CN113999889B

CN113999889B - A dry glucose test strip using Prussian blue nanozyme and preparation method thereof

Info

Publication number: CN113999889B
Application number: CN202111172687.3A
Authority: CN
Inventors: 童鎏; 顾宁; 苏恩本; 张宇; 陆颖琪; 宰云峰; 黄力
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2021-10-08
Filing date: 2021-10-08
Publication date: 2024-11-12
Anticipated expiration: 2041-10-08
Also published as: CN113999889A

Abstract

The invention discloses a dry glucose test strip using Prussian blue nanozyme and a preparation method thereof, and belongs to the field of clinical diagnosis technology. The invention adopts Prussian blue nanozyme to replace the natural horseradish peroxidase commonly used in dry chemical test strips. The artificially synthesized Prussian blue nanomaterial has the characteristic of catalyzing the reaction of peroxide and substrate. Compared with natural enzymes, artificial enzymes have the advantages of easy storage, convenient preparation, industrial mass production, low cost, good thermal stability, etc., and can meet the needs of clinical applications.

Description

Dry glucose test strip adopting Prussian blue nano enzyme and preparation method thereof

Technical Field

The invention relates to a dry glucose test strip adopting Prussian blue nano enzyme, belonging to the technical field of clinical diagnosis.

Background

Most of routine biochemical substances in clinic produce hydrogen peroxide after a series of enzyme catalytic reactions are set, so that the hydrogen peroxide can be subjected to color reaction with a substrate under the action of peroxidase, and quantitative tests on corresponding concentration indexes can be completed. In emergency department or bedside diagnosis, the sampling amount of the sample, the simplicity of the analysis process operation and the reporting time of the test result have higher requirements. The dry chemical method of drying the substances participating in the biochemical reaction on the membranes stacked layer by layer can meet the requirements of fingertip blood detection, is simple and convenient to operate, obtains the result 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 most widely used and most consumed enzyme in dry test strips. The industrial production of horseradish peroxidase usually takes natural horseradish as raw material, and the product is obtained by water extraction, first classification by ammonium sulfate, gel purification by calcium phosphate, classification by ethanol, second classification by ammonium sulfate and crystallization refining. Natural horseradish peroxidase has the following drawbacks: (1) The price is high, the market amount of horseradish peroxidase is huge, the market period is long depending on import, the storage condition is strict, and the logistics cost is high; (2) large differences between enzyme catalytic activity batches; (3) The protein structure of the enzyme has poor heat stability, the reagent strip can cause the reduction of enzyme performance in the drying process, and the processing technology of the reagent strip has strict requirements.

Therefore, it is desirable to provide a dry glucose test strip that does not use natural horseradish peroxidase.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing a dry glucose test strip adopting Prussian blue nano particles to replace natural horseradish peroxidase, so as to solve the problems of poor thermal stability, high price and severe preparation conditions of the dry glucose test strip using horseradish peroxidase in the prior art.

The invention also solves the technical problem of providing a preparation method of the dry 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 glucose test strip adopting Prussian blue nano-enzyme 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 treatment solution (I) on a reaction film, and drying to obtain the reaction film containing the Prussian blue nano enzyme;

Or Prussian blue nano enzyme, an adhesive and a film forming material are added into a buffer solution to prepare a film forming treatment solution (II), so that the film forming treatment solution (II) is adsorbed on a 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), so that the film forming treatment solution (III) is adsorbed on a reaction film;

(3) Sequentially superposing a diffusion film, a blood filtering film and a reaction film from top to bottom to obtain a dry glucose test strip adopting Prussian blue nano-enzyme;

wherein, the combination mode of the film treatment liquid and the film can be soaking or scribing by a scribing instrument; the film treated by the soaking method can be dried in an oven at 45 ℃ for 2 hours, and the film treated by the scribing method can be dried at 37 ℃ for 1 hour.

In the step (1), the preparation method of the Prussian blue nano-enzyme comprises the following steps:

(S1) dispersing PVP and FeCl ₃·6H₂ O in ultrapure water, heating in a water bath at 60 ℃ and stirring for mixing, wherein the molar ratio of PVP to FeCl ₃·6H₂ O is 10:0.05-10:0.15, and the content of PVP in the ultrapure water is 0.100-0.125mmol/mL;

(S2) dispersing K ₄[Fe(CN)₆]·3H ₂ O in ultrapure water according to 0.003-0.006mL/mL,

(S3) dropwise adding the K ₄[Fe(CN)₆]·3H ₂ O solution into the PVP and FeCl ₃·6H₂ O solution prepared in the step (S1) by using a double-channel micro-flow injection pump at the dropwise adding rate of 20-60mL/h, continuously stirring under the condition of a constant-temperature water bath at 60 ℃ after the dropwise adding, closing a power supply of the water bath kettle after the reaction is finished, and slowly cooling the water bath temperature to room temperature;

(S4) filtering unreacted PVP and ions by using a MILLPORE TTF system tangential flow, and finally filtering out the agglomerates by using a filter membrane with the diameter of 0.22 mu m to obtain the Prussian blue nano-enzyme.

In the step (1), the particle size of the Prussian blue nano-enzyme is 50-150nm, preferably 110nm; the concentration of the Prussian blue nano-enzyme in the buffer solution is 0.5-1mg/mL, preferably 0.8mg/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 surface active agents contained in the membrane treatment liquid (I), the membrane treatment liquid (II) and the membrane treatment liquid (III) are Triton X-100 or CHAPS, wherein the volume fraction of 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 contained in the membrane treatment liquid (I), the membrane treatment liquid (II) and the membrane treatment liquid (III) 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), EDTA is contained in the membrane treatment liquid (I) and the membrane treatment liquid (II), and the concentration of EATA is 0.5-1.5 g/L.

In the step (1), the adhesive is hydroxypropyl cellulose or 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 step (1), the peroxide substrate is: the content of the peroxide substrate in the buffer is 4-10 g/L, wherein 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 step (1), the glucose oxidase concentration is: 250-400KU/L.

Preferably, the reaction membrane is a negatively charged nylon membrane or an asymmetric polyether sulfone membrane, the middle blood filtering membrane is a glass fiber membrane or a polyether sulfone membrane, and the diffusion membrane is a polyester membrane or a nylon membrane.

The invention adopts Prussian blue nano enzyme to replace natural horseradish peroxidase commonly used in dry chemical test strips. The artificially synthesized Prussian blue nanomaterial has the characteristic of catalyzing the reaction of peroxide and a substrate. Compared with natural enzymes, the artificial enzymes are easy to store (only needed by storing in a solution state at normal temperature); the preparation is convenient, the industrial mass production can be realized, and the difference between batches is easy to control relative to the natural enzyme; the cost is low; the heat stability is good, and the process requirement in the processing process of the reagent strip is low. The common application of the reagent strip in the dry chemical test strip can greatly reduce the cost of the reagent strip.

The beneficial effects are that:

The invention discloses a dry glucose test paper adopting Prussian blue nano-enzyme and a preparation method thereof, wherein the Prussian blue nano-enzyme adopted in the invention is convenient to synthesize, the cost is far lower than that of natural enzyme, the cost of a reagent strip is reduced, the thermal stability is good, the service time of the reagent strip can be effectively prolonged, the requirement on the storage environment is relaxed, and the requirement on the production process of the reagent strip is also reduced. At present, the dry chemical method reagent strips applied to clinic in emergency department mainly depend on import, and the application of Prussian blue in the related field can help to promote the competitiveness of domestic reagents in the related field.

The successful application of artificial enzymes in diagnostic reagents has great market and clinical value, and the current mainstream nano-enzyme application research is focused on the application of the artificial enzymes in a solution system. The invention relates to a dry test strip which is successfully prepared for the first time and can be applied to detecting glucose by peroxide catalysis in the process of diagnosing reaction by adopting artificial nano enzyme.

Drawings

FIG. 1 is a flow chart of the preparation of a glucose test strip (example 1) based on Prussian blue nanoenzyme.

Fig. 2 is a flow chart of the preparation of a glucose test strip (example 2) based on prussian blue nanoenzyme.

FIG. 3 is a scanning electron microscope image of an asymmetric polyethersulfone film used as a reaction layer.

FIG. 4 is a scanning electron microscope image of an asymmetric polyethersulfone film used as a reaction layer.

Fig. 5 is a scanning electron microscope image of prussian blue nanoparticles.

Fig. 6 is a scanning electron microscope image of prussian blue nanoparticles.

Fig. 7 distribution of Prussian blue nanoparticles on the surface of a reaction membrane (polyethersulfone membrane).

Fig. 8 distribution of Prussian blue nanoparticles on the surface of a reaction membrane (polyethersulfone membrane).

Fig. 9 distribution of prussian blue nanoparticles on the surface of a reaction membrane (polyethersulfone membrane).

FIG. 10 shows a scanning electron microscope image of a glass fiber filter membrane and the distribution of Prussian blue nano-enzyme on the glass fiber filter membrane.

FIG. 11 shows a scanning electron microscope image of a glass fiber filter membrane and the distribution of Prussian blue nanoenzyme on the glass fiber filter membrane.

FIG. 12 shows a scanning electron microscope image of a glass fiber filter membrane and the distribution of Prussian blue nanoenzyme on the glass fiber filter membrane.

FIG. 13 reagent strip is a glucose selectivity test.

The test strips of FIG. 14 were validated against clinical specimens.

Reproducibility of the test strips of fig. 15.

Accelerated stability validation of the FIG. 16 strip.

Detailed Description

The following detailed description of the present invention is given by way of specific examples, which are given for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Preparation of Prussian blue nanoenzyme: 1.11g (10 mmol) of PVP (K-30) was weighed and dispersed in 80mL of ultrapure water by ultrasound, 27.03mg (0.1 mmol) of FeCl ₃·6H ₂ O was weighed after uniform dispersion and added to PVP aqueous solution by ultrasound for 10min, and then transferred into a 250mL three-necked flask in a 60℃thermostat water bath and stirred at a rate of 1200rpm for 30min. In addition, 42.24mg (0.1 mmol) of K ₄[Fe(CN)₆]·3H ₂ O was weighed and dissolved in 20mL of ultrapure water, and after being dispersed uniformly, it was transferred to a 20mL syringe, and fixed on a WZS-50F6 double-micro-flow injection pump, and the dropping rate was selected to be 40mL/h. After the dripping is finished, stirring is continued for 1h 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 slowly cooled to the room temperature. After completion of the reaction unreacted PVP and ions were filtered off with MILLPORE TTF systems tangential flow (MWCO: 50 kDa), and finally the agglomerates were filtered off with a 0.22 μm filter.

Example 1:

The preparation method of glucose test paper adopting Prussian blue artificial enzyme. In the example, prussian blue nano-particles, glucose oxidase and a substrate are on the same reaction film; see FIG. 1

(1) Preparing a membrane treatment solution (1L) containing Prussian blue nano enzyme:

Prussian blue 0.8g/L

Citric acid-sodium citrate buffer (ph=3.5-4.5)

Citric acid 7g/L

Sodium citrate 19.6g/L

BSA 25g/L

EDTA 1g/L

Sodium benzoate 1g/L

Triton X-100.1% (volume fraction)

10% Gantrez 5% (volume fraction)

Glucose oxidase 250-400KU/L

4-Aminoantipyrine (4-AAP) 4.2g/L

N-Ethyl-N (2-hydroxy-3-propylsulfo) -3, 5-dimethylaniline (MAOS) 4.6g/L

(2) Treating the reaction membrane by using a membrane treatment liquid, and fixing Prussian blue nano particles and other necessary reactants on the membrane; either a dipping mode or a scribing mode can be adopted. The parameter of scribing is preferably 5.5 mu L/cm, scribing speed: 50mm/s.

(3) The film treated with the above was put on a shelf of a suitable size and dried at 37℃for 1 hour.

(4) And cutting the dried reaction film into a proper size, and assembling the proper size with the blood filtering film and the diffusion film into a test strip.

Example 2:

The preparation method of glucose test paper adopting Prussian blue artificial enzyme. The materials and reagents used in this example are the same as those in example 1, except that the Prussian blue nanoparticles of this example are adsorbed on the filtration membrane alone, and the glucose oxidase and the substrate are on a single reaction membrane; see fig. 2. The filtering membrane containing Prussian blue nano particles can be independently prepared and stored, has good stability, and can be applied to any biochemical index requiring peroxide catalysis as a filtering membrane with peroxide activity.

Example 3:

Electron microscopy characterization of Prussian blue artificial enzyme test paper reaction membrane in example 1. Fig. 3 to 6 are scanning electron microscope images (fig. 5 and 6) of a blank asymmetric polyethersulfone film (fig. 3 and 4) and prussian blue nanoparticles. From the figure, the Prussian blue nano-particles have the particle size near 110nm, uniform particle size distribution and complete cube structure, and have perfect morphology, which is the basis of nano-enzyme activity. The polyethersulfone membrane fiber ropes are clear, and the pore size distribution is distributed in a gradient from 200um in diameter on one side to 10um on the other side. The asymmetric structure facilitates the liquid sample from the upper layer with large aperture to infiltrate the membrane material, and the surface of the lower layer with small aperture is very compact and flat, which is beneficial to the collection of light reflection signals. Fig. 7-9 are scanning electron microscope images of Prussian blue nanoparticles distributed on a polyethersulfone membrane at different scales. The Prussian blue nano particles are embedded on the surface of the polyethersulfone membrane. The Prussian blue nano particles successfully maintain the finished morphology modification to the surface of the polyether sulfone membrane. This is the basis for the catalytic activity of the artificial enzyme reaction membrane.

Example 4:

Electron microscopy characterization of Prussian blue filtration membranes in example 2. FIG. 10 is an electron microscope of an untreated filtration membrane. The glass fiber structure can effectively filter red blood cells. Fig. 11 and 12 show that 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 filtering membrane, on one hand, blood cells can be adhered in the filtering membrane and do not continuously infiltrate downwards, so that the reaction is interfered. On the other hand, prussian blue nano particles in the filtering membrane can be eluted along with the blood plasma to participate in biochemical reaction in the reaction membrane. A color reaction occurs.

Example 5:

the test strip provided by the invention has selectivity to glucose. Comparison of a sample containing 5.1mM blood glucose with physiological saline without glucose/5% BSA, see FIG. 13, shows that the absorbance of 5.1mM blood glucose gradually decreases over time after the sample is added, indicating that a color reaction is occurring. The absorbance of the control group was not substantially changed with time except for the Prussian blue color originally present in the reagent strip due to wetting immediately after addition. The reagent strip has good selectivity to glucose.

Example 6:

The test strip provided by the invention tests the results of clinical specimens. Clinical whole blood specimens were assigned with a rogowski glucometer. See fig. 14. Eight samples with evenly distributed concentration are taken, 5uL of samples are sampled, the samples are dripped on the test strip prepared in the example 1 for reflection absorbance analysis, and the initial absorbance of the test strip and the light reflection value after 3 minutes of sample addition are recorded and are used as ratios. The ratio was plotted against the corresponding specimen concentration. 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 respect to glucose in whole blood samples for blood glucose samples having a glucose concentration in the range of 2 to 25 mM. The reagent strip prepared by the invention has potential of clinical application to glucose test. Considering the characteristics of relatively simple production mode, lower cost, temperature tolerance and the like of the artificial enzyme, the invention can greatly optimize the production process of the existing clinical blood glucose test strip and reduce the cost.

Example 7:

The test strip of the invention tests repetitive results. Clinical whole blood specimens were assigned with a rogowski glucometer. See fig. 15. Ten measurements were taken of two concentrations near the clinical decision significance and one concentration of middle and high duty, and analysis of Coefficient of Variation (CV) was performed. From the results, the reagent strip adopting Prussian blue nano particles to catalyze the color change has good repeatability, and CV is less than 5%. Meets the clinical use requirement.

Example 8:

The test strip of the invention accelerates the stability test result. And (3) packaging the reagent strips, placing the packaged reagent strips in a 45 ℃ oven, periodically taking out the reagent strips and measuring fresh samples simultaneously with the Rogowski glucometer, examining the deviation, and further judging the acceleration stability of the reagent strips at high temperature. See fig. 16. From the results, the test result shows that the reagent strip adopting Prussian blue nano particles for catalytic discoloration can withstand the environment of 45 ℃ for one month, and the measured value is not changed obviously. The reagent strip has good high temperature tolerance.

Claims

1. A method for preparing a dry glucose test strip using Prussian blue nanozyme, characterized in that:

(1) Adding Prussian blue nanozyme, adhesive, glucose oxidase, and peroxide substrate into a buffer to prepare a membrane treatment solution (I), allowing the membrane treatment solution (I) to be adsorbed on a reaction membrane, drying, and obtaining a reaction membrane containing Prussian blue nanozyme, and stacking a diffusion membrane, a blood filter membrane, and a reaction membrane in sequence from top to bottom to obtain a dry glucose test strip using Prussian blue nanozyme;

Or (2) adding Prussian blue nanozyme and adhesive into a buffer to prepare a membrane treatment solution (II), and allowing the membrane treatment solution (II) to be adsorbed onto the blood filter membrane; then adding glucose oxidase, peroxide substrate, and adhesive into a buffer to prepare a membrane treatment solution (III), and allowing the membrane treatment solution (III) to be adsorbed onto the reaction membrane, and stacking the diffusion membrane, the blood filter membrane, and the reaction membrane in sequence from top to bottom to obtain a dry glucose test strip using Prussian blue nanozyme;

In (1) and (2), the preparation method of the Prussian blue nanozyme is as follows:

(S1) dispersing PVP and FeCl ₃ •6H ₂ O in ultrapure water, heating in a water bath at 60° C. and stirring to mix, wherein the molar ratio of PVP to FeCl ₃ •6H ₂ O is 10:(0.05-0.15), and the content of PVP in ultrapure water is 0.1-0.125 mmol/mL;

(S2) Disperse K ₄ [Fe(CN) ₆ ]•3H ₂ O in ultrapure water at 0.003-0.006 mmol/mL.

(S3) using a dual-channel microfluidic syringe pump, dropwise add the K ₄ [Fe(CN) ₆ ]•3H ₂ O solution to the PVP and FeCl ₃ •6H ₂ O solutions prepared in step (S1) at a dropwise rate of 20-60 mL/h. After the dropwise addition is completed, stirring is continued in a 60° C. constant temperature water bath. After the reaction is completed, the water bath is turned off and the temperature of the reaction solution is slowly lowered to room temperature.

(S4) Using the MILLPORE TTF system to filter out the unreacted PVP and ions by tangential flow, and finally filtering out the aggregates with a 0.22 μm filter membrane to obtain the Prussian blue nanozyme;

In (1) and (2), the particle size of the Prussian blue nanozyme is 50-150 nm; the concentration of the Prussian blue nanozyme in the buffer is 0.5-1 mg/mL;

In (1) and (2), the buffer is a citric acid-sodium citrate buffer, and the pH of the buffer is 3.5-5.0;

In (1) and (2), the membrane treatment solution (I), membrane treatment solution (II) and membrane treatment solution (III) further contain a surfactant, wherein the surfactant is Triton X-100, wherein the volume fraction of the Triton X-100 is 0.02% to 0.1%;

In (1) and (2), the membrane treatment liquid (I), membrane treatment liquid (II) and membrane treatment liquid (III) further contain a preservative, wherein the preservative is sodium benzoate and the content of the preservative is 0.5-1 g/L;

The membrane treatment solution (I) and the membrane treatment solution (II) further contain BSA, and the concentration of the BSA is 20-30 g/L;

The membrane treatment solution (I) and the membrane treatment solution (II) further contain EDTA, and the concentration of the EDTA is 0.5-1.5 g/L;

In (1) and (2), the adhesive is methyl vinyl ether-maleic anhydride copolymer, and the volume fraction of the adhesive is 0.5% to 1%;

In (1) and (2), the peroxide substrate is: MAOS+4-AAP, the content of the peroxide substrate in the buffer is 4-10 g/L, wherein the molar ratio of MAOS to 4-AAP is (1-3):1;

In (1) and (2), the glucose oxidase concentration is 250-400 KU/L;

In (1) and (2), the reaction membrane is a polyethersulfone membrane with an asymmetric structure, the blood filter membrane is a glass fiber membrane, and the diffusion membrane is a polyester membrane.