CN113999889A - Dry-type glucose test strip adopting Prussian blue nanoenzyme and preparation method thereof - Google Patents
Dry-type glucose test strip adopting Prussian blue nanoenzyme and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a dry-type glucose test strip adopting prussian blue nanoenzyme and a preparation method thereof, belonging to the technical field of clinical diagnosis. The invention adopts prussian blue nano-enzyme to replace natural horse radish peroxidase commonly used in dry chemical test paper strips. The artificially synthesized Prussian blue nano material has the characteristic of catalyzing the reaction of peroxide and a substrate. Compared with natural enzymes, the artificial enzyme has the advantages of easy storage, convenient preparation, industrial mass production, low cost, good thermal stability and the like, and can meet the requirements of clinical application.
Description
Technical Field
The invention relates to a dry-type glucose test strip adopting prussian blue nanoenzyme, belonging to the technical field of clinical diagnosis.
Background
Most of clinical routine biochemical substances generate hydrogen peroxide after a set series of enzyme-catalyzed reactions, so that the hydrogen peroxide can generate color reaction with a substrate under the action of peroxidase, and the quantitative test of corresponding concentration indexes is completed. In emergency department or bedside diagnosis, higher requirements are placed on the sample sampling amount, the simplicity of the operation of the analysis process and the report time of the test result. The dry chemical method for drying substances participating in biochemical reaction on the membrane overlapped layer by layer can meet the requirement of fingertip blood detection, is simple and convenient to operate, obtains results within 3 minutes, and is suitable for the fields of emergency department and bedside diagnosis.
Compared with the traditional wet method, the dry test paper needs to be prepared with enzyme solution with better concentration so as to be effectively fixed on the surface of the membrane to meet the corresponding working requirement. Peroxidase is the enzyme which is most widely applied and has the largest consumption in dry chemical test paper strips. The industrial production of horseradish peroxidase usually uses natural horseradish as raw material, and the product is obtained by first classification with ammonium sulfate, purification with calcium phosphate gel, classification with ethanol, second classification with ammonium sulfate and crystallization refining after water extraction. The natural horseradish peroxidase has the following defects: (1) the price is high, the market consumption of the horseradish peroxidase is huge, the horseradish peroxidase is imported, the shelf life is long, the storage condition is strict, and the logistics cost is high; (2) the batch difference of enzyme catalytic activity is large; (3) the protein structure of the enzyme has poor thermal stability, the reagent strip can cause the reduction of the enzyme performance in the drying process, and the processing technology of the reagent strip has strict requirements.
Therefore, there is a need to provide a dry glucose strip that does not use native horseradish peroxidase.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a dry glucose test strip adopting prussian blue nanoparticles to replace natural horseradish peroxidase, so as to solve the problems of poor thermal stability, high price and harsh preparation conditions of the dry glucose test strip using the horseradish peroxidase in the prior art.
The technical problem to be solved by the invention is to provide a preparation method of the dry-type glucose test strip adopting the Prussian blue nano-enzyme.
The technical scheme is as follows: in order to solve the technical problems, the invention adopts the following technical scheme:
a preparation method of a dry-type glucose test strip adopting Prussian blue nanoenzyme comprises the following steps:
(1) adding prussian blue nano-enzyme, an adhesive, a film forming material, glucose oxidase and a peroxide substrate into a buffer solution to prepare a film forming treatment solution (I), adsorbing the film forming treatment solution (I) on a reaction film, and drying to obtain the reaction film containing prussian blue nano-enzyme;
or adding Prussian blue nano enzyme, adhesive and film forming material into buffer solution to prepare film forming treatment liquid (II), and adsorbing the film forming treatment liquid (II) on the blood filtering film; then adding glucose oxidase, a peroxide substrate, an adhesive and a film forming material into a buffer solution to prepare a film forming treatment solution (III), and adsorbing the film forming treatment solution (III) on a reaction film;
(3) sequentially stacking the diffusion membrane, the blood filtration membrane and the reaction membrane from top to bottom to obtain the dry glucose test strip adopting the Prussian blue nanoenzyme;
the combination mode of the membrane treatment liquid and the membrane can be soaking or scribing by adopting a scribing instrument; the membrane treated by soaking can be dried in an oven at 45 ℃ for 2h, and the membrane treated by scribing can be dried at 37 ℃ for 1 h.
In the step (1), the preparation method of the prussian blue nano-enzyme comprises the following steps:
(S1) adding PVP and FeCl3·6H2Dispersing O in ultrapure water, heating in water bath at 60 deg.C, stirring, and mixing, wherein PVP and FeCl are added3·6H2The molar ratio of O is 10:0.05-10:0.15, and the content of PVP in the ultrapure water is 0.100-0.125 mmol/mL;
(S2) adding K4[Fe(CN)6]·3H2Dispersing O in ultrapure water according to the volume ratio of 0.003-0.006mL/mL,
(S3) Using a two-channel micro-fluid injection pump, the K is injected4[Fe(CN)6]·3H2Dropwise adding the O solution to the PVP and FeCl prepared in the step (S1)3·6H2In the O solution, the dropping rate is 20-60mL/h, after the dropping is finished, the stirring is continued under the condition of constant-temperature water bath at 60 ℃, after the reaction is finished, the power supply of the water bath kettle is closed, and the temperature of the water bath is slowly reduced to the room temperature;
(S4) filtering out unreacted PVP and ions by a Millpore TTF system through tangential flow, and finally filtering out aggregates by a 0.22 mu m filter membrane to obtain the Prussian blue nano enzyme.
In the step (1), the particle size of the Prussian blue nano enzyme is 50-150nm, preferably 110 nm; the concentration of the Prussian blue nano enzyme in the buffer solution is 0.5-1mg/mL, and preferably 0.8 mg/mL.
In the step (1), the buffer solution is disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution or citric acid-sodium citrate buffer solution, and the pH value of the buffer solution is 3.5-5.0.
In the step (1), the membrane treatment liquid (I), the membrane treatment liquid (II) and the membrane treatment liquid (III) contain surfactants, wherein the surfactants are Triton X-100 or CHAPS, the volume fraction of the Triton X-100 is 0.02-0.1%, and the content of the CHAPS is 5-10 g/L.
In the step (1), the preservative is contained in the membrane treatment liquid (I), the membrane treatment liquid (II) and the membrane treatment liquid (III), the preservative is one or a mixture of more of benzoic acid, sodium benzoate, sorbic acid, potassium sorbate and calcium propionate, and the content of the preservative is 0.5-1 g/L.
In the step (1), the membrane treatment liquid (I) and the membrane treatment liquid (II) contain BSA, and the concentration of the BSA is 20-30 g/L.
In the step (1), the membrane treatment liquid (I) and the membrane treatment liquid (II) contain EDTA, and the concentration of EATA is 0.5-1.5 g/L.
In the step (1), the adhesive is hydroxypropyl cellulose or a methyl vinyl ether/maleic anhydride copolymer, and the mass fraction of the adhesive is 0.5-1%.
In the step (1), the film-forming material is hydroxypropyl cellulose, methyl vinyl ether/maleic anhydride copolymer, PEG, PVP or PVA, and the mass fraction of the film-forming material is 0.5-3%.
In the step (1), the peroxide substrate is: MAOS +4-AAP, TOOS +4-AAP, CTA +4-AAP, MBTH + DMAB, MBTH + DCHBS or MBTH + ANS, wherein the content of the peroxide substrate in the buffer solution is 4-10 g/L, and the molar ratio of MAOS to 4-AAP, TOOS to 4-AAP, CTA to 4-AAP, MBTH to DMAB, MBTH to DCHBS, MBTH to ANS is (1-3):1
In the step (1), the concentration of the glucose oxidase is as follows: 250- & lt 400 & gtKU/L.
Preferably, the reaction membrane is a negatively charged nylon membrane or a polyethersulfone membrane with an asymmetric structure, the middle blood filtration membrane is a glass fiber membrane or a polyethersulfone membrane, and the diffusion membrane is a polyester membrane or a nylon membrane.
The invention adopts prussian blue nano-enzyme to replace natural horse radish peroxidase commonly used in dry chemical test paper strips. The artificially synthesized Prussian blue nano material has the characteristic of catalyzing the reaction of peroxide and a substrate. Compared with natural enzymes, the artificial enzymes are easy to store (only by storing the solution state at normal temperature); the preparation is convenient, the industrial mass production can be realized, and the batch difference is easy to control relative to natural enzyme; the cost is low; the thermal stability is good, and the process requirement is low in the reagent strip processing process. The general application of the reagent strip in the dry chemical test strip can greatly reduce the cost of the reagent strip.
Has the advantages that:
the Prussian blue nano enzyme adopted in the invention is convenient to synthesize, has the cost far lower than that of natural enzyme, reduces the cost of the reagent strip, has good thermal stability, can effectively prolong the service time of the reagent strip, relaxes the requirement on storage environment, and reduces the requirement on the production process of the reagent strip. At present, dry chemical reagent strips for clinical application in emergency departments mainly depend on import, and the application of Prussian blue in related fields helps to improve the competitiveness of domestic reagents in the related fields.
The successful application of the artificial enzyme in the diagnostic reagent has great market and clinical value, and the mainstream nano-enzyme application research at present focuses on the application of the artificial enzyme in a solution system. The invention relates to a dry chemical test strip which is successfully prepared for the first time and can be applied to the detection of glucose by using peroxide catalysis in the process of carrying out diagnosis reaction by using artificial nano enzyme.
Drawings
Fig. 1 is a flow chart of the preparation of a prussian blue nanoenzyme-based glucose test strip (example 1).
Fig. 2 is a flowchart of the preparation of a prussian blue nanoenzyme-based glucose test strip (example 2).
FIG. 3 scanning electron micrograph of asymmetric polyethersulfone membrane used as reaction layer.
FIG. 4 scanning electron micrograph of asymmetric polyethersulfone membrane used as reaction layer.
Fig. 5 scanning electron micrographs of prussian blue nanoparticles.
Fig. 6 scanning electron micrographs of prussian blue nanoparticles.
Fig. 7 distribution of prussian blue nanoparticles on the surface of the reaction membrane (polyethersulfone membrane).
Fig. 8 distribution of prussian blue nanoparticles on the surface of the reaction membrane (polyethersulfone membrane).
Fig. 9 distribution of prussian blue nanoparticles on the surface of the reaction membrane (polyethersulfone membrane).
FIG. 10 is a scanning electron micrograph of the hemodiafiltration glass fiber membrane and the distribution of Prussian blue nanoenzyme on the glass fiber hemofiltration membrane.
FIG. 11 is a scanning electron micrograph of a hemodiafiltration glass fiber membrane and the distribution of Prussian blue nanoenzyme on the glass fiber hemofiltration membrane.
FIG. 12 is a scanning electron micrograph of a hemodiafiltration glass fiber membrane and the distribution of Prussian blue nanoenzyme on the glass fiber hemofiltration membrane.
FIG. 13 test strip for glucose selectivity.
FIG. 14 validation of the test strip against clinical specimen.
Figure 15 reproducibility verification of the test strip.
Accelerated stability validation of the reagent strip of FIG. 16.
Detailed Description
The technical solutions of the present invention are further described in detail by the following specific examples, but it should be noted that the following examples are only used for describing the content of the present invention and should not be construed as limiting the scope of the present invention.
Preparing Prussian blue nano enzyme: 1.11g (10mmol) of PVP (K-30) is weighed out and ultrasonically dispersed in 80mL of ultrapure water, 27.03mg (0.1mmol) of FeCl is weighed out after uniform dispersion3·6H2O is added to the PVP aqueous solution and sonicated for 10min, followed by stirring in a 250mL three necked flask in a 60 ℃ thermostatted water bath at 1200rpm for 30 min. In addition, 42.24mg (0.1mmol) of K are weighed out4[Fe(CN)6]·3H2Dissolving O in 20mL of ultrapure water, performing ultrasonic treatment for 5min to uniformly disperse the O, transferring the O into a 20mL syringe, fixing the O on a WZS-50F6 double-channel micro-flow injection pump, and selecting the dropping speed of 40 mL/h. After the dropwise addition, the mixture is continuously stirred for 1 hour at the stirring speed of 1200rpm under the condition of a constant-temperature water bath at 60 ℃, and after the reaction is finished, the water bath kettle is closed and the temperature is slowly reduced to the room temperature. After the reaction was complete, unreacted PVP and ions were filtered off with a Millpore TTF system tangential flow (MWCO:50kDa) and finally agglomerates were filtered off with a 0.22 μm filter.
Example 1:
a preparation method of glucose test paper adopting Prussian blue artificial enzyme. In the example, the prussian blue nanoparticles, glucose oxidase and a substrate are on the same reaction membrane; see FIG. 1
(1) Preparing a membrane treatment solution (1L) containing prussian blue nanoenzyme:
(2) treating the reaction membrane by using membrane treatment liquid, and fixing prussian blue nano-particles and other necessary reactants on the membrane; soaking or scoring may be used. The parameters of the scribing are preferably 5.5 μ L/cm, scribing speed: 50 mm/s.
(3) The membrane treated with the above substances was placed on a shelf of appropriate size and dried at 37 ℃ for 1 h.
(4) Cutting the dried reaction membrane into a proper size, and assembling the reaction membrane, the blood filtering membrane and the diffusion membrane into the test strip.
Example 2:
a preparation method of glucose test paper adopting Prussian blue artificial enzyme. Materials and reagents adopted in the embodiment are the same as those of the comparison document 1, except that the prussian blue nanoparticles are independently adsorbed on a filter membrane, and the glucose oxidase and the substrate are on an independent reaction membrane; see fig. 2. The filtering membrane containing the Prussian blue nano particles can be independently prepared and stored, has good stability, and can be used as a filtering membrane with peroxide activity in any biochemical index needing peroxide catalysis.
Example 3:
electron microscopy characterization of the prussian blue artificial enzyme paper reaction film in example 1. FIGS. 3 to 6 are scanning electron micrographs (FIGS. 5 and 6) of a blank asymmetric polyethersulfone film (FIGS. 3 and 4) and Prussian blue nanoparticles. From the figure, the particle size of the prussian blue nano-particle is around 110nm, the particle size distribution is uniform, the prussian blue nano-particle is in a complete cubic structure and has a perfect form, and the basis of the activity of the nano-enzyme is shown. The polyether sulfone membrane fiber strip is clear, and the pore diameter distribution is in gradient distribution from the diameter of 200um on one surface to the diameter of 10um on the other surface. The asymmetric structure facilitates the liquid sample from the upper layer with large aperture to soak the membrane material, and the lower layer with small aperture has very compact and flat surface, thus being beneficial to the collection of light reflection signals. FIGS. 7-9 are scanning electron micrographs of Prussian blue nanoparticles distributed on polyethersulfone membranes at different scales. The surface of the polyethersulfone membrane is inlaid with prussian blue nano-particles. The prussian blue nanoparticles are proved to successfully maintain the finished morphological modification to the surface of the polyethersulfone membrane. This is the basis for the catalytic activity of artificial enzyme reaction membranes.
Example 4:
electron microscopy characterization of prussian blue filter membrane in example 2. FIG. 10 is an electron microscope of an untreated filter membrane. The glass fiber structure can effectively filter out red blood cells. In the fig. 11 and 12, the prussian blue nano-particles are completely adsorbed on the surface of the glass fiber in the filter membrane. When blood passes through the Prussian blue filter membrane, on one hand, blood cells are adhered to the filter membrane and do not continuously seep down to interfere the reaction. On the other hand, prussian blue nanoparticles in the filtering membrane can be eluted with blood plasma and participate in biochemical reaction in the reaction membrane. A color reaction occurs.
Example 5:
the test strip of the invention has selectivity to glucose. Using a control with saline/5% BSA without glucose and a sample containing 5.1mM blood glucose, as shown in FIG. 13, it was found that 5.1mM blood glucose gradually decreased in absorbance with the passage of time after the addition of the sample, indicating that a color reaction was occurring. In contrast, the control group showed substantially no change in absorbance over time, except for the coloration of Prussian blue originally present in the test strips due to wetting upon addition. The test strip showed good selectivity for glucose.
Example 6:
the test strip provided by the invention is used for testing the results of clinical specimens. Clinical whole blood samples were assigned values using a Roche glucometer. See fig. 14. Eight samples with uniformly distributed concentrations are taken, 5uL of the samples are sampled, the samples are dripped onto the test strip prepared in the example 1 for reflection absorbance analysis, the initial absorbance of the test strip and the light reflection value after the sample is added for 3min are recorded, and the ratio is calculated. The ratio is plotted against the corresponding sample concentration to form a correlation curve. It can be seen that the absorbance ratio measured by the artificial enzyme glucose test strip prepared in example 1 of the present invention has a good correlation with glucose in the whole blood sample for the blood glucose sample with the glucose concentration in the range of 2-25 mM. The reagent strip prepared by the invention has the potential of being clinically applied to glucose test. In consideration of the characteristics of relatively simple and convenient production mode, lower cost, temperature tolerance and the like of the artificial enzyme, the invention can optimize the production process of the existing clinical blood sugar test strip to a greater extent and reduce the cost.
Example 7:
the test paper strip provided by the invention tests the repeatability result. Clinical whole blood samples were assigned values using a Roche glucometer. See fig. 15. Two concentrations near the clinical decision significance and one concentration for the higher-ranked middle-ranked are taken, measured ten times, and the Coefficient of Variation (CV) is analyzed. The results show that the reagent strip adopting prussian blue nanoparticles for catalyzing color change has good repeatability, and CV is less than 5%. Meets the clinical use requirements.
Example 8:
the test strip of the invention accelerates the stability test result. And packaging the reagent strip, placing the packaged reagent strip in a 45 ℃ oven, periodically taking out the reagent strip, simultaneously measuring a fresh sample with a Roche glucometer, inspecting deviation, and further judging the acceleration stability of the reagent strip at high temperature. See fig. 16. From the results, the reagent strip adopting prussian blue nanoparticles to catalyze the color change can endure the environment of 45 ℃ for one month, and the measured value of the reagent strip is not obviously changed. The reagent strip of the invention has good high temperature resistance.
Claims (13)
1. A preparation method of a dry-type glucose test strip adopting Prussian blue nanoenzyme is characterized by comprising the following steps:
(1) adding prussian blue nano-enzyme, an adhesive, a film forming material, glucose oxidase and a peroxide substrate into a buffer solution to prepare a film forming treatment solution (I), adsorbing the film forming treatment solution (I) on a reaction film, and drying to obtain the reaction film containing prussian blue nano-enzyme;
or adding Prussian blue nano enzyme, adhesive and film forming material into buffer solution to prepare film forming treatment liquid (II), and adsorbing the film forming treatment liquid (II) on the blood filtering film; then adding glucose oxidase, a peroxide substrate, an adhesive and a film forming material into a buffer solution to prepare a film forming treatment solution (III), and adsorbing the film forming treatment solution (III) on a reaction film;
(3) and sequentially superposing the diffusion membrane, the blood filtration membrane and the reaction membrane from top to bottom to obtain the dry glucose test strip adopting the Prussian blue nanoenzyme.
2. The method for preparing a dry-type glucose test strip using prussian blue nanoenzyme according to claim 1, wherein the prussian blue nanoenzyme is prepared as follows in step (1):
(S1) adding PVP and FeCl3·6H2Dispersing O in ultrapure water, heating in water bath at 60 deg.C, stirring, and mixing, wherein PVP and FeCl are added3·6H2The molar ratio of O is 10: (0.05-0.15), wherein the content of PVP in the ultrapure water is 0.1-0.125 mmol/mL;
(S2) adding K4[Fe(CN)6]·3H2O is dispersed in ultrapure water at a concentration of 0.003 to 0.006mL/mL,
(S3) Using a two-channel micro-fluid injection pump, the K is injected4[Fe(CN)6]·3H2Dropwise adding the O solution to the PVP and FeCl prepared in the step (S1)3·6H2In the O solution, the dropping rate is 20-60mL/h, after the dropping is finished, stirring is continued under the condition of constant-temperature water bath at 60 ℃, after the reaction is finished, the power supply of the water bath kettle is closed, and the temperature of the reaction solution is slowly reduced to the room temperature;
(S4) filtering out unreacted PVP and ions by a Millpore TTF system through tangential flow, and finally filtering out aggregates by a 0.22 mu m filter membrane to obtain the Prussian blue nano enzyme.
3. The method for preparing the dry-type glucose test strip by using the prussian blue nanoenzyme according to claim 1, wherein in the step (1), the particle size of the prussian blue nanoenzyme is 50-150 nm; the concentration of the Prussian blue nano enzyme in the buffer solution is 0.5-1 mg/mL.
4. The method for preparing a dry glucose strip using prussian blue nanoenzyme as claimed in claim 1, wherein in step (1), the buffer is disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer or citric acid-sodium citrate buffer, and the pH of the buffer is 3.5-5.0.
5. The method for preparing the dry-type glucose test strip by using the Prussian blue nanoenzyme according to claim 1, wherein in the step (1), the membrane treatment solution (I), the membrane treatment solution (II) and the membrane treatment solution (III) contain surfactants, the surfactants are Triton X-100 or CHAPS, the volume fraction of the Triton X-100 is 0.02-0.1%, and the content of the CHAPS is 5-10 g/L.
6. The method for preparing the dry-type glucose test strip by using the prussian blue nanoenzyme according to claim 1, wherein in the step (1), the membrane treatment solution (I), the membrane treatment solution (II) and the membrane treatment solution (III) contain preservatives, the preservatives are one or a mixture of more of benzoic acid, sodium benzoate, sorbic acid, potassium sorbate and calcium propionate, and the content of the preservatives is 0.5-1 g/L.
7. The method for preparing a dry-type glucose test strip using prussian blue nanoenzyme according to claim 1, wherein the membrane treatment solution (i) and the membrane treatment solution (ii) contain BSA, and the concentration of BSA is 20 to 30 g/L.
8. The method for preparing the dry-type glucose test strip by using the prussian blue nanoenzyme according to claim 1, wherein the membrane treatment solution (I) and the membrane treatment solution (II) contain EDTA, and the concentration of EATA is 0.5-1.5 g/L.
9. The method for preparing the dry-type glucose test strip by using the prussian blue nanoenzyme according to claim 1, wherein in the step (1), the adhesive is hydroxypropyl cellulose or a methyl vinyl ether/maleic anhydride copolymer, and the mass fraction of the adhesive is 0.5-1%.
10. The method for preparing the dry-type glucose test strip by using the prussian blue nanoenzyme according to claim 1, wherein in the step (1), the film-forming material is hydroxypropyl cellulose, methyl vinyl ether/maleic anhydride copolymer, PEG, PVP or PVA, and the mass fraction of the film-forming material is 0.5-3%.
11. The method for preparing a dry-type glucose test strip using prussian blue nanoenzyme according to claim 1, wherein in step (1), the peroxide substrate is: MAOS +4-AAP, TOOS +4-AAP, CTA +4-AAP, MBTH + DMAB, MBTH + DCHBS or MBTH + ANS, wherein the content of the peroxide substrate in the buffer solution is 4-10 g/L, and the molar ratio of MAOS to 4-AAP, TOOS to 4-AAP, CTA to 4-AAP, MBTH to DMAB, MBTH to DCHBS, MBTH to ANS is (1-3): 1.
12. The method for preparing a dry-type glucose test strip by using prussian blue nanoenzyme according to claim 1, wherein in the step (1), the glucose oxidase concentration is as follows: 250- & lt 400 & gtKU/L.
13. The method for preparing a dry-type glucose test strip using prussian blue nanoenzyme according to claim 1, wherein the reaction membrane is a negatively charged nylon membrane or a polyethersulfone membrane with an asymmetric structure, the middle blood filtration membrane is a glass fiber membrane or a polyethersulfone membrane, and the diffusion membrane is a polyester membrane or a nylon membrane.
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| CN114533857B (en) * | 2022-02-22 | 2024-04-30 | 常州大学 | Antibacterial self-activated cascade reaction system HMPBNPs@GOx and preparation and application methods thereof |
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