent piece according to claim 1, which is characterized in that dosage of each component in the reagent layer are as follows:
4. dry chemistry reagent piece according to claim 1, which is characterized in that the hydrophilic colloid be selected from polyvinyl alcohol,
Amylopectin, cellulose esters, agarose, polyvinylpyrrolidone, polyacrylamide, hydroxypropyl methyl cellulose, ethylene methacrylic
One of base ether/copolymer-maleic anhydride is a variety of.
5. dry chemistry reagent piece according to claim 1, which is characterized in that chromogen is made of A and B in the color layer,
The A is 4-AA, and B is in phenol, parachlorphenol, P-hydroxybenzoic acid, 3,5- dichloro sodium hydroxybenzenesulfonate
It is one or more, dosage of each component in the color layer are as follows:
0.6~30mmol/m of chromogen2；
10~100g/m of hydrophilic colloid2；
0.1~5g/m of surfactant2。
6. dry chemistry reagent piece according to claim 1, which is characterized in that the diffusion layer is polymerize by hydrophilic macromolecule
Object, surfactant and particulate matter composition；Dosage of each component in diffusion layer are as follows:
15~25g/m of hydrophilic high molecular polymer2；
0.1~5g/m of surfactant2；
200~300g/m of particulate matter2。
7. dry chemistry reagent piece according to claim 6, which is characterized in that in the diffusion layer, the hydrophily high score
Sub- polymer be selected from polyurethane, polyamide, sodium carboxymethylcellulose, polyvinyl alcohol, amylopectin, xanthan gum, sodium alginate, Ah
Draw one of uncle's natural gum, peach gum, chitosan, polyvinylacetate or a variety of；
Surfactant is polyethylene glycol to isooctyl phenyl ether；
The particulate matter is in pigment, crystallite colloid, silica, resin bead, bead, diatomite, cellulose esters
It is one or more.
8. dry chemistry reagent piece according to claim 1, which is characterized in that the support layer is poly terephthalic acid second two
The polymer of alcohol ester, with a thickness of 50~300 microns.
9. the preparation method of dry chemistry reagent piece as described in any one of claims 1 to 8, which is characterized in that including walking as follows
It is rapid:
Developing solution reagent, reaction solution reagent are sequentially coated on support layer, then diffusion liquid reagent is sprayed on support layer,
It is dry, obtain successively include support layer, color layer, reagent layer and diffusion layer dry chemistry reagent piece；
Reagent in the reagent layer include buffer system, hydrophilic colloid, glucose oxidase, peroxidase, activator,
Chelating agent and stabilizer；Dosage of each component are as follows:
Reagent in the color layer includes chromogen, hydrophilic colloid and surfactant.
10. preparation method according to claim 9, which is characterized in that ultraviolet irradiation, electricity are passed through in the surface of the support layer
Dizzy or coating bottom layer treatment.
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| CN110398469A (en) * | 2019-07-16 | 2019-11-01 | 北京知几未来医疗科技有限公司 | It is a kind of for determining the reaction solution and method of body fluid glucose level |
| CN110791548A (en) * | 2019-10-31 | 2020-02-14 | 无锡锦帛诚医疗器械科技有限公司 | Glucose quantitative detection dry tablet for in vitro diagnosis |
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