CN113686936A - Preparation method of nano sensing slurry for printing sucrose detection chip - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
The invention belongs to the technical field of sucrose detection chips, and relates to a preparation method of nano sensing slurry for sucrose detection chip printing. The synthesis steps of the nano sensing slurry are as follows: preparing and synthesizing a cerium oxide nanorod solution; preparing a Cu-Co Prussian blue analogue synthetic solution C, D; and (3) synthesizing a Cu-Co Prussian blue analogue in a cerium oxide nanorod suspension. The composite material is mixed with the conductive carbon printing ink according to a certain proportion through centrifugation and drying, the mixed slurry is enabled to be in a printable state by adding ethanol, and then the biosensor chip is prepared by means of a screen printing technology. The slurry synthesis method is simple, the process is controllable, the commercialization is easy to realize, the working potential can be controlled at a low potential of-0.2V, the detection of the hydrogen peroxide product has a very wide linear range, and the sucrose concentration detection requirements of different markets are met.
Description
Technical Field
The invention belongs to the technical field of sucrose detection chips, and relates to a preparation method of nano sensing slurry for sucrose detection chip printing.
Background
Sucrose is a common disaccharide formed by the dehydrocondensation of a molecule of the hemiacetal hydroxyl group of glucose and a molecule of the hemiacetal hydroxyl group of fructose. Sucrose is commonly present in roots, stems, leaves and fruits of the plant kingdom, is particularly rich in sugarcane, beet and maple juice, is a main component of edible sugar, and is also an important food and sweet seasoning. Besides being used as a sweetening agent, the sucrose also serves as a nutrient for yeast, and during the fermentation process, the sucrose content is too high to inhibit the fermentation reaction, and conversely, the sucrose content is too low to cause the death of microorganisms, so that the fermentation effect is not achieved. Therefore, the effective detection equipment and method can accurately detect the concentration of the sucrose, and have great significance for food analysis, fermentation industry and human health.
At present, common sucrose detection methods in the market comprise a chromatographic method, a spectrophotometric method, a qualitative chemical method, a dioptric method, an optical rotation method and the like, and the methods either need expensive and heavy equipment or have the disadvantages of time consumption, complex operation and the like, and cannot meet the requirements of quick and accurate detection at the same time. Electrochemical biosensing is a novel technology, and target molecules and reaction signals thereof are converted into electric signals such as capacitance, current, potential, conductivity and the like through the specific recognition effect among the biomolecules, so that the target analytes are rapidly and accurately detected.
Disclosure of Invention
The invention provides a novel preparation method of nano sensing slurry for printing a sucrose detection chip, aiming at the problems in the traditional sucrose detection. The invention aims to prepare nano sensing slurry mixed by cerium oxide, Cu-Co Prussian blue analogue composite material and conductive carbon printing ink, and the nano sensing slurry can be used for printing electrodes.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
a preparation method of nano sensing slurry for printing a sucrose detection chip comprises the following specific steps:
(1) preparing a cerium oxide nano material: dissolving cerium chloride heptahydrate serving as a cerium source in deionized water to prepare a solution A; polyvinylpyrrolidone (PVP) is used as a surfactant and dissolved in deionized water to prepare a solution B. Mixing and stirring A, B solution until the solution is clear and transparent, adding ammonia water to control pH, transferring the suspension into a hydrothermal kettle, taking out the synthesized nano cerium oxide suspension at a high temperature for a period of time, centrifuging and cleaning to obtain nano cerium oxide precipitate, and putting the material into an oven for a period of time to obtain cerium oxide powder;
(2) preparation of Cu-Co Prussian blue analogue synthetic solution C, D: preparing Prussian blue analogue synthetic liquid C which is a mixed solution of potassium hexacyanocobaltate (III) and deionized water; the Prussian blue analogue synthetic liquid D is prepared by mixing copper chloride dihydrate with deionized water, and sodium citrate is contained in the synthetic liquid D as a surfactant.
(3) Synthesis of Cu-Co Prussian blue analogue in cerium oxide nanorod suspension: and (2) dispersing the cerium oxide powder synthesized in the step (1) in a beaker of deionized water, placing the beaker in a water bath kettle at a certain temperature, taking out the synthetic liquid C, D prepared in the step (2) with the same volume, filling the synthetic liquid C, D in an injection pump, dropping the synthetic liquid into the beaker at the same speed, and synthesizing the nano-composite solution at a micro-speed.
(4) Preparing nano sensing slurry: and (4) centrifuging and drying the nano composite solution composite material synthesized in the step (3), mixing the nano composite solution composite material with conductive carbon printing ink according to a certain proportion to obtain mud-like slurry, and adding ethanol to enable the mixed slurry to reach the viscosity for printing.
The typical steps of the nano sensing slurry prepared by the steps are as follows:
preparation of chip electrode: and (4) printing a substrate electrode on a substrate material by a screen printing technology, taking silver chloride ink as printing reference electrode ink, taking carbon ink as printing counter electrode ink, and taking the nano sensing slurry prepared in the step (4) as printing working electrode ink. The base material may be one of PVC, PET, alumina, ceramic, gold, silver or platinum.
Preparation of enzyme solution: mixing sucrose hydrolase, mutarotase and glucose oxidase with a solvent respectively, adding a proper amount of a cross-linking agent into the prepared enzyme solution, and transferring the enzyme solution into a centrifugal tube for later use respectively; the solvent is one of deionized water and phosphate buffer; the cross-linking agent is one of bis-epoxy hexane, bis-imine methyl ester and glutaraldehyde; the enzyme solution obtained by mixing has a sucrose hydrolase concentration of 0.1-10u/μ L, a glucose mutarotase concentration of 0.1-10u/μ L, and a glucose oxidase concentration of 0.1-10u/μ L.
Preparing a sucrose biosensor chip: wetting the surface of a working electrode of the chip with the solution for a period of time, wiping off the redundant solution with a paper towel, dripping the three enzyme solutions on the surface of the working electrode in sequence, and transferring the working electrode into a refrigerator for storage and standby after the surface of the chip is completely dried. The solution is one of deionized water and phosphate buffer; the chip storage temperature is 2-8 ℃.
As a further improvement of the invention, in the step (1), the ion concentration range of the solution A is 0.001-0.1M, the ion concentration range of the solution B is 0.001-0.01M, the pH value of the mixed solution after adding ammonia water is 7-12, the synthesis temperature is 90-180 ℃, the synthesis time is 8-24h, the centrifugation rate is 6000-12000 r/min, the centrifugation time is 5-20min, the drying temperature is 50-80 ℃, and the drying time is 6-12 h.
As a further improvement of the invention, in the step (2), the concentration of the anion in the synthetic liquid C is 0.005-0.03M, the concentration of the cation in the synthetic liquid D is 0.005-0.03M, and the concentration of the sodium citrate in the synthetic liquid D is 0.01-0.05M.
As a further improvement of the invention, in the step (3), the cerium oxide nanorods are uniformly dispersed in deionized water, the concentration of the cerium oxide is controlled to be 0.001-0.01 g/mL, the speed of dripping the Prussian blue analogue synthetic solution C, D into the beaker is controlled to be 600 muL/min at 300-55 ℃, and the aging time is 6-24 h.
As a further improvement of the invention, in the step (4), the centrifugation rate is 6000-12000 r/min, the centrifugation time is 5-20min, the drying temperature is 50-80 ℃, the drying time is 6-12h, the mass ratio of the nano composite material to the carbon ink is 1:1-1:50, the drying temperature is 30-80 ℃, the drying time is 0.5-1h, a VT-04E viscosity detector can be used for measuring the viscosity of the printing slurry, and the printing viscosity is controlled to be 50-150 dpa.S.
The screen printing chip has good electrochemical performance, and keeps the characteristics of flexible design, low cost, simple structure, good repeatability, easy integration and miniaturization, thereby being widely concerned. The core of the silk-screen printing chip is slurry, and the nano electro-catalysis material has extremely high sensing performance, so that the Prussian blue analogue (Cu-Co PBA) with high electro-catalysis performance is used as an electrode material, the nano structure of the Prussian blue analogue (Cu-Co PBA) is accurately controlled to be compounded with the cerium oxide nano rod, the enzyme immobilization stability is improved, the good wide-linear-range current response can be realized on the product hydrogen peroxide, the working potential of the sensing chip can be controlled to be-0.2V, the sensing chip has good anti-interference performance, and the sucrose concentration detection requirements of different markets are met.
Generally, materials with high electric points have unique advantages for adsorbing proteins with low isoelectric points including enzymes, wherein sucrose hydrolase (INV) IEP used in a sucrose chip is 5.0, Glucose Oxidase (GOD) IEP is 4.2, and cerium oxide (IEP-9) is used as a metal oxide material with high IEP and can be used as an advantageous condition for fixing various enzymes. Therefore, the nano sensing slurry has high conductivity and high catalytic performance, can effectively realize enzyme fixation, has certain advantages in the conditions of sucrose biosensors needing fixation of various enzymes for detection, has good anti-interference performance due to the existence of Cu-Co PBA, can control the working potential to be low potential of-0.2V, and can realize extremely wide linear range of detection of the product hydrogen peroxide, so that the linear range of sucrose detection is greatly improved, and the sucrose concentration detection requirements of different markets are met. The slurry has simple preparation process and low cost, is suitable for large-scale production and application, and has good market application prospect.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the nano printing sensing slurry synthesized by the invention has unique advantages for sucrose detection, wherein the cerium oxide material has high isoelectric point (IEP-9.0), and has unique advantages for adsorbing proteins with low isoelectric point including enzymes, and the cerium oxide material has good advantages for fixing three enzymes due to sucrose hydrolase (INV) IEP =5.0 and Glucose Oxidase (GOD) IEP = 4.2; meanwhile, the synthesized cerium oxide material belongs to a nanometer size, the nanometer material does not have a wide analyte detection range, and the problems of species interference, low signal output and lag signal response can be effectively solved, moreover, due to the existence of the Cu-Co PBA nanometer material, the working potential can be controlled at a low potential of-0.2V, the cerium oxide material has good anti-interference performance, the extremely wide linear range of the detection of the product hydrogen peroxide can be realized, the linear range of the detection of the sucrose is greatly improved, and the combination of the cerium oxide material and the sucrose can meet the concentration detection requirement of the fermentation market.
2. The invention relates to a preparation process of a sensor chip based on a silk-screen printing technology and a sucrose three-enzyme system, and a current enzyme biosensor is the most mature biosensor for industrial application. Mainly based on detecting the electroactive substances in biological recognition or chemical reaction, the potential of a fixed working electrode provides driving force for electroactive electron transfer reaction, the change of current along with time is detected, and the current directly measures the speed of the electron transfer reaction and reflects the speed of biomolecule recognition. The method has the characteristics of good stability, high biological analysis precision, low analysis cost, wide application range and high analysis speed.
Drawings
FIG. 1 is a scanning electron micrograph of cerium oxide synthesized by a hydrothermal method according to example 1.
FIG. 2 is an X-ray diffraction pattern of cerium oxide synthesized by a hydrothermal method in example 1.
FIG. 3 shows the Cu-Co PBA material synthesized in example 1 at different potentials for the final detection product H2O2Timing current diagram of (2).
FIG. 4 shows the Cu-Co PBA materials synthesized in example 1 after driving H with the same concentration2O2Cyclic voltammogram of (a).
FIG. 5 is the Cu-Co PBA versus final detection product H for-0.2V operating potential for example 12O2Linear range chronoamperometry.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
If not specifically stated, the nano-sensing slurry for printing the sucrose detection chip in the following examples is prepared by first synthesizing a cerium oxide material with a nanorod structure by a simple hydrothermal method, using cerium chloride heptahydrate as a cerium source, polyvinylpyrrolidone (PVP) as a surfactant, and ammonia water as a precipitant to adjust pH. Taking out a certain amount of cerium oxide powder to be dispersed in a beaker filled with deionized water, placing the beaker in a water bath kettle at a certain temperature, taking out the synthetic solution A, B of the Cu-Co Prussian blue analogue out of the beaker with the same volume, placing the synthetic solution in an injection pump to be dropped into the beaker at the same speed, synthesizing a nano composite solution at a low speed, and mixing the obtained cerium oxide material and carbon ink according to a certain mass ratio to form the nano sensing slurry capable of drawing wires and having printing characteristics.
The preparation process of the chip comprises the following steps: the prepared mixed ink is used as the ink for printing a working electrode, silver chloride slurry is used as the ink for printing a reference electrode, carbon ink is used as the ink for printing a counter electrode, and the three-electrode biosensor chip is printed on a PVC (polyvinyl chloride) bottom plate material by utilizing a screen printing technology. Mixing the three enzymes with PBS according to a certain proportion, adding 25% (v/v) glutaraldehyde for cross-linking to obtain enzyme solution, sequentially carrying out enzyme solidification on the working electrode of the biosensor chip, drying the surface of the electrode to obtain the sucrose biosensor chip, and storing in a refrigerator at-4 deg.C for later use.
Example 1
A preparation method of nano sensing slurry for printing a sucrose detection chip comprises the following steps:
(1) weighing 0.037258g (0.1 mmol) of cerium chloride heptahydrate, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution A, weighing 2.9g (0.1 mmol) of polyvinylpyrrolidone, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution B, mixing the solution A, B, continuously stirring for 30min, adding about 3mL of ammonia water into the mixed solution to adjust the pH to =7, changing the solution into a pale red flocculent suspension, continuously stirring for 30min, transferring the suspension into a hydrothermal kettle, controlling the temperature of the hydrothermal kettle at 90 ℃, heating for 24h, cooling the product to room temperature after the hydrothermal reaction is finished, taking out the product, adding ethanol, centrifuging for three times, adding deionized water, cleaning for three times, setting the rotating speed of a centrifuge to be 6000r/min, and setting the centrifuging time to be 20 min. And after the reaction is finished, pouring out waste liquid, putting the centrifugal precipitate into an oven, heating at 50 ℃ for 12 hours, taking out the centrifugal precipitate after reaching the heating time, and grinding to obtain cerium oxide powder. The microstructure of cerium oxide powder is shown in figure 1.
(2) Preparing a Prussian blue analogue synthetic solution C which is 100mL of a mixed solution of potassium hexacyanocobaltate (III) and deionized water, wherein the anion concentration is 0.005M; the prepared Prussian blue analogue synthetic solution D is 100mL of mixed solution of copper chloride dihydrate and deionized water, and sodium citrate serving as a surfactant is added into the synthetic solution D, wherein the cation concentration is 0.005M, and the sodium citrate concentration is 0.01M. Each was poured into a 100mL syringe and mounted on a syringe pump for use.
(3) And (3) taking out 20mL of 0.001g/mL cerium oxide nanorod (the powder obtained in the step (1) is added with water and uniformly mixed) solution which is uniformly dispersed in deionized water, putting the beaker into a water bath kettle at the temperature of 25 ℃, and dripping the solution into the beaker by using a syringe pump at the speed of 300 mu L/min to synthesize the nano-composite suspension at a low speed. And (3) cleaning the suspension with deionized water, centrifuging at 6000r/min for 20min, repeating the steps for three times, and drying the suspension at the drying temperature of 50 ℃ for 12h to obtain the powder sample material.
(4) And (3) weighing 1g of the powder sample material obtained in the step (3), mixing the powder sample material with 1g of carbon ink in a mass ratio of (1: 1), adding 0.5mL of ethanol, and stirring in a single direction to enable the ink to meet the printing requirement.
(5) And (3) printing a substrate electrode on the PVC substrate material by a screen printing technology, taking silver chloride ink as printing reference electrode ink, taking carbon ink as printing counter electrode ink, and taking the mixed ink prepared in the step (4) as printing working electrode ink.
(6) 25U of sucrose hydrolase, 25U of mutarotase and 250U of glucose oxidase are respectively dissolved in 100 mu L of phosphate buffer, 1 mu L of 25% (v/v) glutaraldehyde is respectively added into the enzyme solution for crosslinking, and the mixture is transferred into a centrifugal tube for later use.
(7) Wetting the surface of a working electrode of the biosensor chip with a phosphate buffer solution, standing for 3min, dot-absorbing with a piece of dust-free paper, fixing 5 mul of enzyme solution on the surface of the working electrode one by one according to the sequence of sucrose hydrolase, mutarotase and glucose oxidase, and transferring the sucrose biosensor chip into a refrigerator for storage and standby after the surface of the working electrode is completely dried.
By chronoamperometric current testing: the biosensor chip obtained in this example had a high sensitivity of detecting sucrose and glucose, which was 58.12. mu.A.mM-1 ·cm -2The detection limit is as low as 0.1 mu M, and the detection range is 0-1.3M. After the test, the modified electrode is placed in PBS buffer solution with pH of 7.0 for one week at 0 ℃, and the response signal is basically unchanged; after one month, the response signal was 98% of the initial signal; after three months, the response signal was still 95% of the initial signal, indicating that the stability of the slurry was good.
As shown in the SEM representation of FIG. 1, the cerium oxide nanorods synthesized in the step (1) have stable structures and uniform distribution, which shows that the quality of the synthesized material is stable and controllable, and the industrial sensing slurry can be conveniently prepared.
As can be seen from the XRD representation in FIG. 2, the cerium oxide nanorod synthesized in step (1) has high coincidence degree with the diffraction peak of the standard card, and the peak shape is sharp, which indicates that the synthesized cerium oxide nanomaterial has high purity and good crystallinity.
As can be seen from the CV electrochemical characterization of FIG. 3, the oxidation-reduction peaks all show obvious changes with the increase of the concentration of the hydrogen peroxide, which indicates that the material has high responsiveness to hydrogen peroxide and can satisfy the electrochemical detection of sucrose.
As can be seen from the I-t electrochemical characterization of FIG. 4, the sensitivity of the Cu-Co PBA to hydrogen peroxide changes along with the change of the set potential, firstly, the positive and negative potentials are considered, the potential of-0.1V is found to have higher sensitivity compared with the potential of +0.1V, then the influence of the negative potential on the sensitivity is continuously considered, when the potential is increased from-0.1V to-0.3V, the sensitivity is obviously increased, and the potential is controlled to be-0.2V in consideration of the poor anti-interference performance brought by the high potential and the requirement on the high sensitivity.
As can be seen from the I-t electrochemical characterization of FIG. 5, the Cu-Co PBA material shows an extremely wide linear range of 0-1.8M for hydrogen peroxide at a potential of-0.2V, and the material is very advantageous for increasing the linear range of sucrose detection.
Example 2
A preparation method of nano sensing slurry for printing a sucrose detection chip comprises the following steps:
(1) weighing 0.1864g (0.5 mmol) of cerium chloride heptahydrate, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution A, weighing 5.8g (0.2 mmol) of polyvinylpyrrolidone, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution B, mixing the solution A, B, continuously stirring for 30min, adding 4mL of ammonia water into the mixed solution to adjust the pH to =8, changing the solution into a pale red flocculent suspension, continuously stirring for 30min, transferring the suspension into a hydrothermal kettle, controlling the temperature of an oven at 110 ℃, heating for 20h, cooling to room temperature, taking out, transferring into a 50mL centrifuge tube, adding water and ethanol, centrifuging and cleaning for three times, setting the rotation speed of the centrifuge to 7000r/min, and the centrifugation time to 16 min. And after the completion, pouring the waste liquid, putting the material into an oven, heating at 55 ℃ for 10h, taking out the material after reaching the heating time, and grinding the material to obtain cerium oxide powder. The micro-topography is not significantly different from that of figure 1.
(2) Preparing a Prussian blue analogue synthetic solution C which is 100mL of a mixed solution of potassium hexacyanocobaltate (III) and deionized water, wherein the anion concentration is 0.01M; the prepared Prussian blue analogue synthetic solution D is 100mL of mixed solution of copper chloride dihydrate and deionized water, and sodium citrate serving as a surfactant is added into the synthetic solution D, wherein the cation concentration is 0.01M, and the sodium citrate concentration is 0.02M. Each was poured into a 100mL syringe and mounted on a syringe pump for use.
(3) And (3) taking out 25mL of 0.002g/mL cerium oxide nanorod (the powder obtained in the step (1) is added with water and uniformly mixed) solution which is uniformly dispersed in deionized water, putting the beaker into a water bath kettle at the temperature of 30 ℃, and dropping the solution into the beaker by using a syringe pump at the speed of 400 mu L/min to synthesize the nano-composite suspension at a low speed. And (3) cleaning the suspension with deionized water, centrifuging at 7000r/min for 16min, repeating the steps for three times, and drying the suspension at the drying temperature of 55 ℃ for 10h to obtain the powder sample material.
(4) And (3) weighing 1g of the powder sample material obtained in the step (3), mixing with 10g of carbon ink in a mass ratio of (1: 10), adding 0.8mL of ethanol, and stirring in a single direction to enable the ink to meet the printing requirement.
(5) And (3) printing a substrate electrode on the PVC substrate material by a screen printing technology, taking silver chloride ink as printing reference electrode ink, taking carbon ink as printing counter electrode ink, and taking the mixed ink prepared in the step (4) as printing working electrode ink.
(6) 30U of sucrose hydrolase, 30U of mutarotase and 300U of glucose oxidase are respectively dissolved in 100 mu L of phosphate buffer, 1 mu L of 25% (v/v) glutaraldehyde is respectively added into the enzyme solution for crosslinking, and the solution is transferred into a centrifugal tube for later use.
(7) Wetting the surface of a working electrode of the biosensor chip with a phosphate buffer solution, standing for 3min, dot-absorbing with a piece of dust-free paper, fixing 5 mul of enzyme solution on the surface of the working electrode one by one according to the sequence of sucrose hydrolase, mutarotase and glucose oxidase, and transferring the sucrose biosensor chip into a refrigerator for storage and standby after the surface of the working electrode is completely dried.
Through the timing ampere current test, the following results are obtained: the biosensor chip obtained in this example has high detection sensitivity to sucrose and glucose, which is 56.24. mu.A.. multidot.mM -1 ·cm -2The detection limit is as low as 0.2 mu M, and the detection range is 0-1.3M. After the experiment, the modified electrode is placed in PBS buffer solution with pH of 7.0 for one week at the temperature of 0 ℃, and the response signal is basically unchanged; after one month, the response signal was 97% of the initial signal; after three months, the response signal was still 93% of the initial signal, indicating that the stability of the slurry was good.
Example 3
A preparation method of nano sensing slurry for printing a sucrose detection chip comprises the following steps:
(1) weighing 0.74516g (2 mmol) of cerous chloride heptahydrate, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution A, weighing 14.5g (0.5 mmol) of polyvinylpyrrolidone, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution B, mixing the solution A, B, continuously stirring for 30min, adding 5mL of ammonia water into the mixed solution to enable the pH to be =9, changing the solution into a pale red flocculent suspension, continuously stirring for 30min, transferring the suspension into a hydrothermal kettle, controlling the temperature of an oven at 135 ℃, heating for 16h, cooling to room temperature, taking out, transferring into a 50mL centrifuge tube, adding water and ethanol, centrifuging and cleaning for three times, setting the rotation speed of the centrifuge at 8000r/min, and the centrifugation time at 12 min. And after the completion, pouring the waste liquid, putting the material into an oven, heating at 60 ℃ for 9 hours, taking out the material after reaching the heating time, and grinding the material to obtain cerium oxide powder. The micro-topography is not significantly different from that of figure 1.
(2) Preparing a Prussian blue analogue synthetic solution C which is 100mL of a mixed solution of potassium hexacyanocobaltate (III) and deionized water, wherein the anion concentration is 0.015M; the prepared Prussian blue analogue synthetic solution D is 100mL of mixed solution of copper chloride dihydrate and deionized water, and sodium citrate serving as a surfactant is added into the synthetic solution D, wherein the cation concentration is 0.015M, and the sodium citrate concentration is 0.03M. Each was poured into a 100mL syringe and mounted on a syringe pump for use.
(3) And taking 30mL of 0.005g/mL cerium oxide nanorod solution uniformly dispersed in deionized water out of the beaker, placing the beaker in a 35 ℃ water bath, and dropping the cerium oxide nanorod solution into the beaker by using an injection pump at the speed of 450 mu L/min to synthesize the nano-composite suspension at a low speed. And (3) cleaning the suspension with deionized water, centrifuging at 8000r/min for 12min, repeating for three times, and drying at 60 ℃ for 9h to obtain the powder sample material.
(4) And (3) weighing 1g of the powder sample material obtained in the step (3), mixing with 25g of carbon ink in a mass ratio of (1: 25), adding 1.5mL of ethanol, and stirring in a single direction to enable the ink to meet the printing requirement.
(5) And (3) printing a substrate electrode on the PVC substrate material by a screen printing technology, taking silver chloride ink as printing reference electrode ink, taking carbon ink as printing counter electrode ink, and taking the mixed ink prepared in the step (4) as printing working electrode ink.
(6) 35U of sucrose hydrolase, 35U of mutarotase and 350U of glucose oxidase are respectively dissolved in 100 mu L of phosphate buffer, 1 mu L of 25% (v/v) glutaraldehyde is respectively added into the enzyme solution for crosslinking, and the solution is transferred into a centrifugal tube for later use.
(7) Wetting the surface of a working electrode of the biosensor chip with a phosphate buffer solution, standing for 5min, dot-blotting with a piece of dust-free paper, fixing 5 mul of enzyme solution on the surface of the working electrode one by one according to the sequence of sucrose hydrolase, mutarotase and glucose oxidase, and transferring the sucrose biosensor chip into a refrigerator for storage and standby after the surface of the working electrode is completely dried.
Through the timing ampere current test, the following results are obtained: the biosensor chip obtained in this example had a high detection sensitivity of 55.62. mu.A.mM for sucrose and glucose-1 ·cm -2The detection limit is as low as 0.2 mu M, and the detection range is 0-1.4M. After the experiment, the modified electrode is placed in PBS buffer solution with pH of 7.0 for one week at the temperature of 0 ℃, and the response signal is basically unchanged; after one month, the response signal was 97% of the initial signal; after three months, the response signal was still 94% of the initial signal, indicating that the slurry stability was good.
Example 4
A preparation method of nano sensing slurry for printing a sucrose detection chip comprises the following steps:
(1) weighing 1.8629g (5 mmol) of cerous chloride heptahydrate, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution A, weighing 23.2g (0.8 mmol) of polyvinylpyrrolidone, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution B, mixing the solution A, B, continuously stirring for 30min, adding 6mL of ammonia water into the mixed solution to enable the pH to be =10, enabling the solution to become a pale red flocculent suspension, continuously stirring for 30min, transferring the suspension into a hydrothermal kettle, controlling the temperature of an oven at 150 ℃, heating for 12h, cooling to room temperature, taking out, transferring into a 50mL centrifuge tube, adding water and ethanol, centrifuging and cleaning for three times, setting the rotation speed of the centrifuge to 10000r/min, and setting the centrifugation time to 8 min. And after the completion, pouring the waste liquid, putting the material into an oven, heating at 70 ℃ for 8h, taking out the material after reaching the heating time, and grinding the material to obtain cerium oxide powder. The micro-topography is not significantly different from that of figure 1.
(2) Preparing a Prussian blue analogue synthetic solution C which is 100mL of a mixed solution of potassium hexacyanocobaltate (III) and deionized water, wherein the anion concentration is 0.02M; the prepared Prussian blue analogue synthetic solution D is 100mL of a mixed solution of copper chloride dihydrate and deionized water, and sodium citrate serving as a surfactant is added into the synthetic solution D, wherein the cation concentration is 0.02M, and the sodium citrate concentration is 0.04M. Each was poured into a 100mL syringe and mounted on a syringe pump for use.
(3) And taking 40mL of 0.008g/mL cerium oxide nanorod solution uniformly dispersed in deionized water out of a beaker, placing the beaker in a water bath kettle at 45 ℃, and dropping the solution into the beaker by using an injection pump at the speed of 500 mu L/min to synthesize the nano-composite suspension at a low speed. And (3) cleaning the suspension with deionized water, centrifuging at 10000r/min for 8min, repeating the steps for three times, and drying the suspension at the drying temperature of 70 ℃ for 8h to obtain a mixed material powder sample.
(4) And (3) weighing 1g of the powder sample material obtained in the step (3), mixing with 35g of carbon ink in a mass ratio of (1: 35), adding 2mL of ethanol, and stirring in a single direction to enable the ink to meet the printing requirement.
(5) And (3) printing a substrate electrode on the PVC substrate material by a screen printing technology, taking silver chloride ink as printing reference electrode ink, taking carbon ink as printing counter electrode ink, and taking the mixed ink prepared in the step (4) as printing working electrode ink.
(6) 40U of sucrose hydrolase, 40U of mutarotase and 400U of glucose oxidase are respectively dissolved in 100 mu L of phosphate buffer, 1 mu L of 25% (v/v) glutaraldehyde is respectively added into the enzyme solution for crosslinking, and the solution is transferred into a centrifuge tube for later use.
(7) Wetting the surface of a working electrode of the biosensor chip with a phosphate buffer solution, standing for 5min, dot-blotting with a piece of dust-free paper, fixing 5 mul of enzyme solution on the surface of the working electrode one by one according to the sequence of sucrose hydrolase, mutarotase and glucose oxidase, and transferring the sucrose biosensor chip into a refrigerator for storage and standby after the surface of the working electrode is completely dried.
Through the timing ampere current test, the following results are obtained: the biosensor chip obtained in this example had a high detection sensitivity of 59.32. mu.A.mM for sucrose and glucose-1 ·cm -2The detection limit is as low as 0.3 mu M, and the detection range is 0-1.3M. After the experiment, the modified electrode is placed in PBS buffer solution with pH of 7.0 for one week at the temperature of 0 ℃, and the response signal is basically unchanged; after one month, the response signal was 97% of the initial signal; after three months, the response signal was still 93% of the initial signal, indicating that the stability of the slurry was good.
Example 5
A preparation method of nano sensing slurry for printing a sucrose detection chip comprises the following steps:
(1) weighing 3.7258g (10 mmol) of cerous chloride heptahydrate, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution A, weighing 29g (1 mmol) of polyvinylpyrrolidone, dissolving in 100mL of deionized water, stirring the solution until the solution is clear and transparent to obtain a solution B, mixing the solution A, B, continuously stirring for 30min, adding 7mL of ammonia water into the mixed solution to enable the pH to be =12, enabling the solution to become a pale red flocculent suspension, continuously stirring for 30min, transferring the suspension into a hydrothermal kettle, controlling the temperature of an oven at 180 ℃, heating for 8h, cooling to room temperature, taking out, transferring into a 50mL centrifuge tube, adding water and ethanol, centrifuging and cleaning for three times, setting the rotation speed of the centrifuge to 12000r/min, and setting the centrifugation time to 5 min. And after the completion, pouring the waste liquid, putting the material into an oven, heating at 80 ℃ for 6h, taking out the material after reaching the heating time, and grinding the material to obtain cerium oxide powder. The micro-topography is not significantly different from that of figure 1.
(2) Preparing a Prussian blue analogue synthetic solution C which is 100mL of a mixed solution of potassium hexacyanocobaltate (III) and deionized water, wherein the anion concentration is 0.03M; the prepared Prussian blue analogue synthetic solution D is 100mL of a mixed solution of copper chloride dihydrate and deionized water, and sodium citrate serving as a surfactant is added into the synthetic solution D, wherein the cation concentration is 0.03M, and the sodium citrate concentration is 0.05M. Each was poured into a 100mL syringe and mounted on a syringe pump for use.
(3) And taking 40mL of 0.01g/mL cerium oxide nanorod solution uniformly dispersed in deionized water out of a beaker, placing the beaker in a water bath kettle at 55 ℃, and dropping the solution into the beaker by using an injection pump at the speed of 600 mu L/min to synthesize the nano-composite suspension at a low speed. And (3) cleaning the suspension with deionized water, centrifuging at 12000r/min for 6min, repeating the steps for three times, and drying the suspension at the drying temperature of 60 ℃ for 6h to obtain a mixed material powder sample.
(4) And (3) weighing 1g of the powder sample material obtained in the step (3), mixing with 50g of carbon ink in a mass ratio of (1: 50), adding 2.5mL of ethanol, and stirring in a single direction to enable the ink to meet the printing requirement.
(5) And (3) printing a substrate electrode on the PVC substrate material by a screen printing technology, taking silver chloride ink as printing reference electrode ink, taking carbon ink as printing counter electrode ink, and taking the mixed ink prepared in the step (4) as printing working electrode ink.
(6) 40U of sucrose hydrolase, 40U of mutarotase and 400U of glucose oxidase are respectively dissolved in 100 mu L of phosphate buffer, 1 mu L of 25% (v/v) glutaraldehyde is respectively added into the enzyme solution for crosslinking, and the solution is transferred into a centrifuge tube for later use.
(7) Wetting the surface of a working electrode of the biosensor chip with a phosphate buffer solution, standing for 5min, dot-blotting with a piece of dust-free paper, fixing 5 mul of enzyme solution on the surface of the working electrode one by one according to the sequence of sucrose hydrolase, mutarotase and glucose oxidase, and transferring the sucrose biosensor chip into a refrigerator for storage and standby after the surface of the working electrode is completely dried.
Through the timing ampere current test, the following results are obtained: the biosensor chip obtained in this example had a high detection sensitivity of 55.32. mu.A.mM for sucrose and glucose-1 ·cm -2The detection limit is as low as 0.4 mu M, and the detection range is 0-1.5M. After the experiment, the modified electrode is placed in PBS buffer solution with pH of 7.0 for one week at the temperature of 0 ℃, and the response signal is basically unchanged; after one month, the response signal was 96% of the initial signal; after three months, the response signal was still 92% of the initial signal, indicating that the slurry stability was good.
The invention relates to a preparation method of nano sensing slurry for printing a sucrose detection chip, belonging to the technical field of biological analysis. The synthesis steps of the nano sensing slurry are as follows: preparing and synthesizing a cerium oxide nanorod solution; preparing a Cu-Co Prussian blue analogue synthetic solution A, B; and (3) synthesizing a Cu-Co Prussian blue analogue in a cerium oxide nanorod suspension. The composite material is mixed with the conductive carbon printing ink according to a certain proportion through centrifugation and drying, the mixed slurry is enabled to be in a printable state by adding ethanol, and then the biosensor chip is prepared by means of a screen printing technology. The slurry synthesis method is simple, the process is controllable, the commercialization is easy to realize, the adsorption performance of proteins including enzymes with low isoelectric points can be good based on the characteristics of high isoelectric points such as cerium oxide, the problem that multiple enzymes need to be fixed in the sucrose detection process is certain, and based on the existence of Cu-Co PBA, the low potential of-0.2V of the working potential can be controlled, the excellent anti-interference performance is achieved, the extremely wide linear range of hydrogen peroxide detection can be realized, the linear range of sucrose detection is greatly improved, and the sucrose concentration detection requirements of different markets can be met.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.
Claims (5)
1. A preparation method of nano sensing slurry for printing a sucrose detection chip is characterized by comprising the following specific steps:
(1) preparation of cerium oxide nano material
Dissolving cerium chloride heptahydrate serving as a cerium source in deionized water to prepare a solution A; dissolving polyvinylpyrrolidone as a surfactant in deionized water to prepare a solution B; mixing the solution A and the solution B, stirring until the solution A and the solution B are clear and transparent, adding ammonia water to control the pH value, transferring the solution into a hydrothermal kettle, taking out the solution after hydrothermal synthesis reaction, cooling, centrifuging, cleaning to obtain nano cerium oxide precipitate, and drying to obtain cerium oxide powder;
(2) preparation of Cu-Co Prussian blue analogue synthetic liquid
Mixing potassium hexacyanocobaltate (III) with deionized water to obtain Prussian blue analogue synthetic liquid C; mixing copper chloride dihydrate with deionized water, and adding sodium citrate to obtain Prussian blue analogue synthetic solution D;
(3) synthesis of nanocomposite solutions
Dispersing the cerium oxide powder obtained in the step (1) in deionized water, heating in a water bath, then respectively taking the same volume of the synthetic liquid C, D prepared in the step (2), respectively filling the synthetic liquid into an injection pump, dripping the synthetic liquid at the same speed, synthesizing a nano compound solution at a low speed, and carrying out an aging reaction after dripping;
(4) preparation of nano sensing slurry
And (4) centrifuging and drying the nano composite solution subjected to the aging reaction in the step (3), mixing the nano composite solution with conductive carbon ink to obtain mud-shaped slurry, and adding ethanol to enable the mixed slurry to reach the viscosity required by printing.
2. The method for preparing the nano sensing paste for printing the sucrose detection chip according to claim 1, wherein in the step (1), the concentration range of the ions in the solution A is 0.001-0.1M, the concentration range of the ions in the solution B is 0.001-0.01M, the pH value is 7-12 after ammonia water is added, the hydrothermal synthesis temperature is 90-180 ℃, and the hydrothermal synthesis time is 8-24 h; the centrifugation speed is 6000-12000 r/min, the centrifugation time is 5-20min, the drying temperature is 50-80 ℃, and the drying time is 6-12 h.
3. The method for preparing the nano-sized sensing paste for printing the sucrose detection chip as recited in claim 1, wherein in the step (2), the concentration of the anion in the synthetic fluid C is 0.005 to 0.03M, the concentration of the cation in the synthetic fluid D is 0.005 to 0.03M, and the concentration of the sodium citrate in the synthetic fluid D is 0.01 to 0.05M.
4. The method for preparing nano sensing paste for printing sucrose detection chip as defined in claim 1, wherein the concentration of cerium oxide powder in deionized water in step (3) is 0.001-0.01 g/mL, the dropping speed of the synthetic solution C and the synthetic solution D is 300-.
5. The method for preparing the nano sensing paste for printing the sucrose detection chip as recited in claim 1, wherein the centrifugation rate in step (4) is 6000-12000 r/min, the centrifugation time is 5-20min, the drying temperature is 50-80 ℃, the drying time is 6-12h, the mass ratio of the nano composite material to the carbon ink is 1:1-1:50, and the viscosity of the printing paste is 50-150 dpa.S.
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