WO2016134531A1 - 基于浓度梯度的多层膜干化学检测试条 - Google Patents

基于浓度梯度的多层膜干化学检测试条 Download PDF

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Publication number
WO2016134531A1
WO2016134531A1 PCT/CN2015/073387 CN2015073387W WO2016134531A1 WO 2016134531 A1 WO2016134531 A1 WO 2016134531A1 CN 2015073387 W CN2015073387 W CN 2015073387W WO 2016134531 A1 WO2016134531 A1 WO 2016134531A1
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layer
concentration gradient
reagent
test strip
dry chemical
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PCT/CN2015/073387
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English (en)
French (fr)
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曾嵘斌
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曾嵘斌
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Priority to PCT/CN2015/073387 priority Critical patent/WO2016134531A1/zh
Priority to US15/307,009 priority patent/US9664672B2/en
Priority to EP15882969.7A priority patent/EP3264081B1/en
Publication of WO2016134531A1 publication Critical patent/WO2016134531A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/525—Multi-layer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90283—Oxidoreductases (1.) acting on superoxide radicals as acceptor (1.15)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the invention relates to a chemical detection article, in particular to a concentration gradient based multilayer film dry chemical test strip for rapid quantitative detection in the field.
  • Electrochemical methods such as CN101762628A "a dry enzyme biochip/test strip" is made by making a dry enzyme working electrode, a reference electrode, a test liquid diffusion net film, a hydrophobic insulating varnish on a PET substrate, and measuring the signal needs. Electrochemical instrument readout.
  • the dry chemical test strip (paper) of the colorimetric method developed earlier, and the two-layer structure, the three-layer structure, and the latest multi-layer structure were developed from the initial layer structure.
  • a layer of structure such as the initial pH test paper for measuring pH and potassium iodide starch test paper for measuring oxides, etc., are mainly used for qualitative experiments, and the maximum value that can be obtained is semi-quantitative.
  • the reagent carrier for biochemical analysis is the simplest one-layer structure.
  • On the support layer plastic substrate there is a reagent layer cellulose sheet, and all reagents are pre-solidified in the cellulose sheet.
  • urine biochemical analysis reagent strips such as CN1888900A "Thirteen urine test strips sorted by pH segmentation", which are used to assign thirteen different papers to the reagents used in the thirteen test items, and are attached to the plastic substrate in the urine.
  • the component to be tested reacts directly with the reagent of the pre-solid phase on the cellulose sheet, The concentration of the component to be tested is measured by a reflectance photometer to calculate the concentration of the component to be tested.
  • the three-layer structure is provided with a porous film filter layer on the reagent layer, which functions to filter out impurities in the sample and function as a protective reagent layer.
  • a porous film filter layer on the reagent layer, which functions to filter out impurities in the sample and function as a protective reagent layer.
  • Commonly used is a reagent strip for the determination of glucose by a micro method.
  • the measurement path of the reagent carrier of the three-layer structure passes through a transparent plastic substrate without passing through the uppermost filter layer, thus eliminating the influence of interference components in the sample and ensuring that the reaction is performed.
  • the stability and accuracy of the determination of the composition such as CN1225450A "test paper and analyte determination head", blood glucose color test paper preparation and supporting testing equipment.
  • the multilayer film method is a dry chemical multilayer film reagent carrier.
  • the multilayer film method from the manufacturing process mainly has two types of vertical and horizontal.
  • the first type is a multi-layer dry film method represented by Johnson & Johnson and Fuji Co., which uses a coating technology of photographic film to coat reagent layers, auxiliary reagent layers, light diffusing layers and distribution layers on a transparent support layer.
  • the change in optical density is measured from the reverse side of the support layer.
  • the second type is Roche's dry chemical product.
  • US4604264 applies the reagent to the multifilament fiber or fabric. The whole blood is laterally filtered through the glass fiber and then reacted with the upper reagent layer to measure the optical density through the upper covered transparent film. Variety.
  • the concentration distribution of the artificial control reagent (paper) on the test strip (paper) in the dry chemical test strip (paper) is used to achieve: the test liquid (paper) diffuse and penetrate into the test strip to form a similar Analytical detection method based on the principle of chemical titration to achieve on-site rapid quantitative detection of sample liquid.
  • CN2564584 Bacterial drug sensitivity test for detecting the minimum inhibitory concentration of antibiotics" method using a plurality of different quantitative antibiotic concentration gradient filter strips fixed on the substrate, characterized by the longitudinal discontinuous exponential form of the antibiotic on the plastic carrier Decrement and the same concentration in the lateral concentration method (as shown in Figure 5).
  • CN201309942 The method of "comb-type drug-sensitive test strip” is somewhat similar to the method of CN2564584, which is characterized in that the antibiotic is longitudinally discontinuously exponentially and laterally concentrated on the comb of the plastic carrier (as shown in Fig. 6).
  • Stepwise concentration gradient detecting strip and preparation method thereof adopts a method in which a plurality of equal-area sections are equally divided on one side of a carrier, and a set shape and an area are symmetrically coated in the center of each equal-area section.
  • the drug block; the dose of each drug block exhibits a stepwise change of one or more segments, that is, the concentration of the drug block in different segments is different. but
  • the coating thickness and concentration of each drug block arranged in the same segment are the same, and the area size of each drug block is stepped up or down in order, which is characterized by the distribution of the concentration gradient by the area of the antibiotic agent coated on the substrate.
  • the above patent report achieves a stepwise distribution of antibiotic agent concentration, mainly used in microbial susceptibility testing, which is a test for microbial resistance experiments, and in the course of the experiment, antibiotics are spread on the medium to form antibiotics on the surface of the medium.
  • the distribution of the gradient of the concentration of the drug was cultured, and the minimum inhibitory concentration (MIC value, Minimal inhibitory concentration) of the bacteria was read, and the results could not be obtained quickly on site.
  • the object of the present invention is to provide a multi-layer dry chemical test strip based on concentration gradient which can be used for rapid and accurate quantitative analysis and on-site continuous and stepless change of concentration distribution.
  • the invention discloses a multi-layer dry chemical detection test strip based on a concentration gradient, comprising a substrate, an indicator layer, a reagent layer and a diffusion layer arranged in sequence from bottom to top, wherein the test strip further comprises a concentration gradient layer.
  • the concentration gradient layer is disposed on an upper surface or a lower surface of the reagent layer; a first reagent is uniformly coated on the reagent layer; and a second reagent is coated on the concentration gradient layer, Concentration gradient of the second reagent In the detection strip width direction is 0, in the detection strip length direction is a constant or a function of the length variable; the developer layer is evenly coated with a developer.
  • the concentration gradient layer is any of polyvinyl alcohol, gelatin, and a mixture of polyvinyl alcohol and gelatin.
  • the concentration gradient layer is a porous coating or a preformed porous membrane.
  • the diffusion layer is a hydrophilic film.
  • a siphon structure is also disposed on the hydrophilic film.
  • the reagent layer is a high molecular polymer material.
  • the reagent layer is a porous coating or a preformed porous membrane.
  • the indicator layer is a high molecular polymer material.
  • the indicator layer is a porous coating or a preformed porous membrane.
  • the substrate is a transparent material having a thickness of 0.1 to 0.3 mm.
  • the lower surface of the substrate is further provided with a scale.
  • the present invention has the following beneficial effects:
  • the invention discloses a multi-layer dry chemical detection test strip based on a concentration gradient, and adopts a concentration gradient layer, which can continuously and continuously change the concentration distribution of the reagent on the test strip, and all the detection and analysis processes can be directly performed on the test strip.
  • the test results can be obtained without direct reading on the spot, and the temperature and time constraints are not required during the test.
  • the operation is simple, the detection means is open, and the operation process does not require special training. It is suitable for on-site inspection by professionals and non-professionals.
  • Figure 1 is a schematic view showing the structure of a dry chemical test strip of the present invention.
  • FIG. 2 is a reagent distribution diagram of a concentration gradient layer in the present invention.
  • Fig. 3 is a view showing the inspection result of the test strip in the present invention.
  • Fig. 4 is a diagram showing the distribution of antibiotic concentration of the minimum inhibitory concentration test plate for detecting antibiotics in the prior art bacterial drug sensitivity test.
  • Fig. 5 is a diagram showing the concentration distribution of antibiotics in the prior art comb-shaped drug-sensitive test strips.
  • Fig. 6 is a diagram showing the distribution of antibiotic concentration of the prior art stepwise concentration gradient detecting strip.
  • Fig. 7 is a schematic view showing the antibiotic coating of the prior art stepwise concentration gradient detecting strip.
  • Figure 8 is a schematic view of the scale of the lower surface of the substrate in the present invention.
  • a water hardness test strip (paper) is prepared based on the concentration gradient method for rapidly detecting the hardness index of water bodies of various sources.
  • a water hardness test strip (paper) prepared by two structural methods is prepared, and the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the first preparation method is as follows: the uppermost layer of the test strip is a diffusion layer 5, the hydrophilic porous cellulose membrane is coated, the multilayer structure is prepared by coating, and the diffusion layer 5 is a reagent.
  • reagent layer 3 is made of porous polyvinyl alcohol material, reagent for reagent layer 3 is used to prepare the reaction solution as follows - Tris 10-15 g / liter, polyvinyl alcohol emulsion and reagent solution 1:1 -4:1 ratio mixing coating preparation reagent layer; reagent layer 3 below is concentration gradient layer 4, concentration gradient layer 4 is made of porous polyvinyl alcohol material, and the polyvinyl alcohol material is uniformly coated into a porous film in advance, The complexing agent ethylenediaminetetraacetic acid (EDTA) is applied to the polyvinyl alcohol porous film in a linear decreasing concentration gradient manner or a stepwise decreasing concentration gradient to prepare a concentration gradient layer 4; the concentration gradient layer 4 is indicated below Agent layer 2, indicator layer 2 is made of porous polyethylene Material, the developer used for the indicator layer 2 is as follows - acid chrome blue K 1-2 g / liter; under the indicator layer 2 is the substrate 1, the substrate is 0.15-0.2 mm thick grass green transparent PET sheet or
  • the second preparation method is: the diffusion layer 5 is composed of a hydrophilic nylon mesh membrane, the multilayer structure is prepared by using a ready-made film layer multi-layer lamination method; the diffusion layer 5 is a reagent layer 3 below, and the finished polyvinyl alcohol is porous.
  • the membrane was immersed in the above reagent solution for 5 minutes, and dried in the dark.
  • the lower layer of the reagent layer 3 was a concentration gradient layer 4, and the finished polyvinyl alcohol porous membrane was coated with ethylenediaminetetraacetic acid (EDTA) in a linear decreasing concentration gradient.
  • EDTA ethylenediaminetetraacetic acid
  • the concentration gradient layer 4 is an indicator layer 2 below, and the finished polyvinyl alcohol porous film is immersed in the above-mentioned color developing agent for 5 minutes, and is dried in the dark; the substrate under the indicator layer 2 is 0.15-0.2 mm thick. Grass green transparent PET sheet or polycarbonate sheet; the layers are sequentially fixed to the substrate by tensioning of the nylon web of the diffusion layer 5.
  • the water hardness test strip (paper) prepared by the previous multilayer film preparation method is prepared by adding a certain amount of test liquid to the diffusion layer 5; using the latter multilayer film preparation Method for producing water hardness test strips (paper),
  • the sampling process is to quickly insert the test strip (paper) into the test solution for three seconds, and then dry the water droplets; after the sampling is completed, the result is observed after 3 minutes, and the water hardness test strip (paper) is close to the substrate.
  • the concentration gradient layer or the reagent layer exhibits a transition from red-purple (demarcation line A) to blue-violet, due to the absorption of light through the grass green transparent substrate, viewed from the direction of the substrate to be rendered by brick red-blue-violet (demarcation line) A) - Blue transition, read the A value with a ruler, as shown in Figure 3 (a), or by color scanning to obtain a scan curve similar to that shown in Figure 3 (c), read the inflection point A value.
  • an acid value test strip (paper) is prepared based on a concentration gradient method for rapidly detecting the acid value index of various oils and fats.
  • a water hardness test strip (paper) prepared by two structural methods is prepared, and the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the first preparation method is as follows: the uppermost layer of the test strip is a porous cellulose membrane used for the diffusion layer 5 in a coated form, the multilayer structure is prepared by a coating method; and the diffusion layer 5 is a reagent layer 3
  • the reagent layer 3 is made of a porous polyvinyl alcohol material, and the reagent used for the reagent layer 3 is prepared as follows: the protective agent KD is 2-4 g/liter, and the polyvinyl alcohol emulsion is mixed with the reagent solution at a ratio of 1:1 to 4:1.
  • the coating preparation reagent layer; the reagent layer 3 is a concentration gradient layer 4, and the concentration gradient layer 4 is made of a porous polyvinyl alcohol material, and the polyvinyl alcohol and the protective agent KD1 material are mixed in advance and uniformly coated into a porous film, and the titrant is used.
  • Potassium hydroxide is applied to the polyvinyl alcohol porous film in a linear decreasing concentration gradient manner or a stepwise decreasing concentration gradient to prepare a concentration gradient layer 4; the concentration gradient layer 4 is below the indicator layer 2, and the indicator layer 2 Using porous polyvinyl alcohol material, means The developer used in the agent layer 2 is as follows - phenolphthalein 1-2 g / liter; under the indicator layer 2 is the substrate 1, the substrate is a colorless transparent PET sheet having a thickness of 0.15 - 0.2 mm or a carbonate sheet .
  • the second preparation method is: the diffusion layer 5 is composed of a hydrophilic nylon mesh membrane, the multilayer structure is prepared by using a ready-made film layer multi-layer lamination method; the diffusion layer 5 is a reagent layer 3 below, and the finished polyvinyl alcohol is porous. The membrane is immersed in the above reagent solution for 5 minutes, and dried in the dark.
  • the lower layer of the reagent layer 3 is a concentration gradient layer 4, and the finished polyvinyl alcohol porous membrane is coated with a titrant potassium hydroxide in a linear decreasing concentration gradient; concentration Below the gradient layer 4 is an indicator layer 2, which is formed by immersing the finished polyvinyl alcohol porous film in the above-mentioned color developing agent for 5 minutes, and drying in the dark; the substrate under the indicator layer 2 is colorless with a thickness of 0.15-0.2 mm.
  • a transparent PET sheet or a polycarbonate sheet; the layers are sequentially fixed to the substrate by tensioning of the nylon web of the diffusion layer 5.
  • the acid value test strip (paper) prepared by the previous multilayer film preparation method When detecting the acid value of the oil and fat, the acid value test strip (paper) prepared by the previous multilayer film preparation method is used, and the sampling process is to add a certain amount of the treated test liquid to the diffusion layer 5;
  • the acid value test strip (paper) prepared by the film preparation method is prepared by rapidly inserting the test strip (paper) into the treated test liquid for three seconds, and then taking out the liquid bead; after the sampling is completed, the sample is allowed to stand.
  • the acid value test strip (paper) observed a transition from colorless-light red (demarcation line A)-red, and the A value was read with a scale, as shown in Fig. 3(a), or by color.
  • the scan yields a scan curve similar to that shown in Figure 3(b), reading the inflection point A value.
  • a blood glucose test strip (paper) was prepared based on a concentration gradient method for rapid detection of glucose content in blood.
  • the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the multilayer structure is prepared by coating, and the test strip is at the top.
  • One layer is a diffusion layer 5 using a hydrophilic porous cellulose film, in the form of coating, the lower layer of the diffusion layer 5 is a reagent layer 3, and the reagent layer 3 is a porous film prepared by using polyvinylpyrrolidone and a polysulfone material, and a polysulfone material.
  • reagent layer 3 reagent preparation reaction solution is as follows - 10IU / L glucose oxidase, 0.037mol / L citric acid, 0.126mol / L disodium hydrogen phosphate, 10g / L trehalose, 5g / L glycine, 3g / LBSA and 10g / L PEG4000
  • the mixed solution is applied to the porous membrane preparation reagent layer; under the reagent layer 3 is a concentration gradient layer 4, and the concentration gradient layer 4 is made of a porous polyvinyl alcohol material, and the polyvinyl alcohol material is uniformly coated into a porous membrane in advance, and the antioxidant is used.
  • BHT horseradish
  • Concentration gradient layer 4 was prepared by using a mixture of oxide enzyme, 0.037 mol/L citric acid, 0.126 mol/L disodium hydrogen phosphate, 10 g/L trehalose, 5 g/L glycine, 3 g/LBSA and 10 g/L PEG 4000 hydrochloride; concentration
  • concentration is below the indicator layer 2
  • the indicator layer 2 is made of porous polyvinyl alcohol material
  • the developer used for the indicator layer 2 is as follows - 1.0 g / L TMB
  • the indicator layer 2 is below the substrate 1
  • a colorless transparent PET sheet having a thickness of 0.15 to 0.2 mm or a polyacrylate sheet is used.
  • the sampling process is to add a certain amount of blood to the diffusion layer 5; after the sampling is completed, the result is observed after standing for 3 minutes, and the blood glucose detecting strip (paper) exhibits a colorless-blue transition, and the color can be seen.
  • Demarcation line A read the A value with the ruler, as shown in Fig. 3(a), or by color scanning to obtain a scan curve similar to that shown in Fig. 3(b), and read the inflection point A value.
  • the alanine aminotransferase test strip was prepared based on the concentration gradient method for rapid detection of the content of alanine aminotransferase in the blood.
  • the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the multilayer structure is prepared by coating, and the test strip is at the top.
  • One layer is a diffusion layer 5 using a hydrophilic porous cellulose film, which is prepared by coating; the diffusion layer 5 is a concentration gradient layer 4, the concentration gradient layer 4 is a porous polyvinyl alcohol film, and the concentration gradient layer 4 is a valley.
  • the alanine aminotransferase inhibitor (GBH) is applied to the polyvinyl alcohol porous membrane in a linearly decreasing concentration gradient or in a continuous exponentially varying concentration gradient, and the reaction solution is uniformly coated - Good's buffer:
  • Concentration gradient layer 4 was prepared by mixing 200 mM, MgCl 2 * 6H 20 : 2.0 g / L, KH 2 PO 4 : 20 mM and trehalose: 0.5%; reagent layer 3 was below concentration gradient layer 4, and porous polyvinyl alcohol material was used for reagent layer 3
  • the polyvinyl alcohol material is uniformly coated into a porous film, and the reaction solution is uniformly coated on the polyvinyl alcohol porous film - alanine: 1000 ⁇ -ketoglutarate: 20 mM, peroxidase: 100 U/mi, Pyruvate oxidase: 100 U/ml, flavin adenine Glycolate (FAD): 0.2 mg/ml
  • the sampling process is to add a certain amount of test liquid to the diffusion layer 5.
  • the result is observed after standing for 30 minutes, and the alanine aminotransferase test strip (paper) is rendered by colorless-blue.
  • you can see the color boundary line A read the A value with the ruler, as shown in Fig. 3(a), or by color scanning to obtain a scan curve similar to that shown in Fig. 3(b), and read the inflection point A value.
  • a bone alkaline phosphatase test strip was prepared based on a concentration gradient method for rapid detection of bone alkaline phosphatase in blood.
  • the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the multilayer structure is prepared by coating, and the test strip is at the top.
  • One layer is a diffusion layer 5 using a hydrophilic porous cellulose film, which is prepared by coating; the diffusion layer 5 is a concentration gradient layer 4, the concentration gradient layer 4 is a porous polyvinyl alcohol film, and the concentration gradient layer 4 is a bone.
  • the alkaline phosphatase inhibitor (BALH) is applied to the polyvinyl alcohol porous membrane in a linear decreasing concentration gradient or in a continuous exponentially varying concentration gradient to prepare a concentration gradient layer 4;
  • the concentration gradient layer 4 is a reagent Layer 3
  • the reagent layer 3 is made of a porous polyvinyl alcohol material, and the polyvinyl alcohol material is uniformly coated into a porous film in advance, and the reaction liquid is uniformly coated on the polyvinyl alcohol porous film - Tris.
  • the indicator layer 2 is made of a porous polyvinyl alcohol material, and the developer used for the indicator layer 2 is as follows - bromochlorophosphonium phosphate (BCIP) 1.0 mmol / L; the indicator layer 2 is the substrate 1
  • the substrate is a colorless transparent PET sheet or a polycarbonate sheet having a thickness of 0.15-0.2 mm.
  • the sampling process is to add a certain amount of the treated blood test solution to the diffusion layer 5, and after standing for 30 minutes after the sampling is completed, the result is observed, and the bone alkaline phosphatase enzyme test is performed.
  • the strip (paper) is rendered by a colorless-purple transition, the color boundary line A can be seen, the A value is read with a scale, as shown in Fig. 3(a), or a color scan is obtained to obtain a scan similar to that shown in Fig. 3(b). Curve, read the inflection point A value.
  • a superoxide dismutase test strip (paper) was prepared based on a concentration gradient method for rapid detection of superoxide dismutase content in a biological sample.
  • the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the multilayer structure is prepared by coating, and the test strip is at the top.
  • One layer is a diffusion layer 5 using a hydrophilic porous cellulose film, which is prepared by coating; the lower layer of the diffusion layer 5 is a reagent layer 3, the reagent layer 3 is a porous polyvinyl alcohol film, and the reagent layer 3 is additionally uniformly coated.
  • Reaction liquid - hypoxanthine 200 mM and hydroxylamine hydrochloride: 2.0 / L
  • the mixture was prepared to prepare the reagent layer 3; the reagent layer 3 was below the concentration gradient layer 4, and the concentration gradient layer 4 was made of porous polyvinyl alcohol material, and the polyethylene was preliminarily
  • the alcohol material is uniformly coated into a porous film, and the xanthine oxidase (XO) is applied to the polyvinyl alcohol porous film in a linear decreasing concentration gradient or in a continuous exponentially changing concentration gradient, and is porous in polyvinyl alcohol.
  • XO xanthine oxidase
  • the reaction solution was uniformly coated on the membrane - Tris: 5-10/L, trehalose: 10 g/L, glycine: 5 g/L, BSA: 3 g/L, PEG 4000: 10 g/L
  • the concentration gradient layer 4 is prepared by the mixed solution; the indicator layer 2 is below the concentration gradient layer 4,
  • the indicator layer 2 is made of a porous polyvinyl alcohol material, and the developer used for the indicator layer 2 is as follows - p-aminobenzenesulfonic acid reagent: 2.0 / L and a-naphthylamine: 1.0 / L; under the indicator layer 2 is a substrate 1.
  • the substrate is a colorless transparent PET sheet or a polycarbonate sheet having a thickness of 0.15-0.2 mm.
  • the sampling process is to add a certain amount of biological sample to the diffusion layer 5 to process the test solution, and after standing for 30 minutes after the sampling is completed, the result is observed, and the superoxide dismutase detection test strip ( Paper) presents a transition from colorless-purple red, can see the color boundary line A, read the A value with the ruler, as shown in Fig. 3(a), or by color scanning to obtain a scan curve similar to that shown in Fig. 3(b). , read the inflection point A value.
  • a residual chlorine detection test strip was prepared based on the concentration gradient method for rapidly detecting the residual chlorine content in different water samples.
  • the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the multilayer structure is prepared by coating, and the test strip is at the top.
  • One layer is the diffusion layer 5 Prepared with a hydrophilic porous cellulose membrane in a coated form; under the diffusion layer 5 is a concentration gradient layer 4, the concentration gradient layer 4 is a porous polyvinyl alcohol film, and the concentration gradient layer 4 is linear with an antioxidant (BHT).
  • BHT antioxidant
  • the concentration gradient layer 4 is prepared by a decreasing concentration gradient or a continuous exponentially varying concentration gradient on the polyvinyl alcohol porous membrane; the reagent layer 3 is below the concentration gradient layer 4, and the porous polyvinyl alcohol material is used for the reagent layer 3
  • the polyvinyl alcohol material is uniformly coated into a porous film in advance, and the reaction liquid is uniformly coated on the polyvinyl alcohol porous film - KH2PO4: 20 mM to prepare the reagent layer 3; the reagent layer 3 is below the indicator layer 2, the indicator Layer 2 is made of a porous polyvinyl alcohol material, and the developer used for the indicator layer 2 is as follows - TMB: 0.1%; the underside of the indicator layer 2 is the substrate 1, and the substrate is made of a colorless transparent PET sheet having a thickness of 0.15 - 0.2 mm.
  • the sampling process is to add a certain amount of biological sample to the diffusion layer 5 to process the test solution.
  • the result is observed after standing for 10 minutes, and the residual chlorine detection test strip (paper) is presented by Color-blue transition, can see the color boundary line A, read the A value with the ruler, as shown in Figure 3 (a), or by color scanning to obtain a scan curve similar to that shown in Figure 3 (b), read the inflection point A value.
  • a hydrogen peroxide detection test strip was prepared based on a concentration gradient method for rapidly detecting the content of hydrogen peroxide in water samples from different sources.
  • the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the multilayer structure is prepared by coating, and the test strip is at the top.
  • One layer is a diffusion layer 5 using a hydrophilic porous cellulose film, which is prepared by coating; the diffusion layer 5 is a concentration gradient layer 4, the concentration gradient layer 4 is a porous polyvinyl alcohol film, and the concentration gradient layer 4 is resistant.
  • the oxidant (BHT) is applied to the porous polyvinyl alcohol membrane in a linearly decreasing concentration gradient or in a continuous exponentially varying concentration gradient, and the reaction solution is uniformly coated - 0.037 mol/L citric acid and 0.126 mol.
  • the concentration gradient layer 4 is prepared by mixing the /L disodium hydrogen phosphate solution; the reagent layer 3 is below the concentration gradient layer 4, and the porous polyvinyl alcohol material is used for the reagent layer 3, and the polyvinyl alcohol material is uniformly coated into a porous film in advance, and is concentrated.
  • a reagent layer 3 by uniformly coating a reaction liquid - a mixture of 0.05 g / L horseradish peroxidase, 10 g / L trehalose, 5 g / L glycine, 3 g / LBSA and 10 g / L PEG 4000 on the porous film of vinyl alcohol;
  • indicator layer 2 Below layer 3 is indicator layer 2, and indicator layer 2 is used more
  • the polyvinyl alcohol material, the developer used for the indicator layer 2 is as follows - 2.0 g / L TMB hydrochloride; the indicator layer 2 is the substrate 1 below, and the substrate is a colorless transparent PET sheet having a thickness of 0.15-0.2 mm Or polycarbonate sheet.
  • the sampling process is to add a certain amount of biological sample to the diffusion layer 5 to process the test solution, and after standing for 10-30 minutes after the sampling is completed, the observation result, the hydrogen peroxide detection test strip (paper) ) rendering a transition from colorless to blue, seeing the color boundary line A, reading the A value with a ruler, as shown in Figure 3(a), or by color scanning to obtain a scan curve similar to that shown in Figure 3(b). Read the inflection point A value.
  • an organophosphorus test strip was prepared based on a concentration gradient method for rapidly detecting the residual organic phosphorus content in agricultural products.
  • the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the multilayer structure is prepared by coating, and the test strip is at the top.
  • One layer is a diffusion layer 5 using a hydrophilic porous cellulose film, which is prepared by coating; the lower layer of the diffusion layer 5 is a reagent layer 3, the reagent layer 3 is a porous polyvinyl alcohol film, and the reagent layer 3 is uniformly coated with a reaction liquid.
  • the agent layer 3; the reagent layer 3 is a concentration gradient layer 4, and the concentration gradient layer 4 is made of a porous polyvinyl alcohol material, and the polyvinyl alcohol material is uniformly coated into a porous film in advance, and the acetylcholinesterase is linearly decreasing in concentration gradient.
  • the method is applied to the polyvinyl alcohol porous membrane preparation reagent layer 3 in a manner of a continuous exponentially changing concentration gradient; the indicator layer 2 is under the concentration gradient layer 4, and the indicator layer 2 is made of a porous polyvinyl alcohol material, an indicator layer.
  • the color developer used is as follows - 2,6-dichloroindophenol acetate: 10 g / L; the indicator layer 2 is the substrate 1 below, and the substrate is a colorless transparent PET sheet having a thickness of 0.15-0.2 mm or Polycarbonate sheet.
  • the sampling process is to add a certain amount of biological sample to the diffusion layer 5 to process the test liquid.
  • a urea detection test strip (paper) is prepared based on a concentration gradient method for rapidly detecting the content of urea in biological samples and water samples from different sources.
  • the test strip (paper) is composed of five basic functional layers: a substrate 1, an indicator layer 2, a reagent layer 3, a concentration gradient layer 4, and a diffusion layer 5.
  • the multilayer structure is prepared by coating, and the test strip is at the top.
  • One layer is a diffusion layer 5 using a hydrophilic porous cellulose film, which is prepared by coating; the lower layer of the diffusion layer 5 is a reagent layer 3, the reagent layer 3 is a porous polyvinyl alcohol film, and the reagent layer 3 is uniformly coated with a reaction liquid.
  • the reagent layer 3 is a concentration gradient layer 4, and the concentration gradient layer 4 is made of a porous polyvinyl alcohol material.
  • the polyvinyl alcohol material is uniformly coated into a porous film in advance, and the citric acid is linearly decreasing in a concentration gradient manner or continuously.
  • An exponentially varying concentration gradient is applied to the polyvinyl alcohol porous membrane to prepare a concentration gradient 2; the concentration gradient layer 4 is below the indicator layer 2, the indicator layer 2 is made of a porous polyvinyl alcohol material, and the indicator layer 2 is used for color development.
  • the agent is as follows - bromothymol blue: 1.5%; under the indicator layer 2 is the substrate 1 Using a colorless and transparent substrate thickness of 0.15-0.2mm PET sheet or polycarbonate sheet.
  • the sampling process is to add a certain amount of sample test solution to the diffusion layer 5, and after standing for 30 minutes after the sampling is completed, the result is observed, and the urea test strip (paper) exhibits a transition from yellow to blue. , can see the color boundary line A, read the A value with the ruler, as shown in Figure 3 (a), or by color scanning to obtain a scan curve similar to that shown in Figure 3 (b), read the inflection point A value.

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Abstract

一种基于浓度梯度的多层膜干化学检测试条，包括从下至上依次设置的基片(1)、指示剂层(2)、试剂层(3)以及扩散层(5)，该检测试条还包括浓度梯度层(4)，所述浓度梯度层(4)设置于所述试剂层(3)的上表面或下表面；在所述试剂层(3)上均匀涂布有第一试剂；在所述浓度梯度层(4)上涂布有第二试剂，所述的第二试剂的浓度梯度∇ρ在检测试条宽度方向为0，在检测试条长度方向为常数或为长度变量的函数，在所述指示剂层(2)上均匀涂布有显色剂。该基于浓度梯度的多层膜干化学检测试条能够使试剂在检测试条上的浓度分布呈连续无级变化，全部检测、分析过程可直接在试条上进行，并且能够在现场不借助仪器直读得到检测结果。

Description

基于浓度梯度的多层膜干化学检测试条 技术领域
本发明涉及化学检测制品，特别涉及一种用于现场快速定量检测的基于浓度梯度的多层膜干化学检测试条。
背景技术
随着人民生活水平的日益提高，人们越来越关注自身健康安全以及生活居住，离不开分析检验，包括医疗临床检验，食品安全检测，环境质量检测等，这些以化学分析和生化分析方法的检测从以前的手工实验的方法正在朝着全自动分析仪器自动分析检测和现场检测(如床边检测POCT，Point of care testing)发展，极大地方便了分析操作人员的工作负担。分析检验按分析试剂类型可以分为液体试剂和干化学试剂两种方法。采用液体试剂(湿化学)方法有足够的准确度和精密度，但需昂贵的仪器和专业技术人才，并且操作繁琐、不能快速测定出结果，不适合现场检验(如医院急诊室和小批量样品的检测的需求)；而干化学试剂以其操作简便、测定快速、灵活以及无需专业人才等优点很好的补充了湿化学法的不足之处，其测定准确度和精密度已经接近湿化学测定方法，其中尤以干化学检测试条(纸)(干化学)广泛地应用于临床检验，食品安全检测，环境质量检测领域，提供了极大的便利。“干化学”是与传统的“湿化学”(即溶液化学)相对比较而言的。它是以被检测样品中的液体作为反应媒介，待测物直接与固化于载体上的干粉试剂反应的一种方式。它与传统湿化学的最大区别就在于参与化学反应的媒介不同。随着生物化学中酶的分离、提纯存储等技术的发展，传感器、光度计和电极技术的进步，以及计算机应用的普及，干化学技术在近20年里得到了长足的进步干化学检测试条(纸)目前用于临床、食品安全和环境质量领域的检测常用的是比色方法和电化学方法。
电化学方法如CN101762628A“一种干酶法生物芯片/试条”是在PET基片上制作干酶工作电极、参比电极，测试液扩散网膜，疏水性绝缘漆来制成，测量的信号需要电化学仪器读出。
比色方法的干化学检测试条(纸)发展较早，从开始的一层结构发展而来二层结构、三层结构和现在最新的多层结构。
一层结构如最初的测量酸碱度的pH试纸和测量氧化物的碘化钾淀粉试纸等，它们主要用于定性实验，最多只能得到的值是半定量的。
在一层结构的基础上发展到用于生化分析的试剂载体最简单的是二层结构，在支持层塑料基片上有一试剂层纤维素片，在纤维素片中预固相了全部试剂。常见的是尿生化分析试剂条，如CN1888900A“pH分段排序的十三项尿试纸”，将十三个检测项目所用试剂赋予十三个不同纸片，粘贴在塑料基片上，尿液中的待测成分与预固相在纤维素片上的试剂直接反应， 通过反射光度计测定其颜色深浅的变化，从而计算待测成份的浓度，这种结构只能对待测成分进行定性或半定量测定，这种试纸也需要仪器进行结果的读取，而且测量线性范围不大，遇到有使用药物情况下会干扰实验结果，这样就限制了它在其它需要准确定量的领域的应用。
三层结构在试剂层上加一多孔胶膜过滤层，其作用是将样品中的杂质过滤掉，并起保护试剂层作用。常见的是微量法测定葡萄糖的试剂条，三层结构的试剂载体的测定光路是通过透明的塑料基片，而不经过最上面的过滤层，这样消除了样品中干扰成分的影响，保证了待测成分测定的稳定性和准确性，如CN1225450A“试纸和分析物测定头”中，血糖比色试纸制备和配套的检测仪器。
三层结构中试剂层只有一层，有许多分析测试中所有的试剂不能预先混合，而且复杂测试液需要预处理，这就限制了三层结构的干化学检测试条(纸)应用领域。为了解决三层结构的缺陷，现在最新技术——多层膜法，即干化学的多层膜试剂载体。从制造工艺来分多层膜法主要有纵向和横向两类。第一类是以强生公司和富士公司为代表的多层膜干片法，利用感光胶片的涂层技术将试剂层、辅助试剂层、光漫射层和分布层等试剂涂布在透明支持层上，从支持层的反面测定光密度的变化。如US5508173。第二类是罗氏公司的干化学产品，US4604264将试剂涂布在多丝的纤维或织物上，全血通过玻璃纤维横向过滤后与上试剂层接触反应，通过上部覆盖的透明薄膜测定光密度的变化。
上述这些干化学检测试条(纸)很难做到定量检测，并且有的生产工艺复杂，对材料设备要求高，有的可能产生某些缺陷，如在加样端容易产生一定的气泡、过量液体等，加样和测定在同一侧样品可能造成干扰，在测量时有的检测项目需要温度和时间控制。而且目前应用的干式生化测定仪为大型设备，将所有的生化项目集中在一台机器上测定。如强生VITROS系列(VITR0S-250，VITR0S-950)半自动及全自动干式生化仪及生化试剂；VITR0S-250，950在医院应用较多。当送检的样本数量少时开机不方便。在现场检测的情况下如急诊项目如急腹症需要马上测定，如果有能直接定量分析试纸可以满足急需。这对一些需要现场检测场所意义巨大。
为了实现现场快速准确的定量分析检测，采用在干化学检测试条(纸)人为控制试剂在试条(纸)上的浓度分布，以达到：待测液在试条(纸)扩散渗透形成类似于化学滴定原理的分析检测方法，实现对样液的现场快速定量检测。
CN2564584“细菌药物敏感性试验检测抗菌素的最小抑菌浓度检测板”的方法采用的是多种不同定量的抗生素浓度梯度滤膜条固定在基片上，特点是抗生素在塑料载体上纵向非连续指数形式递减和在横向浓度是相同的阶梯度浓度法(如图5所示)。
CN201309942“梳状药敏试条”的方法有些类似与CN2564584的方法，特点是抗生素在塑料载体的梳齿上纵向非连续指数形式递减和横向浓度(如图6所示)。
CN1667414“阶梯式浓度梯度检测条及其制备方法”的方法采用的是在载体的一面等长地划分有若干个等面积区间，在每个等面积区间中心对称地涂布有设定形状及面积的药物块；各药物块的药量呈现出一段或多段的阶梯性变化，即：在不同段中的药物块的浓度不同。但 是在同一段中排列的各个药物块的涂布厚度和浓度相同，各药物块的面积大小按顺序阶梯递增或递减，其特点是浓度梯度的分布是通过抗生素药剂涂布在基片上的面积大小确定的，即在试条(纸)纵向抗生素药呈非连续阶梯式分布(如图8所示)，在试条(纸)横向抗生素药浓度呈现凸峰分布(如图7)，采用这种方式的还有美国专利US20130244316A1“Paper strip for determining minimum inhibitory concentrations of antibiotics”和US4778758“Device for susceptibility testing of microorganisms”。
上述专利报导实现了抗生素药剂浓度的阶梯式分布，主要运用在微生物药敏实验，是微生物耐药实验的检测，而且在实验过程中是通过抗生素在培养基上的扩散，在培养基表面形成抗生素药剂浓度梯度变化的分布，经过培养，读到细菌最小抑菌浓度(MIC值，Minimal inhibitory concentration)，不能现场快速的得到结果。
发明内容
针对现有技术的不足之处，本发明的目的在于提供一种可用于现场快速准确定量分析检测、浓度分布连续无级变化的基于浓度梯度的多层膜干化学检测试条。
为实现上述目的，本发明采用的技术方案是：
本发明一种基于浓度梯度的多层膜干化学检测试条，包括从下至上依次设置的基片、指示剂层、试剂层以及扩散层，其特征在于，该检测试条还包括浓度梯度层，所述浓度梯度层设置于所述试剂层的上表面或下表面；在所述试剂层上均匀涂布有第一试剂；在所述浓度梯度层上涂布有第二试剂，所述的第二试剂的浓度梯度
Figure PCTCN2015073387-appb-000001
在检测试条宽度方向为0，在检测试条长度方向为常数或为长度变量的函数；在所述指示剂层上均匀涂布有显色剂。
所述浓度梯度层采用聚乙烯醇、明胶以及聚乙烯醇和明胶混合物中的任一种。
所述浓度梯度层为多孔涂层或预制的多孔膜。
所述扩散层为亲水性薄膜。
所述亲水性薄膜上还设置有虹吸结构。
所述试剂层为高分子聚合物材料。
所述试剂层为多孔涂层或预制的多孔膜。
所述指示剂层为高分子聚合物材料。
所述指示剂层为多孔涂层或预制的多孔膜。
所述基片为透明材料，厚度为0.1-0.3mm。
所述基片下表面还设置有标尺。
与现有技术相比，本发明具有的有益效果是：
本发明基于浓度梯度的多层膜干化学检测试条，采用了浓度梯度层，能够使试剂在检测试条上的浓度分布呈连续无级变化，全部检测、分析过程可直接在试条上进行，并且能够在现场不借助仪器直读得到检测结果，同时在检测过程中不要温度和时间的限制条件，操作简便，检测手段开放，操作过程无需专门训练，适合专业人士和非专业人士现场检验。
附图说明
图1为本发明的干化学检测试条结构示意图。
图2为本发明中的浓度梯度层的试剂分布图。
图3为本发明中的检测试条检验结果图。
图4为现有技术细菌药物敏感性试验检测抗菌素的最小抑菌浓度检测板抗生素浓度分布图。
图5现有技术梳状药敏试条抗生素浓度分布图。
图6现有技术阶梯式浓度梯度检测条的抗生素浓度分布图。
图7现有技术阶梯式浓度梯度检测条的抗生素涂布示意图。
图8本发明中的基片下表面的标尺示意图。
图中：1、基片，2、指示剂层，3、试剂层，4、浓度梯度层，5、扩散层。
具体实施方式
下面结合附图对本发明作进一步说明。
实施例1：
如图1，图3和图8示，基于浓度梯度法制备水质硬度检测试条(纸)，用于快速检测各种来源的水体中硬度指标。制备两种结构方式制作的水质硬度检测试条(纸)，试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，第一种制备方式为：所述检测试条最上面一层为扩散层5采用亲水的多孔纤维素膜采用涂布的形式，多层结构采用涂层方式制备；扩散层5下面则为试剂层3，试剂层3采用多孔聚乙烯醇材料，试剂层3所用试剂制备反应液如下——三羟甲基氨基甲烷(Tris)10-15克/升，聚乙烯醇乳液与试剂液1：1-4∶1的比例混匀涂层制备试剂层；试剂层3下面为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，将络合剂乙二胺四乙酸(EDTA)以线性递减的浓度梯度的方式或者阶梯状递减的浓度梯度的方式涂布于聚乙烯醇多孔膜，制备浓度梯度层4；浓度梯度层4下面为指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——酸性铬兰K 1-2克/升；指示剂层2下面为基片1，基片采用0.15-0.2mm厚度的草绿色透明PET片材或者据碳酸酯片材。第二种制备方式为：扩散层5由亲水的尼龙网膜构成，多层结构采用现成的膜层多层层压方式制备；扩散层5下面为试剂层3，为成品的聚乙烯醇多孔膜浸渍上述试剂液中5分钟后避光干燥而成，试剂层3下面为浓度梯度层4，为成品的聚乙烯醇多孔膜以线性递减的浓度梯度的方式涂布乙二胺四乙酸(EDTA)；浓度梯度层4下面为指示剂层2，为成品的聚乙烯醇多孔膜浸渍上述显色剂中5分钟后避光干燥而成；指示剂层2下面的基片采用0.15-0.2mm厚度的草绿色透明PET片材或者聚碳酸酯片材；将各层按顺序通过扩散层5的尼龙网膜的绷压固定在基片上。当检测水体总硬度时，采用前一种多层膜制备方式制作的水质硬度检测试条(纸)，取样过程是在扩散层5上加一定量的测试液；采用后一种多层膜制备方式制作的水质硬度检测试条(纸)， 取样过程是将试条(纸)迅速的插入测试液三秒内拿出，吸干上面水珠后；取样完成后静置3分钟后观察结果，水质硬度检测试条(纸)贴近基片的浓度梯度层或试剂层呈现由红色-紫色(分界线A)-蓝紫色的过渡，由于光线经过草绿色透明基片的吸收作用，从基片方向观察到呈现由砖红色-蓝紫色(分界线A)-蓝色的过渡，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(c)的扫描曲线，读取拐点A值。
实施例2：
如图1，图3和图8示，基于浓度梯度法制备酸价检测试条(纸)，用于快速检测各种油脂的酸价指标。制备两种结构方式制作的水质硬度检测试条(纸)，试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，第一种制备方式为：所述检测试条最上面一层为扩散层5采用的多孔纤维素膜采用涂布的形式，多层结构采用涂层方式制备；扩散层5下面则为试剂层3，试剂层3采用多孔聚乙烯醇材料，试剂层3所用试剂制备反应液如下——保护剂KD 2-4克/升，聚乙烯醇乳液与试剂液1：1-4∶1的比例混匀涂层制备试剂层；试剂层3下面为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇材料，采取预先将聚乙烯醇与保护剂KD1材料混合后均匀涂布成多孔膜，将滴定剂氢氧化钾以线性递减的浓度梯度的方式或者阶梯状递减的浓度梯度的方式涂布于聚乙烯醇多孔膜，制备浓度梯度层4；浓度梯度层4下面为指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——酚酞1-2克/升；指示剂层2下面为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者据碳酸酯片材。第二种制备方式为：扩散层5由亲水的尼龙网膜构成，多层结构采用现成的膜层多层层压方式制备；扩散层5下面为试剂层3，为成品的聚乙烯醇多孔膜浸渍上述试剂液中5分钟后避光干燥而成，试剂层3下面为浓度梯度层4，为成品的聚乙烯醇多孔膜以线性递减的浓度梯度的方式涂布滴定剂氢氧化钾；浓度梯度层4下面为指示剂层2，为成品的聚乙烯醇多孔膜浸渍上述显色剂中5分钟后避光干燥而成；指示剂层2下面的基片采用0.15-0.2mm厚度的无色透明PET片材或者聚碳酸酯片材；将各层按顺序通过扩散层5的尼龙网膜的绷压固定在基片上。检测油脂的酸价时，采用前一种多层膜制备方式制作的酸价检测试条(纸)，取样过程是在扩散层5上加一定量的经处理的测试液；采用后一种多层膜制备方式制作的酸价检测试条(纸)，取样过程是将试条(纸)迅速的插入经处理的测试液三秒后拿出，吸干上面液珠后；取样完成后静置3分钟后观察结果，酸价检测试条(纸)观察到呈现由无色-淡红色(分界线A)-红色的过渡，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(b)的扫描曲线，读取拐点A值。
实施例3：
如图1，图3和图8所示，基于浓度梯度法制备血糖检测试条(纸)，用在临床快速检测血液中葡萄糖的含量。试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，多层结构采用涂层方式制备，所述检测试条最上面一层为扩散层5采用亲水的多孔纤维素膜，采用涂布的形式，扩散层5下面则为试剂层3，试剂层3采用聚乙烯吡咯烷酮和聚砜材料制备的多孔膜，聚砜材料的多孔膜也负责吸附去除血细胞，试剂层 3所用试剂制备反应液如下——10IU/L葡萄糖氧化酶、0.037mol/L柠檬酸、0.126mol/L磷酸氢二钠、10g/L海藻糖、5g/L甘氨酸、3g/LBSA和10g/LPEG4000的混合液涂布于多孔膜制备试剂层；试剂层3下面为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，将抗氧化剂(BHT)以线性递减的浓度梯度的方式或者阶梯状递减的浓度梯度的方式涂布于聚乙烯醇多孔膜，另外聚乙烯醇多孔膜上再均匀涂布反应液——20IU/L辣根过氧化物酶、0.037mol/L柠檬酸、0.126mol/L磷酸氢二钠、10g/L海藻糖、5g/L甘氨酸、3g/LBSA和10g/LPEG4000盐酸盐混合液制备浓度梯度层4；浓度梯度层4下面指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——1.0g/L TMB；指示剂层2下面为为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者据聚酸酯片材。检测血糖时，取样过程是在扩散层5上加一定量的血液；取样完成后静置3分钟后观察结果，血糖检测试条(纸)呈现由无色-蓝色的过渡，能够看到颜色分界线A，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(b)的扫描曲线，读取拐点A值。
实施例4：
如图1，图3和图8示，基于浓度梯度法制备谷丙转氨酶检测试条(纸)，用在临床快速检测血液中谷丙转氨酶的含量。试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，多层结构采用涂层方式制备，所述检测试条最上面一层为扩散层5采用亲水的多孔纤维素膜，采用涂布的形式制备；扩散层5下面则为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇膜，浓度梯度层4将谷丙转氨酶抑制剂(GBH)以线性递减的浓度梯度的方式或者呈连续指数型变化的浓度梯度的方式涂布于聚乙烯醇多孔膜，另外再均匀涂布反应液——Good′s缓冲液：200mM、MgCl2*6H20：2.0g/L、KH2PO4：20mM和海藻糖：0.5％混合液制备浓度梯度层4；浓度梯度层4下面为试剂层3，试剂层3采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，在聚乙烯醇多孔膜上再均匀涂布反应液——丙氨酸：1000α一酮戊二酸钠：20mM、过氧化物酶：100U/mi、丙酮酸氧化酶：100U/ml、黄素腺嘌呤二核苷酸(FAD)：0.2mg/ml、硫胺素焦磷酸(TPP)：0.8mg/ml和TMB：0.1％混合液制备试剂层3；试剂层3下面指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——1.0g/LTMB；指示剂层2下面为为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者聚碳酸酯片材。检测血液中谷丙转氨酶的含量时，取样过程是在扩散层5上加一定量的测试液，取样完成后静置30分钟后观察结果，谷丙转氨酶检测试条(纸)呈现由无色-蓝色的过渡，能够看到颜色分界线A，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(b)的扫描曲线，读取拐点A值。
实施例5：
如图1，图3和图8所示，基于浓度梯度法制备骨碱性磷酸酶检测试条(纸)，用在临床快速检测血液中骨碱性磷酸酶的含量。试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，多层结构采用涂层方式制备，所述检测试条最上面 一层为扩散层5采用亲水的多孔纤维素膜，采用涂布的形式制备；扩散层5下面则为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇膜，浓度梯度层4将骨碱性磷酸酶抑制剂(BALH)以线性递减的浓度梯度的方式或者呈连续指数型变化的浓度梯度的方式涂布于聚乙烯醇多孔膜，制备浓度梯度层4；浓度梯度层4下面为试剂层3，试剂层3采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，在聚乙烯醇多孔膜上再均匀涂布反应液——三羟甲基氨基甲烷(Tris)：5-10/L、凝集素：UA-3：0.5％、MgCl2*6H20：5mmol/L和硝基氯化四氮唑兰(NBT)1.0mmol混合液制备试剂层3；试剂层3下面指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——溴氯吲哚磷酸盐(BCIP)1.0mmol/L；指示剂层2下面为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者聚碳酸酯片材。检测血液中骨碱性磷酸酶的含量时，取样过程是在扩散层5上加一定量的处理过的血液测试液，取样完成后静置30分钟后观察结果，骨碱性磷酸酶酶检测试条(纸)呈现由无色-紫色的过渡，能够看到颜色分界线A，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(b)的扫描曲线，读取拐点A值。
实施例6：
如图1，图3和图8所示，基于浓度梯度法制备超氧化物歧化酶检测试条(纸)，用于快速检测生物样本中超氧化物歧化酶含量。试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，多层结构采用涂层方式制备，所述检测试条最上面一层为扩散层5采用亲水的多孔纤维素膜，采用涂布的形式制备；扩散层5下面则为试剂层3，试剂层3采用多孔聚乙烯醇膜，试剂层3另外再均匀涂布反应液——次黄嘌呤：200mM和盐酸羟胺：2.0/L、混合液制备试剂层3；试剂层3下面为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，将黄嘌呤氧化酶(XO)以线性递减的浓度梯度的方式或者呈连续指数型变化的浓度梯度的方式涂布于聚乙烯醇多孔膜，在聚乙烯醇多孔膜上再均匀涂布反应液——三羟甲基氨基甲烷(Tris)：5-10/L、海藻糖：10g/L、甘氨酸：5g/L、BSA：3g/L、PEG4000：10g/L混合液制备浓度梯度层4；浓度梯度层4下面为指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——对氨基苯磺酸试剂：2.0/L和a-萘胺：1.0/L；指示剂层2下面为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者聚碳酸酯片材。检测生物样本中超氧化物歧化酶的含量时，取样过程是在扩散层5上加一定量的生物样本处理测试液，取样完成后静置30分钟后观察结果，超氧化物歧化酶检测试条(纸)呈现由无色-紫红色的过渡，能够看到颜色分界线A，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(b)的扫描曲线，读取拐点A值。
实施例7：
如图1，图3和图8所示，基于浓度梯度法制备余氯检测试条(纸)，用于快速检测不同水样中余氯的含量。试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，多层结构采用涂层方式制备，所述检测试条最上面一层为扩散层5采 用亲水的多孔纤维素膜，采用涂布的形式制备；扩散层5下面则为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇膜，浓度梯度层4将抗氧化剂(BHT)以线性递减的浓度梯度的方式或者呈连续指数型变化的浓度梯度的方式涂布于聚乙烯醇多孔膜制备浓度梯度层4；浓度梯度层4下面为试剂层3，试剂层3采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，在聚乙烯醇多孔膜上再均匀涂布反应液——KH2PO4：20mM制备试剂层3；试剂层3下面为指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——TMB：0.1％；指示剂层2下面为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者聚碳酸酯片材。检测水样中余氯的含量时，取样过程是在扩散层5上加一定量的生物样本处理测试液，取样完成后静置10分钟后观察结果，余氯检测试条(纸)呈现由无色-蓝色的过渡，能够看到颜色分界线A，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(b)的扫描曲线，读取拐点A值。
实施例8：
如图1，图3和图8所示，基于浓度梯度法制备过氧化氢检测试条(纸)，用于快速检测不同来源的水样中过氧化氢的含量。试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，多层结构采用涂层方式制备，所述检测试条最上面一层为扩散层5采用亲水的多孔纤维素膜，采用涂布的形式制备；扩散层5下面则为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇膜，浓度梯度层4将抗氧化剂(BHT)以线性递减的浓度梯度的方式或者呈连续指数型变化的浓度梯度的方式涂布于聚乙烯醇多孔膜，另外再均匀涂布反应液——0.037mol/L柠檬酸和0.126mol/L磷酸氢二钠混合液制备浓度梯度层4；浓度梯度层4下面为试剂层3，试剂层3采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，在聚乙烯醇多孔膜上再均匀涂布反应液——0.05g/L辣根过氧化物酶、10g/L海藻糖、5g/L甘氨酸、3g/LBSA和10g/LPEG4000混合液制备试剂层3；试剂层3下面为指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——2.0g/L TMB盐酸盐；指示剂层2下面为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者聚碳酸酯片材。检测水样中过氧化氢的含量时，取样过程是在扩散层5上加一定量的生物样本处理测试液，取样完成后静置10-30分钟后观察结果，过氧化氢检测试条(纸)呈现由无色-蓝色的过渡，能够看到颜色分界线A，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(b)的扫描曲线，读取拐点A值。
实施例9：
如图1，图3和图8所示，基于浓度梯度法制备有机磷检测试条(纸)，用于快速检测农产品中残留的有机磷的含量。试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，多层结构采用涂层方式制备，所述检测试条最上面一层为扩散层5采用亲水的多孔纤维素膜，采用涂布的形式制备；扩散层5下面则为试剂层3，试剂层3采用多孔聚乙烯醇膜，试剂层3均匀涂布反应液——海藻糖：10g/L、甘氨酸：5g/L、BSA：3g/L、PEG4000：10g/L、柠檬酸：0.04mol/L和磷酸氢二钠：0.15mol/L混合液制备试 剂层3；试剂层3下面为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，将乙酰胆碱酯酶以线性递减的浓度梯度的方式或者呈连续指数型变化的浓度梯度的方式涂布于聚乙烯醇多孔膜制备试剂层3；浓度梯度层4下面为指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——2，6-二氯靛酚乙酸酯：10g/L；指示剂层2下面为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者聚碳酸酯片材。检测农产品中有机磷的含量时，取样过程是在扩散层5上加一定量的生物样本处理测试液，取样完成后静置10-30分钟后观察结果，有机磷检测试条(纸)呈现由橙红色-蓝色的过渡，能够看到颜色分界线A，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(c)的扫描曲线，读取拐点A值。
实施例10：
如图1，图3和图8所示，基于浓度梯度法制备尿素检测试条(纸)，用于快速检测生物样本和不同来源的水样中尿素的含量。试条(纸)由基片1、指示剂层2、试剂层3、浓度梯度层4及扩散层5五个基本功能层组成，多层结构采用涂层方式制备，所述检测试条最上面一层为扩散层5采用亲水的多孔纤维素膜，采用涂布的形式制备；扩散层5下面则为试剂层3，试剂层3采用多孔聚乙烯醇膜，试剂层3均匀涂布反应液——尿素酶：20KU/L、PBS(pH6.8)：5.0umol/L、海藻糖：1％、甘氨酸：5g/L、BSA：3g/L和PEG4000：10g/L、的制备试剂层3；试剂层3下面为浓度梯度层4，浓度梯度层4采用多孔聚乙烯醇材料，采取预先将聚乙烯醇材料均匀涂布成多孔膜，将柠檬酸以线性递减的浓度梯度的方式或者呈连续指数型变化的浓度梯度的方式涂布于聚乙烯醇多孔膜制备浓度梯度2；浓度梯度层4下面为指示剂层2，指示剂层2采用多孔聚乙烯醇材料，指示剂层2所用显色剂如下——溴百里酚蓝：1.5％；指示剂层2下面为基片1，基片采用0.15-0.2mm厚度的无色透明PET片材或者聚碳酸酯片材。检测血液中尿素的含量时，取样过程是在扩散层5上加一定量的样本测试液，取样完成后静置30分钟后观察结果，尿素检测试条(纸)呈现由黄色-蓝色的过渡，能够看到颜色分界线A，用标尺读取A值，如图3(a)，或者经颜色扫描得到类似与如图3(b)的扫描曲线，读取拐点A值。
上述各实施例仅是对本发明技术方案的具体描述，而不能解释为对本发明的限制。本领域的技术人员应该理解，在不脱离本发明技术方案的实质精神和保护范围的情况下，对本发明所作的任何形式或细节的各种修改及等同替换，均在本发明的保护范围内。

Claims (10)

  1. 一种基于浓度梯度的多层膜干化学检测试条，该检测试条包括从下至上依次设置的基片(1)、指示剂层(2)、试剂层(3)以及扩散层(5)，其特征在于，该检测试条还包括浓度梯度层(4)，所述浓度梯度层(4)设置于所述试剂层(3)的上表面或下表面；在所述试剂层(3)上均匀涂布有第一试剂；在所述浓度梯度层(4)上涂布有第二试剂，所述的第二试剂的浓度梯度
    Figure PCTCN2015073387-appb-100001
    在检测试条宽度方向为0，在检测试条长度方向为常数或为长度变量的函数；在所述指示剂层(2)上均匀涂布有显色剂。
  2. 根据权利要求1所述基于浓度梯度的多层膜干化学检测试条，其特征在于，所述浓度梯度层(4)的材料为聚乙烯醇、明胶以及聚乙烯醇和明胶混合物中的任一种。
  3. 根据权利要求2所述基于浓度梯度的多层膜干化学检测试条，其特征在于，所述浓度梯度层(4)为多孔涂层或预制的多孔膜。
  4. 根据权利要求1所述基于浓度梯度的多层膜干化学检测试条，其特征在于，所述扩散层(5)为亲水性薄膜。
  5. 根据权利要求4所述基于浓度梯度的多层膜干化学检测试条，其特征在于，所述亲水性薄膜上还设置有虹吸结构。
  6. 根据权利要求1所述基于浓度梯度的多层膜干化学检测试条，其特征在于，所述试剂层(3)为高分子聚合物材料。
  7. 根据权利要求6所述基于浓度梯度的多层膜干化学检测试条，其特征在于，所述试剂层(3)为多孔涂层或预制的多孔膜。
  8. 根据权利要求1所述基于浓度梯度的多层膜干化学检测试条，其特征在于，所述指示剂层(2)为高分子聚合物材料；所述指示剂层(2)为多孔涂层或预制的多孔膜。
  9. 根据权利要求1所述基于浓度梯度的多层膜干化学检测试条，其特征在于，所述基片(1)为透明材料，厚度为0.1-0.3mm。
  10. 根据权利要求9基于浓度梯度的多层膜干化学检测试条，其特征在于，所述基片(1)下表面还设置有标尺。
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