CN101598727A - The drying chemical reagent paper of quantitatively determining human blood urea content - Google Patents
The drying chemical reagent paper of quantitatively determining human blood urea content Download PDFInfo
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Abstract
The invention belongs to technical field of in-vitro clinical diagnostic reagent, be specially the drying chemical reagent paper of urea content in a kind of quantitatively determining human blood.Test paper is made up of upper, middle and lower three supporting layers and each test layer, can be divided into holding area and test section.The test section is followed successively by diffusion layer, reagent layer and developer layer from top to bottom.Open a well on the upper supporting layer, test sample book infiltrates into reagent layer after well enters diffusion layer, in reagent layer generation enzymatic reaction, generate ammonia by urease catalyzes urea, under alkaline environment, the ammonia that reaction generates diffuses to color layer and ammonia indicator generation chromogenic reaction.Can measure the optical density variation of color layer by reflectance spectrophotomete by the instrument connection of lower supporting layer.This test strips is used for quantitatively determining human blood content of urea, and for the diagnosis of renal function provides foundation, suitable hospital emergency rooms is promoted the use of.
Description
Technical field
The invention belongs to technical field of in-vitro clinical diagnostic reagent, be specifically related to a kind of drying chemical reagent paper that is applicable to urea content in the quantitatively determining human blood.
Background technology
Urea is the dead end product of human body protein metabolism, and vivo acid resolves into 2-ketoacid and NH through deamination
3, NH
3In liver cell, enter urea cycle and CO
2Generate urea.Liver is the main organ that generates urea.The growing amount of urea depends on protein intake in the diet, tissue protein kalabolism and liver function situation.The urea that generates in the liver is mainly through renal excretion, and fraction is discharged through sweat.It mainly is through glomerular filtration, and excretes with urine.If renal function suffers damage, glomerular filtration rate(GFR reduces, and urea concentration will increase in the blood.
Urea content is one of most important observation index of renal function in the blood, is routine clinical and the indispensable test item of emergency treatment.Be used to diagnose some kidney trouble and metabolic disturbance diseases.
The Determination of Urea method adopts traditional wet chemical method for a long time, as Diacetylmonoxime method, urase one indoxyl method, urase-proline deaminase coupling method etc.These methods have enough accuracy and precision, but need the expensive instrument and the professional technique talent, and complex operation, can not fast measuring go out the result, are not suitable for the demand of hospital emergency rooms.Dry chemistry reagent is easy and simple to handle with it, measure fast, flexibly and need not the weak point that advantage such as professional has well been replenished wet chemistry method, its accuracy of measurement and precision are extensively adopted by hospital emergency rooms near the wet chemical determination method.
At present, the employed import dry chemical product that is of hospital emergency biochemical test can be divided into two classes according to the difference on its product structure:
The first kind is to be the dry chemistry reagent of the employing coating technology of representative with Johnson ﹠ Johnson and Fuji.Johnson ﹠ Johnson and Fuji have applied to the clinical diagnosis field with the coating technology in the film production field, and all ingredients required in the assaying reaction is coated on the transparent plastic sheet according to the bottom-up layering uniformly respectively of the precedence of reaction.Measuring dry plate with urea is example, respectively is diffusion layer, optical diffusion layer, enzyme reagent layer, semi-transparent rete and developer layer from top to bottom.The final purpose that arrives quantitative measurement by the variation of Instrument measuring color layer light signal.This technique functions comes from the beginning of the seventies in last century, makes a concerted effort to develop (Johnson﹠amp now by Eastman organic chemistry goods company and Kodak company; Johnson).The two utilizes the speciality in each comfortable chemical reagent and coating technology field respectively, go through the research and development of several years, glucose, urea and uric acid test dry plate have successively successfully been developed, U.S.Pat 3992158 has elaborated form, structure, test philosophy and process of its test dry plate or the like, developed dry plate testing tool Eastman Kodak Ektachem400 in 1980, tested the dry plate projects with 12 and introduce to the market in 1981 the most finally.So far, Johnson﹠amp; Johnson successively releases dry type Biochemical Analyzers such as Vitros 250 analysers, Vitros 950, and test event is nearly individual surplus in the of 30, comprising some immunoassay projects.
Second class is to be the dry chemical product of representative with Luo Shi, and it adopts the transverse dispersion pattern, is reaction carriers with various membrane materials, such as glass fibre or various fabrics etc.After dripping test sample book, sample transverse dispersion to the test zone generation change in optical signal that reacts.Its distinguishing feature is can be to measure sample with the whole blood, and shortcoming is that the application of sample amount is bigger.
Urea test dry plate or the test strips of Johnson ﹠ Johnson, Fuji and Luo Shi, its test philosophy all is to have adopted urease methods, promptly utilize urease behind the alkaline environment decomposing urea, to produce ammonia, by indicator (a kind of pH indicator) and the ammonia generation chromogenic reaction that generates, the indirect content of urea that determines.Its difficult point that is different from other test events is how to avoid testing alkaline components in the link to the interference of indicator.Johnson ﹠ Johnson, Fuji and Luo Shi have adopted hydrophobic breathable layer as solution, specifically be that one deck hydrophobic breathable layer is set between test paper layer and color layer, effect is to isolate the interference of the alkaline components of responding layer to developer, and the ammonia of guarantee reagent layer generation simultaneously can diffuse to color layer by hydrophobic breathable layer and react.Because its difference and other special constructions of testing dry plates have caused the difficulty on the production technology.The difference that U.S.Pat7022285B2 has disclosed the hydrophobic breathable layer performance directly influences the precision of test result, becomes the crucial restraining factors of urea test dry plate performance.
In the above product, the complex manufacturing that has is such as the Luo Shi test strips; The equipment and technology that the needs that have are special is such as Johnson ﹠ Johnson and Fuji's dry plate.Particularly its reagent costs an arm and a leg, and only uses at large hospital, is difficult to be small-middle hospital that particularly small hospital accepts, and has hindered the widespread usage of dry chemical product.And matching used with it dry type Biochemical Analyzer mostly is big-and-middle-sized instrument, no matter on price and portability certain limitation arranged all.Therefore, the more rational dry chemistry reagent of development research small portable dry type Biochemical Analyzer and price has more practical significance for China's small-middle hospital, more helps dry chemical assay method applying in clinical diagnosis.
Urea test paper bar of the present invention adopts particular structure, need not hydrophobic breathable layer, has both simplified processing technology, has lowered cost, has reduced the influence of hydrophobic breathable layer to test again.
Summary of the invention
The object of the present invention is to provide a kind of easy to use, but and the dry chemistry test paper slip of urea content in the quantitative determination blood of human body.
The drying chemical reagent paper of quantitatively determining human blood urea content provided by the invention, it is a long strips, and an end is test section 1, and the other end is a handheld terminal 2, as shown in Figure 1.It is made up of upper supporting layer 4, middle supporting layer 5, lower supporting layer 6, diffusion layer 7, reagent layer 8 and color layer 9, as shown in Figure 2.Wherein, have well 10, have air hole 11 and instrument connection 12 in the relevant position of middle supporting layer 5 and lower supporting layer 6 in the test section of upper supporting layer 4; Diffusion layer 7 and reagent layer 8 are superimposed, and between the upper supporting layer 4 and middle supporting layer 5 at well 10 and air hole 11 places, color layer 9 is between the middle supporting layer 5 and lower supporting layer 6 at air hole 11 and instrument connection 12 places; Upper supporting layer 4, middle supporting layer 5 and lower supporting layer 6 stick together mutually by the glue-line that carries, and corresponding diffusion layer 7, reagent layer 8 and color layer 9 are adhered fixed in described relevant position.
The drying chemical reagent paper of said structure is mainly used in and measures the human serum urea content.If the mensuration whole blood sample then will be provided with one deck filtering layer so that the elimination red blood cell between above-mentioned diffusion layer 7 and reagent layer 8.
Among the present invention, described upper supporting layer, middle supporting layer and lower supporting layer material can adopt composite fibre materials, as: polypropylene, polyethylene terephthalate, tygon, Polyvinylchloride, polystyrene or dacron etc. wherein are best with the polyester material.Single-sheet thickness is 50～350 μ m, and width is 5～14mm, and length is 15～50mm.Well 10, air hole 11 and instrument connection 12 are circular hole, and equal diameters, size are 4～8mm.Well 10 is used for dripping the used sample of test; The effect of air hole 11 is to isolate reagent layer 8 and color layer 9, avoids the contact of the two and after dripping sample, and the ammonia that produces at reagent layer 8 can diffuse to color layer 9 by air hole 11 chromogenic reactions take place; Instrument connection 12 is used for observation and measures the color layer change in color.
Among the present invention, the effect of diffusion layer 7 is that it can be penetrated into reagent layer 8 rapidly and uniformly with the sample that drips.The material that is fit to do diffusion layer comprises various filter paper, glass fibre, nonwoven fabrics, nylon membrane or synthon etc., preferred composite fibre materials, and only material is dacron or nylon fiber material, is good with the screen cloth form wherein.
Among the present invention, reagent layer 8 is a responding layer.After dripping sample, sample diffuses to reagent layer 8, and the urease catalyzes urea that is fixed in the reagent layer 8 generates NH
3And CO
2, under alkaline environment, the NH of generation
3Discharge from reagent layer 8, diffuse to color layer and developer generation chromogenic reaction through air hole 11.The material that is fit to do reagent layer has various filter paper, nonwoven fabrics, synthetic film and fabric etc., and pore size is 0.2～15 μ m.Among the present invention, color layer 9 be coated with can with NH
3The developer of chromogenic reaction takes place, and this developer comprises following one or more pH indicator: bromophenol blue, bromcresol green, bromcresol purple or bromthymol blue etc.The weight concentration of developer is 0.5%～10%.The material that is fit to do color layer comprises synthetic film, filter paper, fabric or nonwoven fabrics etc.The synthetic membrane material that preferably has plastic backings.Also developer directly can be coated on the diaphragm.Among the present invention, the reagent in the reagent layer 7 comprises buffer system, urease and enzyme stabilizers etc.Wherein buffer system is Tris-HCl or phosphate buffer, and concentration is 0.02～0.5M, and the pH value is 7.5～9.5; Urease concentration is 20ku/L～200ku/L; Enzyme stabilizers is disaccharide class, polysaccharide or bovine serum albumin(BSA), and weight concentration is 0.2%-2.0%.
The drying chemical reagent paper of the quantitatively determining human urea content that the present invention proposes, can be used for testing the chemical constitution that can produce ammonia by reaction, as: creatinine, blood ammonia or the like, difference is the peculiar difference of reagent layer composition, and during test, test sample book infiltrates into reagent layer after well enters diffusion layer, in reagent layer generation enzymatic reaction, generate ammonia by urease catalyzes urea, under alkaline environment, the ammonia that reaction generates diffuses to color layer and ammonia indicator generation chromogenic reaction.Can measure the optical density variation of color layer by reflectance spectrophotomete by the instrument connection of lower supporting layer.This test strips is used for quantitatively determining human blood content of urea, and for the diagnosis of renal function provides foundation, suitable hospital emergency rooms is promoted the use of.
Be urea content in the quantitative measurement blood, the inventor has also developed reflectance spectrophotomete (separate case is applied for a patent), with the supporting use of dry chemistry test paper slip of the present invention.This instrument can be controlled at temperature of reaction 37 ± 0.5 ℃, and the mensuration wavelength is 630nm, reach the reaction time after, instrument reads reflection density automatically, and light signal is converted into urea concentration by built-in typical curve, and report and stores test results automatically, and printable.
Description of drawings
Fig. 1 is a drying chemical reagent paper structural diagrams of the present invention.
Fig. 2 is drying chemical reagent paper structure elucidation figure of the present invention.
Fig. 3 is a drying chemical reagent paper diagram of the present invention.Wherein, (A) be application of sample before, (B) be application of sample after.
Number in the figure: 1 is the test section, and 2 is the holding area, and 3 is plastic film, and 4 is upper supporting layer, and 5 is middle supporting layer, and 6 is lower supporting layer, and 7 is diffusion layer, and 8 is reagent layer, and 9 is color layer, and 10 is well, and 11 is bleeder vent, and 12 is instrument connection.
Embodiment
Embodiment:
Embodiment 1: upper, middle and lower supporting layer material adopts the PVC material, and thickness is respectively 0.14mm, 0.12mm, 0.14mm; Length is 20cm, and width is 8cm.Wherein go up the lower supporting layer single face with viscose glue, middle supporting layer is two-sided with viscose glue.At one end get well, air hole and instrument connection respectively, bore dia is 4mm, and pitch-row is 8mm.Diffusion layer adopts nylon net cloth, and reagent layer adopts fabric, and color layer adopts the nitrocellulose filter of band plastic backings, and width is 8mm.Diffusion layer, reagent layer and color layer are pasted the relevant position of upper, middle and lower supporting layer respectively, promptly be positioned at well, air hole and instrument connection place.The alignment of upper supporting layer and middle supporting layer sticks together then.The enzyme reaction solution and the colour developing liquid that prepare are dropped to reagent layer and color layer respectively, enzyme reaction solution 12 μ l/ holes, colour developing liquid 8 μ l/ holes.
The prescription of enzyme reaction solution reagent is as follows:
Urease 20KU/L
Tris-HCl 0.2mM
The color-developer reagent prescription:
Bromophenol blue weight concentration 1.5%
Test paper behind the specking is put into 60 ℃ of drying boxes, take out after 60 minutes, supporting layer and lower supporting layer alignment edge sticks together in will going up, and it cuts into the wide test strips of 8mm with the hobboing cutter machine, puts into drying.
The urea standard items of preparation variable concentrations drip 10 μ l standard items in well, measure with baffled photometer, read the OD value at 3min place.
Embodiment 2: upper, middle and lower supporting layer material adopts the PET material, and thickness is respectively 0.12mm, 0.15mm, 0.12mm; Length is 20cm, and width is 8cm.Wherein go up the lower supporting layer single face with viscose glue, middle supporting layer is two-sided with viscose glue.At one end get well air hole and instrument connection respectively, bore dia is 4mm, and pitch-row is 8mm.Diffusion layer adopts the polyester screen cloth, and reagent layer adopts filter paper material, and color layer adopts the nitrocellulose filter of band plastic backings, and width is 8mm.Diffusion layer, reagent layer and color layer are pasted the relevant position of upper, middle and lower supporting layer respectively, promptly be positioned at well, air hole and instrument connection place.The alignment of upper supporting layer and middle supporting layer sticks together then.The enzyme reaction solution and the colour developing liquid that prepare are dropped to reagent layer and color layer respectively, enzyme reaction solution 10 μ l/ holes, colour developing liquid 10 μ l/ holes.
The prescription of enzyme reaction solution reagent is as follows:
Urease 80KU/L
Tris-HCl 0.5mM
Color-developer reagent is weight concentration 2% a bromophenol blue solution.
Test paper behind the specking is put into 60 ℃ of drying boxes, take out after 50 minutes, and supporting layer and lower supporting layer alignment edge stick together in will going up, and it cuts into the wide test strips of 8mm with the hobboing cutter machine, puts into drying.
The urea standard items of preparation variable concentrations drip 5 μ l standard items in well, measure with baffled photometer, read the OD value at 3min place.
Claims (9)
1. the drying chemical reagent paper of a quantitatively determining human blood urea content is long strips, and the one end is test section (1), and the other end is handheld terminal (2), it is characterized in that being made up of upper supporting layer (4), middle supporting layer (5), lower supporting layer (6); Wherein, upper supporting layer (4), middle supporting layer (5) and lower supporting layer (6) stick together by the glue-line that carries; Have well (10) in the test section of upper supporting layer (4), middle supporting layer (5) and lower supporting layer (6) relevant position have air hole (11) and instrument connection (12); Diffusion layer (7), reagent layer (8) and color layer (9) are arranged between well and the instrument connection; Diffusion layer (7) and reagent layer (8) are superimposed, are arranged between upper supporting layer (4) and the supporting layer (5); Color layer (9) is arranged between supporting layer (5) and the lower supporting layer (6); Reagent layer (8) contains the required reagent that responds, and this reagent mainly comprises buffer system, urease, enzyme stabilizers; Wherein buffer system is Tris-HCL or phosphate buffer, and concentration is 0.02～0.5M, and the pH value is 7.5～9.5; Urease concentration is 20ku/L～200ku/L; Enzyme stabilizers is disaccharide class or polysaccharide, and weight concentration is 0.2%～2.0%; The reagent of color layer (9) is following one or more pH indicator: bromophenol blue, bromcresol green, bromcresol purple, bromthymol blue, weight concentration are 0.5%～10%.
2. the drying chemical reagent paper of quantitatively determining human urea content according to claim 1 is characterized in that being coated with on the upper supporting layer (1) the wide plastic film of one deck (3), and plastic film (3) is gone up with glue-line, in order to adhere on the upper supporting layer (1).
3. the drying chemical reagent paper of quantitatively determining human urea content according to claim 1 and 2 is characterized in that also being provided with between described diffusion layer (7) and the reagent layer (8) filtering layer in order to filtering whole blood.
4. according to the drying chemical reagent paper of claim 1,2, one of 3 described quantitatively determining human urea contents, it is characterized in that forming a cavity by middle supporting layer (5) between reagent layer (8) and the color layer (9), in order to the ammonia of temporary reaction generation, so that cause the chromogenic reaction of color layer (9).
5. according to the drying chemical reagent paper of claim 1,2, one of 3 described quantitatively determining human urea contents, it is characterized in that described upper, middle and lower supporting layer material is a composite fibre materials, every layer thickness is 50～300 μ m, and width is 5～14mm, and length is 15～25mm; Described well (10), air hole (11) and instrument connection (12) are circular hole, and diameter is 4～8mm.
6. according to the drying chemical reagent paper of claim 1,2, one of 3 described quantitatively determining human urea contents, the material that it is characterized in that described diffusion layer (7) is synthon, nonwoven fabrics, glass fibre, nylon membrane or filter paper.
7. according to the drying chemical reagent paper of claim 1,2, one of 3 described quantitatively determining human urea contents, it is characterized in that described diffusion layer (7) adopts the screen cloth form.
8. according to the drying chemical reagent paper of claim 1,2, one of 3 described quantitatively determining human urea contents, it is characterized in that the material of described reagent layer (8) is synthetic film, filter paper, fabric or nonwoven fabrics.
9. according to the drying chemical reagent paper of claim 1,2, one of 3 described quantitatively determining human urea contents, it is characterized in that the material of described color layer (9) is synthetic film, filter paper, fabric or nonwoven fabrics.
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