CN114045327A - Biological sensing membrane and preparation method and application thereof - Google Patents

Biological sensing membrane and preparation method and application thereof Download PDF

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CN114045327A
CN114045327A CN202111313677.7A CN202111313677A CN114045327A CN 114045327 A CN114045327 A CN 114045327A CN 202111313677 A CN202111313677 A CN 202111313677A CN 114045327 A CN114045327 A CN 114045327A
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membrane
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CN114045327B (en
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罗建泉
宋思青
张昊
万印华
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Institute of Process Engineering of CAS
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Abstract

The invention provides a biosensing membrane and a preparation method and application thereof. The biosensor film comprises a modified carbon nanotube layer, a base film, a fixing layer, and Prussian blue and enzyme fixed on the fixing layer, which are sequentially arranged. The biological sensing film has good conductivity by using the modified carbon nanotube layer; and by selecting a specific fixing layer, the Prussian blue is targeted and uniformly deposited and the enzyme molecules are efficiently and stably fixed, and meanwhile, the Prussian blue and the enzyme are uniformly distributed on the biological sensing film, are not overlapped and agglomerated, so that the electron transfer efficiency of the biological sensing film is remarkably improved, and the biological sensing film has better sensing performance. The biosensor film can realize high-sensitivity detection of analytes in a sample through the synergistic effect of the modified carbon nanotube layer, the base film, the fixing layer and the prussian blue and the enzyme fixed on the fixing layer.

Description

Biological sensing membrane and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biosensing materials, and particularly relates to a biosensing membrane and a preparation method and application thereof.
Background
The electrochemical sensor can accurately and rapidly detect a large amount of analytes through the multifunctional combination of target molecule screening, specificity identification, signal transduction and reading, and is widely applied to the fields of medical care, environmental monitoring, biological safety and the like in recent years. The enzyme is used as a biocatalyst, has specific selectivity to a substrate and high catalytic efficiency, and can realize high-sensitivity detection of analytes in complex samples.
Biological enzymes are easy to denature and inactivate under extreme conditions, and in the prior art, the enzymes are mostly fixed on electrodes so as to improve the signal transmission efficiency and sensitivity of the enzymes in actual biological analysis monitoring. For example, CN108918625A discloses a method for preparing a biosensor film, a biosensor film and a monitoring device. The preparation method of the biosensing membrane comprises the steps of carrying out electrochemical activation modification on oxidoreductase, then using a chemical cross-linking agent for cross-linking treatment, and coating the electrode surface with the modified oxidoreductase, so as to form the biosensing membrane. The biosensing membrane is stable and durable, can be used for multiple detection, and is particularly suitable for being used as a biosensing membrane of a living body monitoring device.
However, enzyme-based electrochemical sensors still face constant challenges: firstly, the conventional electrochemical sensor mainly comprises a two-dimensional plane electrode, the specific surface area is small and the active sites are few due to the two-dimensional plane and non-open pore structure of the electrode, the enzyme immobilization and catalysis efficiency is influenced, and meanwhile, the in-situ separation of an analyte and an interferent is difficult to realize. Compared with a two-dimensional plane electrode, the three-dimensional porous electrode is beneficial to the infiltration of electrolyte, the contact area between an analyte and the electrode is increased, and in addition, the larger specific surface area is beneficial to the improvement of the stability of immobilized enzyme. The membrane, as a three-dimensional porous matrix, has the advantages of protecting the enzyme molecules from the external environment. And convective mass transfer effects can accelerate the reaction of the enzyme with the substrate to increase sensitivity. In addition, the porous structure of the membrane is also beneficial to enhancing the catalysis and amplifying the detection signal.
Because the surface of the conductive membrane and the surface of the electrocatalyst represented by prussian blue lack active functional groups, the traditional enzyme immobilization method is to soak the conductive membrane in a high-concentration enzyme solution, and cross-link the conductive membrane with glutaraldehyde after drying ("polysaccharide-driven membrane separation and in-situ electrochemical detection based on3D continuous chemical gradient membrane", Wu H et al, biosens. bioelectrron. 2021, 171: 112722). However, it is difficult to achieve stable loading of the bio-enzyme on the membrane, resulting in high leakage or low activity of the enzyme, which in turn affects the sensitivity, stability, reproducibility and lifetime of the sensing membrane. The prior art discloses a biosensing membrane, which is based on the unique structure of a gradient hollow fiber membrane, and assembles size-adjustable enzyme-loaded nanoparticles into a pore-restricted space of a three-dimensional gradient conductive membrane by a mild controllable filtration embedding method, so as to obtain higher enzyme loading and detection sensitivity ("modulated assembly of enzyme-loaded nanoparticles in 3D porous fiber electrode for electrochemical sensing", Wu H, etc., chem.eng.j., 2021, 421: 129721). Although the physical embedding method improves the stability of the immobilized enzyme, the enzyme-loaded nanoparticle and the conductive film lack a firmer chemical interaction, and the enzyme is still in a shedding problem.
Therefore, the development of a biosensing membrane with high enzyme loading, high sensitivity and good stability is a technical problem to be solved in the field.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a biosensing membrane and a preparation method and application thereof. The Prussian blue is subjected to targeted uniform deposition on the surface of the membrane by modifying the base membrane through a specific fixing layer, meanwhile, a phenol group on a phenol group-containing compound in the fixing layer can be oxidized into a quinone group, the enzyme is fixed on the biological sensing membrane through a covalent bond, and the stability and the reproducibility of the sensing membrane are enhanced by abundant quinone group covalent immobilized enzymes, so that a large amount of enzyme required by the traditional adsorption and crosslinking methods is avoided; meanwhile, the Prussian blue and the enzyme are uniformly distributed on a sensing interface, so that the electron transfer efficiency of the biological sensing membrane is remarkably improved, and the biological sensing membrane has better sensing performance.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a biosensing membrane, which comprises a modified carbon nanotube layer, a base membrane, a fixed layer, and prussian blue and an enzyme which are fixed on the fixed layer, wherein the modified carbon nanotube layer, the base membrane and the fixed layer are sequentially arranged; the fixed layer includes a combination of a phenol-containing compound and ferric chloride.
According to the invention, the modified carbon nanotube layer enables the biosensing film to have excellent conductivity; the fixing layer adopts the combination of the compound containing the phenolic group and the ferric trichloride, so that conditions are provided for the subsequent stable fixation of the Prussian blue and the enzyme, the target uniform deposition of the Prussian blue nano-particles on the surface of the base film can be realized, the agglomeration of the Prussian blue nano-particles is avoided, the contact area of the Prussian blue and an analyte can be increased, and the electron transfer resistance can be reduced; meanwhile, the Prussian blue and the enzyme are uniformly distributed on a sensing interface and are not overlapped or agglomerated, so that the electron transfer efficiency of the biological sensing membrane is remarkably improved, and the biological sensing membrane has better sensing performance.
As the preferable technical scheme of the invention, the modified carbon nanotube layer is a polyvinyl alcohol modified carbon nanotube layer.
Preferably, the mass ratio of the polyvinyl alcohol to the carbon nanotubes in the modified carbon nanotube layer is 1: (4.5 to 5.5), for example, the ratio of 1: 4.5, 1: 5. 1: 5.5, and the like.
In the invention, the carbon nano tube is modified by adopting polyvinyl alcohol and glutaraldehyde under an acidic condition, so that the stability of the carbon nano tube on the surface of the membrane is improved, and the carbon nano tube cannot fall off; if the carbon nano tube is not modified, the carbon nano tube can continuously fall off in the subsequent modification process, and the subsequent determination of enzyme activity and sensitivity is influenced.
Preferably, the base membrane includes an inorganic porous support membrane and/or a polymeric porous support membrane.
Preferably, the inorganic porous support membrane comprises any one of an alumina ceramic membrane, a silica ceramic membrane, or a titania ceramic membrane, or a combination of at least two thereof.
Preferably, the material of the high molecular porous support membrane comprises any one or a combination of at least two of polyacrylonitrile, nylon, polyvinylidene fluoride, polyether sulfone or aromatic polyamide.
Preferably, the phenolic group-containing compound comprises tannic acid and/or polydopamine.
Preferably, the fixing layer further comprises 3-aminopropyltriethoxysilane and/or polyethyleneimine.
Preferably, the fixing layer comprises a combination of tannic acid, 3-aminopropyltriethoxysilane, and ferric chloride.
In the invention, the tannic acid is a plant polyphenol with good hydrophilicity, and is rich in catechol groups, so that the tannic acid can be adhered to the surface of a base film through various interactions, and meanwhile, the tannic acid and 3-aminopropyltriethoxysilane can form a coating with a spherical structure through Michael addition reaction or Schiff base reaction, so that the adhesiveness of the tannic acid is further improved; the iron trichloride is coordinated with the catechol group of the tannic acid and the amino group of the 3-aminopropyltriethoxysilane, so that the acid-base stability of the immobilized layer is improved, conditions are provided for subsequent Prussian blue targeted uniform deposition, and meanwhile, the phenol group on the tannic acid can be oxidized to form a quinone group, so that the immobilized enzyme can be used for subsequent immobilization.
Preferably, the mass ratio of the tannic acid, the 3-aminopropyltriethoxysilane and the ferric trichloride in the fixed layer is (0.005-4.5): (0.02-4.5): 1, for example, may be 0.005: 0.02: 1. 0.5: 1: 1. 0.8: 0.5: 1. 1: 0.8: 1. 2: 1.5: 1. 3: 1: 1. 4: 2: 1. 0.8: 0.6: 1. 0.83: 0.83: 1. 0.67: 0.33: 1. 0.88: 0.55: 1. 4: 4: 1, preferably (0.8-1): (0.5-0.6): 1.
in the invention, the tannic acid, the 3-aminopropyltriethoxysilane and the ferric trichloride are in a specific ratio, the sensitivity and the enzyme carrying amount of the biosensor membrane have the best effect, and the sensitivity and the enzyme carrying amount of the biosensor membrane are reduced due to the fact that the tannic acid or the 3-aminopropyltriethoxysilane is too high or too low in quality.
Preferably, the prussian blue is prussian blue nanoparticles formed by the reaction of potassium ferricyanide and ferric trichloride in the fixed layer.
Preferably, the mass ratio of the potassium ferricyanide to the ferric trichloride is (0.8-6.5): 1, for example, may be 0.8: 1. 1: 1. 1.5: 1. 2: 1. 2.5: 1. 3: 1. 4: 1. 5: 1.6:1, and more preferably (0.8 to 3): 1.
in the invention, the mass ratio of the potassium ferricyanide to the ferric trichloride is lower or higher, and the sensitivity of the biological sensing film is reduced.
Preferably, the enzyme is an enzyme that catalyzes the production of hydrogen peroxide.
Preferably, the enzyme comprises any one of or a combination of at least two of glucose oxidase, alcohol oxidase, galactose oxidase, lactate oxidase, nucleoside oxidase, glycerol oxidase, cholesterol oxidase, or superoxide dismutase.
Preferably, the enzyme comprises glucose oxidase or lactate oxidase.
In the invention, the enzyme can catalyze the analyte to generate hydrogen peroxide, and the generated hydrogen peroxide can be further decomposed under the action of the electrocatalyst prussian blue to generate response current, so that the quantitative detection of the analyte is realized.
In a second aspect, the present invention provides a method for preparing the biosensing membrane according to the first aspect, comprising the steps of:
(1) respectively arranging a modified carbon nanotube layer and a fixed layer on two surfaces of a base film; the fixed layer reacts with potassium ferricyanide to obtain a Prussian blue fixed sensing film;
(2) and (2) mixing the sensing membrane obtained in the step (1) with enzyme, and reacting to obtain the biological sensing membrane.
As a preferable technical solution of the present invention, the method for setting the modified carbon nanotube layer in step (1) includes: filtering and depositing the carbon nano tube dispersion liquid on one surface of the base film to obtain a carbon nano tube layer; and then adding polyvinyl alcohol reaction liquid to react to obtain the modified carbon nanotube layer.
In the present invention, the carbon nanotube dispersion is diluted to an aqueous solution having a concentration of 0.1 to 1g/L, and the concentration may be, for example, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, or the like.
Preferably, the pressure of the filtration deposition is 0.5-1 bar, such as 0.55bar, 0.6bar, 0.65bar, 0.7bar, 0.75bar, 0.8bar, 0.85bar, 0.9bar, 0.95bar, etc.
Preferably, the polyvinyl alcohol reaction solution further comprises glutaraldehyde.
Preferably, the pH value of the polyvinyl alcohol reaction solution is 1 to 5, and may be 1, 2, 3, 4, 5, or the like, for example.
In the invention, polyvinyl alcohol with different mass fractions is selected as polyvinyl alcohol in the polyvinyl alcohol reaction solution, and the mass fraction of the polyvinyl alcohol is 0.1-1%, and can be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and the like, for example.
In the present invention, the glutaraldehyde is 0.1 to 1% by mass, and may be, for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or the like.
In the present invention, the pH of the polyvinyl alcohol reaction solution is provided by hydrochloric acid, and the mass fraction of the hydrochloric acid is 25 to 35%, and may be, for example, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or the like.
Preferably, the reaction temperature is 20 to 100 ℃, for example, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and the like.
Preferably, the reaction time is 5-60 min, such as 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, and the like.
In the invention, under an acidic environment, polyvinyl alcohol and glutaraldehyde are adopted to modify the carbon nano tube, and carboxyl on the carbon nano tube reacts with hydroxyl on the polyethylene and aldehyde group on the glutaraldehyde, so that the modified carbon nano tube layer is obtained.
Preferably, the method for disposing the fixed layer in step (1) includes: treating one side of the base film, which is far away from the modified carbon nanotube layer, by using a phenolic compound-containing reaction solution, and reacting to obtain a first fixed layer; and reacting the first fixed layer with ferric trichloride to obtain the fixed layer.
Preferably, the phenolic group-containing compound reaction liquid further comprises 3-aminopropyltriethoxysilane and/or polyethyleneimine.
Preferably, the reaction time for obtaining the first fixed layer is 2-24 h, for example, 4h, 6h, 8h, 10h, 15h, 18h, 20h, 22h, and the like.
Preferably, the reaction pH for obtaining the first immobilization layer is 7.5 to 9.5, and may be, for example, 7.5, 8, 8.5, 9, 9.5, or the like.
Preferably, the reaction temperature for obtaining the first immobilization layer is 25 to 30 ℃, and may be, for example, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ or the like.
Preferably, the reaction time of the first fixed layer and ferric trichloride is 1-10 h, for example, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h and the like.
Preferably, the temperature of the reaction between the first fixed layer and ferric trichloride is 25 to 30 ℃, and may be, for example, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ or the like.
In the invention, the phenolic group-containing compound is dissolved in a Tris-HCl buffer solution to prepare the Tris-HCl buffer solution with the concentration of 0.5-10 g/L for use; dissolving the 3-aminopropyltriethoxysilane in ethanol to prepare a 2-10 g/L3-aminopropyltriethoxysilane ethanol solution for use; dissolving the polyethyleneimine into water to prepare a polyethyleneimine water solution with the concentration of 2-10 g/L for use; the ferric trichloride is dissolved in water and prepared into a ferric trichloride aqueous solution with the concentration of 2-10 g/L for use.
The concentration of potassium ferricyanide is preferably 5 to 40mM, and may be, for example, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, or the like, and more preferably 5 to 20 mM.
In the invention, the sensitivity of the biosensing membrane is reduced due to the fact that the concentration of the potassium ferricyanide is too high or too low.
Preferably, the reaction time in step (1) is 1-10 h, for example, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, etc.
Preferably, the reaction temperature in step (1) is 20-60 ℃, for example, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and the like.
Preferably, the reaction in step (1) has a pH of 1 to 3, and may be, for example, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, etc.
Preferably, the reaction time in the step (2) is 2-60 h, for example, 4h, 8h, 12h, 20h, 25h, 30h, 35h, 40h, 45h, 50h, 55h, and the like.
Preferably, the reaction temperature in step (2) is 4-30 ℃, such as 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃ and the like.
Preferably, the reaction in step (2) has a pH of 3 to 6, and may be, for example, 3.5, 4.0, 4.5, 5.0, 5.5, or the like.
As a preferred technical scheme of the invention, the preparation method comprises the following steps:
(1) filtering and depositing the carbon nano tube dispersion liquid on one surface of the base film under the pressure of 0.5-1 bar to obtain a carbon nano tube layer; then adding reaction liquid of polyvinyl alcohol and glutaraldehyde with the pH value of 1-5, and reacting for 5-60 min at the temperature of 20-100 ℃ to obtain the modified carbon nanotube layer; then, treating one side of the base film, which is far away from the modified carbon nanotube layer, by using a phenolic compound-containing reaction solution, and reacting for 2-24 hours under the conditions that the pH value is 7.5-9.5 and the temperature is 25-30 ℃ to obtain a first fixed layer; adding a ferric trichloride solution, and reacting the first fixed layer with the ferric trichloride solution at the temperature of 20-30 ℃ for 1-10 h to obtain the fixed layer; the fixing layer and potassium ferricyanide react for 1-10 hours under the conditions that the pH value is 1-3 and the temperature is 20-60 ℃ to obtain the Prussian blue fixed sensing film;
(2) and (2) mixing the sensing membrane obtained in the step (1) with enzyme, and reacting for 2-60 hours under the conditions that the pH value is 3-6 and the temperature is 4-30 ℃ to obtain the biological sensing membrane.
In the present invention, the preparation method needs to be realized by adopting steps in a specific sequence. In the invention, if the base membrane is treated by adopting the mixed solution of ferric trichloride and potassium ferricyanide, although enzyme immobilization sites can be provided, the ferric trichloride and the potassium ferricyanide directly react in the solution to form Prussian blue, and the synthesized Prussian blue is greatly agglomerated in the reaction solution, so that quinone sites for enzyme immobilization are covered, and further the response capability to glucose is low.
In a third aspect, the present invention provides a biosensing material comprising the biosensing film according to the first aspect.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has the beneficial effects that:
the biological sensing film provided by the invention has good conductivity by using the modified carbon nanotube layer, avoids the agglomeration of Prussian blue by selecting the specific fixing layer, can stably fix the enzyme, optimizes the spatial distribution of the Prussian blue and the enzyme and improves the sensing performance; as a preferred technical scheme of the invention, the sensitivity of the biosensing membrane is more than 8, the enzyme carrying amount is more than or equal to 365 mug, and the high-sensitivity detection of the analyte in the sample can be realized.
Detailed Description
For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The following examples and comparative examples do not indicate any particular technique or condition, and are carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1
The embodiment provides a biological sensing membrane, which comprises a modified carbon nanotube layer, a base membrane (a nylon membrane, the aperture of which is 0.22 μm), a fixed layer and prussian blue and glucose oxidase fixed on the fixed layer, wherein the modified carbon nanotube layer, the base membrane (the nylon membrane, the aperture of which is 0.22 μm) and the fixed layer are arranged in sequence; the fixed layer comprises a mixture of, by mass, 0.889: 0.555: 1 tannic acid, 3-aminopropyltriethoxysilane and ferric chloride.
The embodiment provides a preparation method of the biosensing membrane, which comprises the following specific steps:
(1) placing the basement membrane in a filter, filtering and depositing 25mL of 1g/L carbon nanotube dispersion liquid under the pressure of 0.65bar, then adding a mixed solution of 1mL of polyvinyl alcohol (mass fraction is 0.5%), 0.5mL of glutaraldehyde (mass fraction is 0.5%) and 0.5mL of hydrochloric acid (mass fraction is 31.5%), and reacting for 5min at 60 ℃ to obtain the basement membrane provided with the modified carbon nanotube layer;
(2) respectively dissolving tannic acid and 3-aminopropyltriethoxysilane in 10mM Tris-HCl buffer solution (pH 8.5) and ethanol to prepare a tannic acid Tris-HCl buffer solution with the concentration of 2g/L and a 3-aminopropyltriethoxysilane ethanol solution with the concentration of 10g/L, and then mixing the tannic acid Tris-HCl buffer solution and the 3-aminopropyltriethoxysilane ethanol solution according to the volume ratio of 8: 1, mixing to obtain a mixed solution; treating one side of the base film, far away from the modified carbon nanotube layer, in the step (1) by using the mixed solution, and reacting for 18h at room temperature and the rotating speed of 150rpm to obtain a first fixed layer; adding a ferric trichloride aqueous solution with the concentration of 2g/L into the solution to react with the first fixed layer at the rotating speed of 150rpm for 1h at room temperature, and washing the solution with deionized water at the rotating speed of 150rpm for 1h to obtain two base films of which the surfaces are respectively provided with a modified carbon nanotube layer and the fixed layer;
(3) mixing the base film obtained in the step (2) with 20mL of acidic potassium ferricyanide aqueous solution (pH is 1.5) with the concentration of 7.4mM, and reacting the fixed layer with the potassium ferricyanide at 150rpm and 35 ℃ for 1h to obtain the Prussian blue fixed sensing film; subsequently, 20mL of glucose oxidase acetate buffer solution (pH 5.010 mM acetate buffer solution) at a concentration of 0.25g/L was added, and the immobilization layer was reacted with glucose oxidase at 4 ℃ for 48 hours, followed by washing with deionized water for 1 hour to obtain the biosensor film.
Example 2
This example provides a biosensor film, which is different from example 1 only in that the concentration of the acidic potassium ferricyanide aqueous solution in step (3) of the preparation method of the biosensor film is 5mM, and other raw materials, steps and parameters are the same as those of example 1.
Example 3
This example provides a biosensor film, which is different from example 1 only in that the concentration of the acidic potassium ferricyanide aqueous solution in step (3) in the preparation method of the biosensor film is 20mM, and other raw materials, steps and parameters are the same as those in example 1.
Example 4
This example provides a biosensing membrane which differs from example 1 only in that glucose oxidase in the biosensing membrane is replaced by lactate oxidase of equal mass, and the other components, amounts and structures are the same as in example 1.
The embodiment provides a preparation method of the biosensing membrane, and the specific steps are the same as those of the embodiment 1.
Example 5
This example provides a biosensor membrane, which is different from example 1 only in that the 3-aminopropyltriethoxysilane ethanol solution is replaced by an equal-concentration and equal-volume polyethyleneimine aqueous solution in step (2) of the preparation method of the biosensor membrane, and other raw materials, steps and parameters are the same as those in example 1.
Example 6
This example provides a biosensing membrane, which is different from example 1 only in that the volume ratio of the tannic acid Tris-HCl buffer solution in step (2) to the 3-aminopropyltriethoxysilane ethanol solution in the preparation method of the biosensing membrane is 5: 1, other raw materials, procedures and parameters were the same as those of example 1.
Example 7
This example provides a biosensing membrane, which is different from example 1 only in that the volume ratio of the tannic acid Tris-HCl buffer solution and the 3-aminopropyltriethoxysilane ethanol solution in step (2) in the preparation method of the biosensing membrane is 2: 1, other raw materials, procedures and parameters were the same as those of example 1.
Example 8
This example provides a biosensing membrane which differs from example 1 only in that tannic acid and 3-aminopropyltriethoxysilane were replaced by polydopamine of equal mass in the fixing layer, and other components, amounts and structures were the same as in example 1.
The embodiment provides a preparation method of the biosensing membrane, and the specific steps are the same as those of the embodiment 1.
Example 9
This example provides a biosensor membrane, which is different from example 1 only in that, in the preparation method of the biosensor membrane, after the base membrane is treated with the Tris-HCl tannin buffer solution and the 3-aminopropyltriethoxysilane ethanol solution in step (2), a mixed solution of ferric chloride and acidic potassium ferricyanide is added to treat the base membrane, and other raw materials, steps and parameters are the same as those in example 1.
Comparative example 1
This comparative example provides a biosensing membrane which is different from example 1 only in that the biosensing membrane is prepared by the method in which the base membrane is not treated with the tannic acid Tris-HCl buffer solution and the 3-aminopropyltriethoxysilane ethanol solution in step (2), and the other raw materials, steps and parameters are the same as those of example 1.
Comparative example 2
This comparative example provides a biosensing membrane, which is different from example 1 only in that after the base membrane is treated with the tannic acid Tris-HCl buffer solution and the 3-aminopropyltriethoxysilane ethanol solution in step (2), the base membrane is treated by adding an equal mass of acidic potassium ferricyanide, and then treated by adding an equal mass of ferric chloride solution, and other raw materials, steps and parameters are the same as those of example 1.
Comparative example 3
This comparative example provides a biosensor film, which is different from example 1 only in that the base film is not treated with an acidic aqueous potassium ferricyanide solution in step (3) of the preparation method of the biosensor film, and other raw materials, steps and parameters are the same as those of example 1, i.e., the finally obtained biosensor film does not contain prussian blue.
Comparative example 4
This comparative example provides a biosensing membrane which is different from example 1 only in that the biosensing membrane is prepared by the method in which the base membrane is not treated with the glucose oxidase acetate buffer solution in step (3), and the other raw materials, steps and parameters are the same as those of example 1, i.e., the finally obtained biosensing membrane does not contain the enzyme.
Comparative example 5
This comparative example provides a biosensing membrane, which is different from example 1 only in that the biosensing membrane is prepared by the method of step (1) without filtering and depositing a carbon nanotube dispersion on a base membrane and without adding a mixed solution of polyvinyl alcohol, glutaraldehyde and hydrochloric acid to perform a reaction, and other steps and parameters are the same as those of example 1, i.e., a biosensing membrane without a modified carbon nanotube layer is finally obtained.
Performance testing
(1) Activity of the enzyme: taking glucose oxidase as an example, phenol, 4-aminoantipyrine, horseradish peroxidase and glucose were dissolved in an acetic acid buffer (10mM, pH 5.0) to prepare substrate solutions in which their concentrations were 40mM, 4mM, 40mM, 100mM, respectively; glucose oxidase was then added to 20mL of the above substrate solution under the action of a magnetic stirrer, and the change in absorbance of the solution at 505nm was recorded. One unit of catalytic activity (U) is defined as the consumption of 1. mu. mol H per minute under the measurement conditions (25 ℃, pH 5.0)2O2The amount of enzyme(s);
lactate oxidase: phenol, 4-aminoantipyrine, horseradish peroxidase and lactic acid were dissolved in an acetic acid buffer (10mM, pH 5.0) to prepare substrate solutions in which their concentrations were 40mM, 4mM, 40mM, 100mM, respectively; lactate oxidase was then added to 20mL of the above substrate solution under the action of a magnetic stirrer, and the change in absorbance of the solution at 505nm was recorded. One unit of catalytic activity (U) is defined as the consumption of 1. mu. mol H per minute under the measurement conditions (25 ℃, pH 5.0)2O2The amount of enzyme(s);
(2) enzyme loading: calculating the enzyme activities of the original enzyme solution, the residual enzyme solution and the eluted enzyme solution according to the method (1), and calculating the amount of the enzyme immobilized on the surface of the biosensing membrane according to the activity balance (all enzyme solutions are diluted to similar concentrations before measurement so as to eliminate the influence of the enzyme concentration on the activity determination);
enzyme loading (μ g) ═ As-Ar-Aw)/As Ms;
wherein, As, Ar and Aw are respectively the catalytic activity (U) of the original enzyme solution, the residual enzyme solution and the eluted enzyme solution, and Ms is the enzyme amount in the original enzyme solution;
(3) sensing performance: an analyte such as glucose or lactic acid was dissolved in a phosphate buffer solution (50mM, pH 6.5, containing 0.1M KCl), and the analyte was added to the three-electrode reaction system every 100 to 300 seconds by amperometry, with the operating voltage set at-0.5V. And finally, obtaining the relation between the response current and the analyte concentration, wherein the slope is the sensitivity of the biological sensing membrane.
The specific test results are shown in table 1:
TABLE 1
Figure BDA0003342801720000141
Figure BDA0003342801720000151
The above table shows that the biosensing membrane provided by the invention is beneficial to the targeted uniform deposition of prussian blue and the efficient and stable fixation of enzyme by selecting a specific fixing layer, and simultaneously optimizes the spatial distribution of prussian blue and enzyme, thereby being more beneficial to electron transfer. Through the synergistic effect of the modified carbon nanotube layer, the base film, the fixed layer and the prussian blue and the enzyme fixed on the fixed layer, the biosensor film can realize the high-sensitivity detection of the analyte in the sample.
As is clear from comparison of example 1 with examples 2 and 3, different concentrations of potassium ferricyanide affect the catalytic sensing effect of the Prussian blue formed, and the preferred concentration of potassium ferricyanide in the present invention is 7.4mM, in which case the enzyme loading amount and sensitivity of the biosensing membrane of the present invention are the best. As can be seen from the comparison between example 1 and examples 4 and 5, the biosensing membrane has excellent sensitivity and enzyme loading capacity by selecting lactate oxidase or combining tannic acid and polyethyleneimine; as can be seen from comparison between example 1 and examples 6 and 7, the mass ratio of tannic acid to 3-aminopropyltriethoxysilane affects the Prussian blue distribution and the number of quinone sites for subsequent enzyme immobilization, and the mass ratio of tannic acid to 3-aminopropyltriethoxysilane is preferably 1.6:1 in the present invention, so that the biosensor membrane of the present invention has the highest enzyme loading and the best sensitivity. As is clear from comparison between example 1 and examples 8 to 9, when a specific combination is not used in the anchor layer or a specific procedure is not followed in the preparation method, the performance of the biosensing membrane is degraded.
As can be seen by comparing example 1 with comparative example 1, the biosensing membrane showed almost no response to glucose, indicating the lack of sites for enzyme immobilization. As can be seen from comparison between example 1 and comparative example 2, when the acidic potassium ferricyanide solution is added first and then the ferric trichloride solution is added, the sensitivity and the enzyme carrying amount of the biosensing membrane are 0, because the acidic potassium ferricyanide solution is added first, so that tannic acid and 3-aminopropyltriethoxysilane are subjected to protonation decomposition and fall off, and further subsequent enzyme immobilization and sensing performances are affected. As can be seen from comparison of example 1 with comparative examples 3 to 5, the sensitivity of the biosensor film is decreased when a certain component is absent in the biosensor film.
In summary, the biosensor film provided by the invention has the advantages of high sensitivity, high enzyme loading and excellent performance through the synergistic effect of the modified carbon nanotube layer, the base film, the fixed layer and the prussian blue and the enzyme fixed on the base film and through the preparation method with specific steps.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The biosensing membrane is characterized by comprising a modified carbon nanotube layer, a base membrane, a fixed layer, and Prussian blue and enzyme which are fixed on the fixed layer, wherein the modified carbon nanotube layer, the base membrane and the fixed layer are sequentially arranged;
the fixed layer includes a combination of a phenol-containing compound and ferric chloride.
2. The biosensing film according to claim 1, wherein the modified carbon nanotube layer is a polyvinyl alcohol-modified carbon nanotube layer;
preferably, the mass ratio of the polyvinyl alcohol to the carbon nanotubes in the modified carbon nanotube layer is 1: (4.5-5.5);
preferably, the base membrane comprises an inorganic porous support membrane and/or a polymeric porous support membrane;
preferably, the inorganic porous support membrane comprises any one of an alumina ceramic membrane, a silica ceramic membrane or a titania ceramic membrane or a combination of at least two of the same;
preferably, the material of the high molecular porous support membrane comprises any one or a combination of at least two of polyacrylonitrile, nylon, polyvinylidene fluoride, polyether sulfone or aromatic polyamide.
3. The biosensing membrane according to claim 1 or 2, wherein the phenolic group-containing compound comprises tannic acid and/or polydopamine;
preferably, the fixing layer also comprises 3-aminopropyltriethoxysilane and/or polyethyleneimine;
preferably, the fixing layer comprises a combination of tannic acid, 3-aminopropyltriethoxysilane, and ferric chloride;
preferably, the mass ratio of the tannic acid, the 3-aminopropyltriethoxysilane and the ferric trichloride in the fixed layer is (0.005-4.5): (0.02-4.5): 1, preferably (0.8-1): (0.5-0.6): 1.
4. the biosensing film according to any one of claims 1 to 3, wherein the Prussian blue is Prussian blue nanoparticles formed by the reaction of potassium ferricyanide and ferric trichloride in the immobilization layer;
preferably, the mass ratio of the potassium ferricyanide to the ferric trichloride is (0.8-6.5): 1, more preferably (0.8 to 3): 1;
preferably, the enzyme is an enzyme that catalyzes the production of hydrogen peroxide;
preferably, the enzyme comprises any one of or a combination of at least two of glucose oxidase, alcohol oxidase, galactose oxidase, lactate oxidase, nucleoside oxidase, glycerol oxidase, cholesterol oxidase, or superoxide dismutase;
preferably, the enzyme comprises glucose oxidase or lactate oxidase.
5. A method for preparing the biosensing film according to any one of claims 1 to 4, wherein the method comprises the following steps:
(1) respectively arranging a modified carbon nanotube layer and a fixed layer on two surfaces of a base film; the fixed layer reacts with potassium ferricyanide to obtain a Prussian blue fixed sensing film;
(2) and (2) mixing the sensing membrane obtained in the step (1) with enzyme, and reacting to obtain the biological sensing membrane.
6. The preparation method of claim 5, wherein the method for disposing the modified carbon nanotube layer in step (1) comprises: filtering and depositing the carbon nano tube dispersion liquid on one surface of the base film to obtain a carbon nano tube layer; then adding polyvinyl alcohol reaction liquid and reacting to obtain the modified carbon nanotube layer;
preferably, the pressure of the filtration deposition is 0.5-1 bar;
preferably, the polyvinyl alcohol reaction solution further comprises glutaraldehyde;
preferably, the pH value of the polyvinyl alcohol reaction solution is 1-5;
preferably, the reaction temperature is 20-100 ℃;
preferably, the reaction time is 5-60 min.
7. The production method according to claim 5 or 6, wherein the setting method of the fixing layer of step (1) comprises: treating one side of the base film, which is far away from the modified carbon nanotube layer, by using a phenolic compound-containing reaction solution, and reacting to obtain a first fixed layer; reacting the first fixed layer with ferric trichloride to obtain the fixed layer;
preferably, the phenolic group-containing compound reaction liquid further comprises 3-aminopropyltriethoxysilane and/or polyethyleneimine;
preferably, the reaction time for obtaining the first fixed layer is 2-24 h;
preferably, the reaction pH value of the obtained first fixed layer is 7.5-9.5;
preferably, the reaction temperature for obtaining the first fixed layer is 25-30 ℃;
preferably, the reaction time of the first fixed layer and ferric trichloride is 1-10 h;
preferably, the reaction temperature of the first fixed layer and ferric trichloride is 25-30 ℃.
8. The method according to any one of claims 5 to 7, wherein the concentration of potassium ferricyanide is 5 to 40mM, more preferably 5 to 20 mM;
preferably, the reaction time in the step (1) is 1-10 h;
preferably, the temperature of the reaction in the step (1) is 20-60 ℃;
preferably, the pH value of the reaction in the step (1) is 1-3;
preferably, the reaction time in the step (2) is 2-60 h;
preferably, the temperature of the reaction in the step (2) is 4-30 ℃;
preferably, the pH value of the reaction in the step (2) is 3-6.
9. The method according to any one of claims 5 to 8, characterized by comprising the steps of:
(1) filtering and depositing the carbon nano tube dispersion liquid on one surface of the base film under the pressure of 0.5-1 bar to obtain a carbon nano tube layer; then adding reaction liquid of polyvinyl alcohol and glutaraldehyde with the pH value of 1-5, and reacting for 5-60 min at the temperature of 20-100 ℃ to obtain the modified carbon nanotube layer; then, treating one side of the base film, which is far away from the modified carbon nanotube layer, by using a phenolic compound-containing reaction solution, and reacting for 2-24 hours under the conditions that the pH value is 7.5-9.5 and the temperature is 25-30 ℃ to obtain a first fixed layer; adding a ferric trichloride solution, and reacting the first fixed layer with the ferric trichloride solution at the temperature of 20-30 ℃ for 1-10 h to obtain the fixed layer; the fixing layer and potassium ferricyanide react for 1-10 hours under the conditions that the pH value is 1-3 and the temperature is 20-60 ℃ to obtain the Prussian blue fixed sensing film;
(2) and (2) mixing the sensing membrane obtained in the step (1) with enzyme, and reacting for 2-60 hours under the conditions that the pH value is 3-6 and the temperature is 4-30 ℃ to obtain the biological sensing membrane.
10. A biosensing material, comprising the biosensing film according to any one of claims 1 to 4.
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