CN114045327B - Biological sensing film and preparation method and application thereof - Google Patents
Biological sensing film and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 77
- 108090000790 Enzymes Proteins 0.000 claims abstract description 77
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical class [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 55
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229960003351 prussian blue Drugs 0.000 claims abstract description 40
- 239000013225 prussian blue Substances 0.000 claims abstract description 40
- 230000008021 deposition Effects 0.000 claims abstract description 7
- 229940088598 enzyme Drugs 0.000 claims description 72
- 239000012528 membrane Substances 0.000 claims description 63
- 238000000034 method Methods 0.000 claims description 41
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 33
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- 238000006243 chemical reaction Methods 0.000 claims description 27
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- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 claims description 23
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- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 18
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 14
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 10
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
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- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 4
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
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- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical group OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/002—Electrode membranes
- C12Q1/003—Functionalisation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
- C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Emergency Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides a biological sensing film, a preparation method and application thereof. The biological sensing film comprises a modified carbon nano tube layer, a base film, a fixing layer and Prussian blue and enzyme which are sequentially arranged on the fixing layer. The biological sensing film has good conductivity by using the modified carbon nano tube layer; and by selecting a specific fixing layer, the targeting uniform deposition of Prussian blue and the efficient stable fixing of enzyme molecules are realized, and the Prussian blue and the enzyme are uniformly distributed on the biological sensing film and are not overlapped or 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 biosensing film can realize high-sensitivity detection of analytes in samples through the synergistic effect of the modified carbon nanotube layer, the base film, the fixing layer and Prussian blue and enzyme fixed on the fixing layer.
Description
Technical Field
The invention belongs to the technical field of biological sensing materials, and particularly relates to a biological sensing film, a preparation method and application thereof.
Background
Electrochemical sensors can accurately and rapidly detect a large number of analytes through the multifunctional combination of target molecule screening, specific recognition, signal transduction and readout, and have been widely applied to the fields of medical care, environmental monitoring, biosafety 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, enzymes are immobilized 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 biosensing film, a biosensing film and a monitoring device. The preparation method of the biological sensing film comprises the steps of carrying out electrochemical activation modification on the oxidation-reduction enzyme, then carrying out crosslinking treatment by using a chemical crosslinking agent, and coating the electrode surface to form the biological sensing film. The biological sensing film is stable and durable, can be detected for multiple times, and is particularly suitable for being used as a biological sensing film of a living body monitoring device.
However, enzyme-based electrochemical sensors still face a constant challenge: firstly, the electrochemical sensor commonly used mainly consists of two-dimensional plane electrodes, the two-dimensional plane and non-open pore structure of the electrodes lead to small specific surface area and few active sites, the immobilization and catalytic efficiency of enzymes are affected, and meanwhile, in-situ separation of analytes and interferents is difficult to realize. Compared with a two-dimensional plane electrode, the three-dimensional porous electrode is favorable for infiltration of electrolyte, the contact area between the analyte and the electrode is increased, and in addition, the larger specific surface area is favorable for improving the stability of the immobilized enzyme. The membrane, as a three-dimensional porous matrix, has the advantages described above, and also protects the enzyme molecules from the external environment. And convective mass transfer can accelerate the reaction of the enzyme with the substrate to increase sensitivity. In addition, the porous structure of the membrane is also advantageous for enhancing catalysis and amplifying detection signals.
Because of the lack of active functional groups on the surfaces of the conductive membrane and the electrocatalyst typified by Prussian blue, the conventional enzyme immobilization method is to soak the conductive membrane in a high concentration enzyme solution, crosslink ("Capillary-driven blood separation and in-situ electrochemical detection based on3D conductive gradient hollow fiber membrane",Wu H with glutaraldehyde after drying, and the like, biosens. Bioelectron.,2021, 171:112722). However, it is difficult to achieve a stable loading of biological enzymes on the membrane, resulting in high leakage or low activity of the enzymes, which in turn affects the sensitivity, stability, reproducibility and lifetime of the sensing membrane. The prior art discloses a biological sensing membrane, which is based on a unique structure of a gradient hollow fiber membrane, and enzyme-carrying nano particles with adjustable size are assembled in a three-dimensional gradient conductive membrane pore limit space by a mild and controllable filtration embedding method, so that higher enzyme carrying amount, detection sensitivity ("Modular assembly of enzyme loaded nanoparticles in 3D hollow fiber electrode for electrochemical sensing",Wu H and the like are obtained, and chem.Eng.J.,2021, 421:129721. Although physical entrapment improves the stability of immobilized enzymes, the lack of more robust chemical interactions between enzyme-loaded nanoparticles and conductive films still faces the problem of enzyme shedding.
Therefore, developing a biosensing membrane with high enzyme-carrying capacity, high sensitivity and good stability is a technical problem to be solved in the field.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a biological sensing film, and a preparation method and application thereof. The targeting uniform deposition of Prussian blue on the surface of the membrane is realized by modifying the base membrane by adopting a specific fixing layer, meanwhile, the phenol group on the phenol group-containing compound in the fixing layer can be oxidized into a quinone group, and enzymes are fixed on the biological sensing membrane through covalent bonds, so that the stability and reproducibility of the sensing membrane are enhanced by the abundant quinone group covalent immobilized enzymes, and a large amount of enzymes required by the traditional adsorption and crosslinking methods are avoided; meanwhile, prussian blue and enzyme are uniformly distributed on a sensing interface, so that the electron transfer efficiency of the biological sensing film is remarkably improved, and the biological sensing film has better sensing performance.
To achieve the purpose, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a biosensing membrane, which comprises a modified carbon nanotube layer, a base membrane, a fixed layer, prussian blue and enzyme, wherein the modified carbon nanotube layer, the base membrane, the fixed layer, and the Prussian blue and the enzyme are sequentially arranged on the fixed layer; the anchoring layer comprises a combination of a phenol group containing compound and ferric trichloride.
In the invention, the modified carbon nano tube layer enables the biological sensing film to have excellent conductive performance; the fixing layer adopts the combination of the phenol group-containing compound and the ferric trichloride, provides conditions for the stable fixing of the Prussian blue and the enzyme, can realize the targeted uniform deposition of Prussian blue nano particles on the surface of the base film, and avoids the agglomeration of the Prussian blue nano particles, so that the contact area of the Prussian blue and an analyte can be increased, and the electron transfer resistance can be reduced; meanwhile, prussian blue and enzyme are uniformly distributed at a sensing interface and 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.
As a preferable technical scheme of the invention, the modified carbon nano tube layer is a polyvinyl alcohol modified carbon nano tube 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, may be 1:4.5, 1:5. 1:5.5, etc.
In the invention, the polyvinyl alcohol and glutaraldehyde are adopted to modify the carbon nano tube under the acidic condition, so that the stability of the carbon nano tube on the surface of the film is improved, and the carbon nano tube cannot fall off; if the carbon nano tube is not modified, the carbon nano tube is continuously fallen off in the subsequent modification process, and the subsequent enzyme activity and sensitivity measurement are affected.
Preferably, the base film comprises an inorganic porous support film and/or a polymeric porous support film.
Preferably, the inorganic porous support membrane comprises any one or a combination of at least two of an alumina ceramic membrane, a silica ceramic membrane, or a titania ceramic membrane.
Preferably, the material of the porous polymer support membrane comprises any one or a combination of at least two of polyacrylonitrile, nylon, polyvinylidene fluoride, polyethersulfone or aromatic polyamide.
Preferably, the phenolic group-containing compound comprises tannic acid and/or polydopamine.
Preferably, the fixing layer further comprises 3-aminopropyl triethoxysilane and/or polyethylenimine.
Preferably, the immobilizing layer comprises a combination of tannic acid, 3-aminopropyl triethoxysilane, and ferric trichloride.
In the invention, the tannic acid is plant polyphenol with good hydrophilicity, and because the tannic acid is rich in catechol groups, the tannic acid can be adhered to the surface of a base film through various interactions, and meanwhile, because the tannic acid and 3-aminopropyl triethoxysilane can form a coating with a spherical structure through Michael addition reaction or Schiff base reaction, the adhesion of the tannic acid is further improved; and ferric trichloride is coordinated with catechol groups of tannic acid and amino groups of 3-aminopropyl triethoxy silane, so that acid-base stability of the immobilized layer is improved, conditions are provided for the subsequent targeting uniform deposition of Prussian blue, and meanwhile, phenolic groups on the tannic acid can be oxidized to form quinone groups, so that the immobilized enzyme can be used for the subsequent immobilized enzyme.
Preferably, the mass ratio of tannic acid, 3-aminopropyl triethoxysilane and ferric trichloride in the fixing layer is (0.005-4.5): (0.02-4.5): 1, for example 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 or the like, is preferably (0.8 to 1): (0.5-0.6): 1.
In the invention, the tannic acid, the 3-aminopropyl triethoxysilane and the ferric trichloride are in a specific proportion, the sensitivity and enzyme loading effect of the biological sensing film are best, and the excessive or insufficient quality of the tannic acid or the 3-aminopropyl triethoxysilane can cause the reduction of the sensitivity and enzyme loading of the biological sensing film.
Preferably, the Prussian blue is Prussian blue nano particles formed by reacting potassium ferricyanide with 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, etc., 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 or a combination of at least two of glucose oxidase, ethanol 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 electro-catalyst 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 a biosensing film according to the first aspect, the method comprising the steps of:
(1) The two surfaces of the base film are respectively provided with a modified carbon nano tube layer and a fixed layer; the fixed layer reacts with potassium ferricyanide to obtain a sensing film for fixing Prussian blue;
(2) And (3) mixing the sensing film obtained in the step (1) with enzyme, and reacting to obtain the biological sensing film.
As a preferred technical solution of the present invention, the method for setting the modified carbon nanotube layer in the 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 the polyvinyl alcohol reaction liquid to react to obtain the modified carbon nano tube 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, for example, 0.55bar, 0.6bar, 0.65bar, 0.7bar, 0.75bar, 0.8bar, 0.85bar, 0.9bar, 0.95bar, etc.
Preferably, the polyvinyl alcohol reaction liquid further comprises glutaraldehyde.
The pH of the polyvinyl alcohol reaction liquid is preferably 1 to 5, and may be, for example, 1,2, 3, 4, 5, or the like.
In the present invention, the polyvinyl alcohol is selected from the polyvinyl alcohol reaction solutions having different mass fractions, and the mass fraction of the polyvinyl alcohol may be 0.1 to 1%, for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, etc.
In the present invention, 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%, etc.
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 may be, for example, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or the like.
Preferably, the temperature of the reaction is 20 to 100 ℃, for example, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, and the like can be used.
Preferably, the reaction time is 5 to 60min, for example, 10min,15min,20min,25min,30min,35min,40min,45min,50min,55min, etc.
In the invention, polyvinyl alcohol and glutaraldehyde are adopted to modify the carbon nano tube in an acidic environment, and carboxyl groups on the carbon nano tube react with hydroxyl groups on polyethylene and aldehyde groups on glutaraldehyde, so that the modified carbon nano tube layer is obtained.
Preferably, the method for setting the fixing layer in the step (1) includes: treating one side of the base film far away from the modified carbon nano tube layer by using a reaction solution containing a phenol group compound, and reacting to obtain a first fixed layer; and the first fixing layer reacts with ferric trichloride to obtain the fixing layer.
Preferably, the phenolic group-containing compound reaction solution further comprises 3-aminopropyl triethoxysilane and/or polyethyleneimine.
Preferably, the reaction time for obtaining the first fixed layer is 2 to 24 hours, and may be, for example, 4 hours, 6 hours, 8 hours, 10 hours, 15 hours, 18 hours, 20 hours, 22 hours, and the like.
Preferably, the reaction pH for obtaining the first immobilized layer is 7.5 to 9.5, and may be, for example, 7.5, 8, 8.5, 9, 9.5, or the like.
The reaction temperature for obtaining the first fixed layer is preferably 25 to 30 ℃, and may be, for example, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, or the like.
Preferably, the reaction time of the first fixing layer and the ferric trichloride is 1-10 h, for example, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h and the like can be adopted.
Preferably, the temperature at which the first fixing layer reacts with ferric trichloride is 25 to 30 ℃, for example, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ or the like.
In the invention, the phenol group-containing compound is dissolved in Tris-HCl buffer solution to prepare the phenol group-containing compound Tris-HCl buffer solution with the concentration of 0.5-10 g/L for use; the 3-aminopropyl triethoxysilane is dissolved in ethanol to prepare a 3-aminopropyl triethoxysilane ethanol solution with the concentration of 2-10 g/L for use; the polyethyleneimine is dissolved in water to prepare polyethyleneimine water solution with the concentration of 2-10 g/L for use; the ferric trichloride is dissolved in water to prepare ferric trichloride aqueous solution with the concentration of 2-10 g/L for use.
The concentration of potassium ferricyanide is preferably 5 to 40mM, for example, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, etc., and more preferably 5 to 20mM.
In the present invention, too high or too low a concentration of potassium ferricyanide results in a decrease in the sensitivity of the biosensing membrane.
Preferably, the reaction time in step (1) is 1 to 10 hours, and may be, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, etc.
Preferably, the temperature of the reaction in the step (1) is 20 to 60℃and may be, for example, 25℃30℃35℃40℃45℃50℃55 ℃.
Preferably, the pH of the reaction in step (1) is 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 to 60 hours, and may be, for example, 4 hours, 8 hours, 12 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, etc.
Preferably, the temperature of the reaction in step (2) is 4 to 30 ℃, for example 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, etc.
Preferably, the pH of the reaction in step (2) is 3 to 6, and may be, for example, 3.5, 4.0, 4.5, 5.0, 5.5, etc.
As a preferable 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 polyvinyl alcohol with pH value of 1-5 and glutaraldehyde reaction liquid, and reacting for 5-60 min at 20-100 ℃ to obtain the modified carbon nano tube layer; then, treating one side of the base film far away from the modified carbon nano tube layer by adopting a reaction solution containing a phenol group compound, 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 ferric trichloride solution, and reacting the first fixed layer with the ferric trichloride solution at 20-30 ℃ for 1-10 h to obtain the fixed layer; the fixed layer reacts with potassium ferricyanide for 1-10 hours under the conditions that the pH value is 1-3 and the temperature is 20-60 ℃ to obtain a sensing film fixed with Prussian blue;
(2) Mixing the sensing film 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 film.
In the present invention, the preparation method is implemented by adopting a specific sequence of steps. In the invention, if the base film is treated by adopting a mixed solution of ferric trichloride and potassium ferricyanide, although enzyme immobilization sites can be provided, ferric trichloride and 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 the quinone site for enzyme immobilization is covered, and the response capability to glucose is lower.
In a third aspect, the present invention provides a biosensing material comprising a biosensing film according to the first aspect.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has the beneficial effects that:
The biological sensing film provided by the invention has good conductivity through the modified carbon nano tube layer, avoids aggregation of Prussian blue through selecting a specific fixing layer, can stably fix enzyme and optimize spatial distribution of Prussian blue and enzyme, and improves sensing performance; as the preferable technical scheme of the invention, the sensitivity of the biological sensing film is more than 8, and the enzyme loading amount is more than or equal to 365 mug, so that the high-sensitivity detection of the analyte in the sample can be realized.
Detailed Description
To facilitate understanding of the present invention, examples are set forth below. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The following examples and comparative examples do not identify specific techniques or conditions, which may be followed by those described in the literature in the field or by the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
The embodiment provides a biological sensing membrane, which comprises a modified carbon nano tube layer, a base membrane (nylon membrane, aperture 0.22 μm), a fixing layer, prussian blue and glucose oxidase which are fixed on the fixing layer, wherein the modified carbon nano tube layer, the base membrane, the fixing layer, the Prussian blue and the glucose oxidase are sequentially arranged; the fixed layer comprises the following components in percentage by mass of 0.889:0.555: tannic acid of 1, 3-aminopropyl triethoxysilane and ferric trichloride.
The embodiment provides a preparation method of the biological sensing film, which comprises the following specific steps:
(1) Placing the base film in a filter, filtering and depositing 25mL of carbon nano tube dispersion liquid at the pressure of 0.65bar, adding 1mL of mixed solution of polyvinyl alcohol (with the mass fraction of 0.5 percent), 0.5mL of glutaraldehyde (with the mass fraction of 0.5 percent) and 0.5mL of hydrochloric acid (with the mass fraction of 31.5 percent), and reacting at the temperature of 60 ℃ for 5min to obtain the base film provided with the modified carbon nano tube layer;
(2) Tannic acid and 3-aminopropyl triethoxysilane are respectively dissolved in 10mM Tris-HCl buffer solution (pH 8.5) and ethanol to prepare tannic acid Tris-HCl buffer solution with concentration of 2g/L and 3-aminopropyl triethoxysilane ethanol solution with concentration of 10g/L, and then the tannic acid Tris-HCl buffer solution and the 3-aminopropyl triethoxysilane ethanol solution are mixed according to the volume ratio of 8:1, mixing to obtain a mixed solution; adopting the mixed solution to treat one side of the base film far away from the modified carbon nano tube layer in the step (1), and reacting for 18 hours at room temperature and the rotating speed of 150rpm to obtain a first fixed layer; adding 2g/L ferric trichloride aqueous solution into the first fixed layer, reacting for 1h at room temperature at the rotating speed of 150rpm, and washing with deionized water at 150rpm for 1h to obtain a base film with two surfaces respectively provided with a modified carbon nano tube layer and a fixed layer;
(3) Mixing the base film obtained in the step (2) with an acidic potassium ferricyanide aqueous solution (pH is 1.5) with the concentration of 7.4mM and 20mL, and reacting the fixed layer with potassium ferricyanide at 150rpm and 35 ℃ for 1h to obtain a sensing film fixed with Prussian blue; subsequently, glucose oxidase acetate buffer solution (ph=5.0.10 mm acetate buffer solution) was added at a concentration of 0.25g/L, and the immobilized layer was reacted with glucose oxidase at 4 ℃ for 48 hours, and rinsed with deionized water for 1 hour, to obtain the biosensing membrane.
Example 2
This example provides a biosensing membrane which differs from example 1 only in that the concentration of the acidic aqueous potassium ferricyanide solution in step (3) in the method of producing a biosensing membrane is 5mM, and other raw materials, steps and parameters are the same as in example 1.
Example 3
This example provides a biosensing membrane which differs from example 1 only in that the concentration of the acidic aqueous potassium ferricyanide solution in step (3) in the method of producing a biosensing membrane is 20mM, and other raw materials, steps and parameters are the same as in example 1.
Example 4
This example provides a biosensing membrane which differs from example 1 only in that glucose oxidase is replaced with equal mass of lactate oxidase in the biosensing membrane, and other components, amounts and structures are the same as in example 1.
This example provides a method for preparing the biosensing membrane, and the specific steps are the same as in example 1.
Example 5
This example provides a biosensing film differing from example 1 only in that in step (2) of the biosensing film production method, the 3-aminopropyl triethoxysilane ethanol solution is replaced with an equal concentration and equal volume of polyethyleneimine aqueous solution, and other raw materials, steps and parameters are the same as in example 1.
Example 6
This example provides a biosensing membrane which differs from example 1 only in that the ratio by volume of Tris-HCl tannate buffer solution to 3-aminopropyl triethoxysilane ethanol solution in step (2) is 5:1, other materials, steps and parameters were the same as in example 1.
Example 7
This example provides a biosensing membrane which differs from example 1 only in that the ratio by volume of Tris-HCl tannate buffer solution to 3-aminopropyl triethoxysilane ethanol solution in step (2) is 2:1, other materials, steps and parameters were the same as in example 1.
Example 8
This example provides a biosensing membrane which differs from example 1 only in that tannic acid and 3-aminopropyl triethoxysilane are replaced by equal mass of polydopamine in the immobilization layer, and the other components, amounts and structures are the same as in example 1.
This example provides a method for preparing the biosensing membrane, and the specific steps are the same as in example 1.
Example 9
This example provides a biosensing membrane which differs from example 1 only in that in the preparation method of the biosensing membrane, after the base membrane is treated with Tris-HCl tannate buffer solution and 3-aminopropyl triethoxysilane ethanol solution in step (2), the base membrane is treated with a mixed solution of ferric trichloride of equal mass and acidic potassium ferricyanide of equal mass, and other raw materials, steps and parameters are the same as in example 1.
Comparative example 1
This comparative example provides a biosensing film differing from example 1 only in that step (2) in the preparation method of the biosensing film does not treat the base film with Tris-HCl tannate buffer solution and 3-aminopropyl triethoxysilane ethanol solution, and other raw materials, steps and parameters are the same as example 1.
Comparative example 2
This comparative example provides a biosensing film differing from example 1 only in that in the preparation method of the biosensing film, step (2) of treating the base film with Tris-HCl tannate buffer solution and 3-aminopropyl triethoxysilane ethanol solution, the base film is treated with an equal mass of acidic potassium ferricyanide, and then with an equal mass of ferric trichloride solution, and other raw materials, steps and parameters are the same as in example 1.
Comparative example 3
This comparative example provides a biosensing film differing from example 1 only in that step (3) of the biosensing film production method does not treat the base film with an acidic aqueous potassium ferricyanide solution, and other raw materials, steps and parameters are the same as example 1, i.e., the finally obtained biosensing film does not contain prussian blue.
Comparative example 4
This comparative example provides a biosensing membrane which differs from example 1 only in that step (3) of the biosensing membrane preparation method does not treat the base membrane with a glucose oxidase acetate buffer solution, and other raw materials, steps and parameters are the same as in example 1, i.e., the finally obtained biosensing membrane contains no enzyme.
Comparative example 5
This comparative example provides a biosensing film differing from example 1 only in that step (1) does not filter and deposit a carbon nanotube dispersion on a base film, and a mixed solution of polyvinyl alcohol, glutaraldehyde and hydrochloric acid is not added for reaction, and other steps and parameters are the same as example 1, to finally obtain a biosensing film containing no modified carbon nanotube layer.
Performance testing
(1) Enzyme activity: taking glucose oxidase as an example, phenol, 4-aminoantipyrine, horseradish peroxidase and glucose were dissolved in an acetate buffer (10 mM, 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 at 505nm of the solution was recorded. One unit of catalytic activity (U) is defined as the amount of enzyme that consumes 1. Mu. Mol of H 2O2 per minute under the assay conditions (25 ℃, pH 5.0);
Lactate oxidase: phenol, 4-aminoantipyrine, horseradish peroxidase and lactic acid were dissolved in acetic acid buffer (10 mM, ph=5.0) to prepare substrate solutions, wherein their concentrations were 40mM, 4mM, 40mM, 100mM, respectively; then, lactate oxidase was added to 20mL of the above substrate solution under the action of a magnetic stirrer, and the change in absorbance at 505nm of the solution was recorded. One unit of catalytic activity (U) is defined as the amount of enzyme that consumes 1. Mu. Mol of H 2O2 per minute under the assay conditions (25 ℃, pH 5.0);
(2) Enzyme loading amount: calculating 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 enzyme immobilized on the surface of the biological sensing film 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 measurement);
Enzyme loading (μg) = (As-Ar-Aw)/as×ms;
Wherein As, ar and Aw are respectively the catalytic activities (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 (50 mM, pH 6.5, containing 0.1M KCl), and was added to the three-electrode reaction system every 100 to 300 seconds by amperometry, with the operating voltage set at-0.5V. Finally, the relation between the response current and the concentration of the analyte is obtained, and the slope is the sensitivity of the biological sensing film.
The specific test results are shown in table 1:
TABLE 1
As can be seen from the table, the biological sensing film provided by the invention is favorable for targeting uniform deposition of Prussian blue and efficient and stable fixation of enzyme by selecting a specific fixing layer, and simultaneously optimizes the spatial distribution of the Prussian blue and is more favorable for electron transfer. The biological sensing film can realize high-sensitivity detection of analytes in samples through the synergistic effect of the modified carbon nanotube layer, the base film, the fixing layer and Prussian blue and enzyme fixed on the fixing layer.
As is clear from comparison of examples 1 with examples 2 and 3, the catalytic sensing effect of Prussian blue formed is affected by different concentrations of potassium ferricyanide, and the preferred concentration of potassium ferricyanide in the present invention is 7.4mM, at this time, the enzyme loading and sensitivity of the biosensing membrane of the present invention are the best. As is clear from comparison of example 1 with examples 4 and 5, the biosensing membrane also has excellent sensitivity and enzyme loading by selecting lactate oxidase or using a combination of tannic acid and polyethyleneimine; as is clear from comparison of examples 1 with examples 6 and 7, the mass ratio of tannic acid to 3-aminopropyl triethoxysilane affects the distribution of Prussian blue and the number of quinone-based sites immobilized by the subsequent enzyme, and the mass ratio of tannic acid to 3-aminopropyl triethoxysilane is preferably 1.6:1, and at this time, the enzyme-carrying amount of the biosensing membrane of the invention is the highest and the sensitivity is the best. As is clear from a comparison of example 1 with examples 8 to 9, the performance of the biosensing film is degraded when a specific combination is not used in the fixed layer or a specific procedure is not performed in the preparation method.
As can be seen from a comparison of example 1 with comparative example 1, the biosensing membrane was almost unresponsive to glucose, indicating the lack of sites for enzyme immobilization. As can be seen from the comparison between the example 1 and the comparative example 2, the sensitivity and enzyme loading amount of the biosensing membrane are 0 when the acidic potassium ferricyanide solution is added first and then the ferric trichloride solution is added, because the acidic potassium ferricyanide solution is added first to cause the tannic acid and the 3-aminopropyl triethoxysilane to be protonized and decomposed to fall off, thereby affecting the immobilization and sensing performance of the subsequent enzyme. As is clear from comparison of example 1 with comparative examples 3 to 5, when a certain component is absent in the biosensing film, the biosensing film sensitivity is lowered.
In summary, the biosensing film of the invention has high sensitivity, high enzyme loading capacity and excellent performance through the synergistic effect of the modified carbon nanotube layer, the base film, the fixing layer and Prussian blue and enzyme fixed on the base film, and through the preparation method of specific steps.
While the foregoing is directed to embodiments of the present invention, other and further details of the invention may be had by the present invention, it should be understood that the foregoing description is merely illustrative of the present invention and that no limitations are intended to the scope of the invention, except insofar as modifications, equivalents, improvements or modifications are within the spirit and principles of the invention.
Claims (30)
1. The biological sensing film is characterized by comprising a modified carbon nano tube layer, a base film, a fixed layer, prussian blue and enzyme which are sequentially arranged, wherein the Prussian blue and the enzyme are fixed on the fixed layer;
the fixing layer comprises a combination of tannic acid, 3-aminopropyl triethoxysilane and ferric trichloride;
the modified carbon nano tube layer is a polyvinyl alcohol modified carbon nano tube layer;
the enzyme comprises glucose oxidase or lactate oxidase;
The mass ratio of the polyvinyl alcohol to the carbon nano tube in the modified carbon nano tube layer is 1: (4.5 to 5.5);
The mass ratio of tannic acid, 3-aminopropyl triethoxysilane and ferric trichloride in the fixed layer is (0.8-1): (0.5-0.6): 1, a step of;
the base film comprises an inorganic porous support film or a high-molecular porous support film.
2. The biosensing membrane according to claim 1, wherein said inorganic porous support membrane comprises any one or a combination of at least two of an alumina ceramic membrane, a silica ceramic membrane or a titania ceramic membrane.
3. The biosensing membrane according to claim 1, characterized in that the material of said polymeric porous support membrane comprises any one or a combination of at least two of polyacrylonitrile, nylon, polyvinylidene fluoride, polyethersulfone or aromatic polyamide.
4. The biosensing membrane of claim 1, wherein said prussian blue is a prussian blue nanoparticle formed by reacting potassium ferricyanide with ferric trichloride in an immobilization layer.
5. The biosensing membrane according to claim 4, wherein a mass ratio of potassium ferricyanide to ferric trichloride is (0.8-6.5): 1.
6. The biosensing membrane according to claim 5, wherein a mass ratio of potassium ferricyanide to ferric trichloride is (0.8-3): 1.
7. A method of producing a biosensing film according to any one of claims 1-6, characterized in that the method of producing comprises the steps of:
(1) The two surfaces of the base film are respectively provided with a modified carbon nano tube layer and a fixed layer; the fixed layer reacts with potassium ferricyanide to obtain a sensing film for fixing Prussian blue;
(2) And (3) mixing the sensing film obtained in the step (1) with enzyme, and reacting to obtain the biological sensing film.
8. The method of claim 7, wherein the method of 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; and then adding the polyvinyl alcohol reaction liquid to react to obtain the modified carbon nano tube layer.
9. The method according to claim 8, wherein the pressure of the filtration deposition is 0.5 to 1bar.
10. The method according to claim 8, wherein the polyvinyl alcohol reaction solution further comprises glutaraldehyde.
11. The method according to claim 8, wherein the pH of the polyvinyl alcohol reaction solution is 1 to 5.
12. The process according to claim 8, wherein the temperature of the reaction is 20 to 100 ℃.
13. The method according to claim 8, wherein the reaction time is 5 to 60 minutes.
14. The method of claim 7, wherein the method of disposing the fixing layer in step (1) comprises: treating one side of the base film far away from the modified carbon nano tube layer by using a reaction solution containing a phenol group compound, and reacting to obtain a first fixed layer; and the first fixing layer reacts with ferric trichloride to obtain the fixing layer.
15. The method according to claim 14, wherein the phenol group-containing compound reaction liquid further comprises 3-aminopropyl triethoxysilane.
16. The method of claim 14, wherein the reaction time to obtain the first anchoring layer is 2 to 24 hours.
17. The method of claim 14, wherein the reaction pH to obtain the first immobilized layer is 7.5 to 9.5.
18. The method of claim 14, wherein the reaction temperature to obtain the first anchoring layer is 25-30 ℃.
19. The method of claim 14, wherein the first anchoring layer is reacted with ferric trichloride for a period of 1 to 10 hours.
20. The method of claim 14, wherein the first anchoring layer is reacted with ferric trichloride at a temperature of 25-30 ℃.
21. The method according to claim 7, wherein the concentration of potassium ferricyanide is 5 to 40mM.
22. The method according to claim 21, wherein the concentration of potassium ferricyanide is 5 to 20mM.
23. The process according to claim 7, wherein the reaction time in step (1) is 1 to 10 hours.
24. The process according to claim 7, wherein the temperature of the reaction in step (1) is 20 to 60 ℃.
25. The process according to claim 7, wherein the pH of the reaction in step (1) is 1 to 3.
26. The process according to claim 7, wherein the reaction time in step (2) is 2 to 60 hours.
27. The process according to claim 7, wherein the temperature of the reaction in step (2) is 4 to 30 ℃.
28. The process according to claim 7, wherein the pH of the reaction in step (2) is 3 to 6.
29. The preparation method according to claim 7, characterized in that the preparation method comprises 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 polyvinyl alcohol with pH value of 1-5 and glutaraldehyde reaction liquid, and reacting for 5-60 min at 20-100 ℃ to obtain the modified carbon nano tube layer; then, treating one side of the base film far away from the modified carbon nano tube layer by adopting a reaction solution containing a phenol group compound, 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 ferric trichloride solution, and reacting the first fixed layer with the ferric trichloride solution at 20-30 ℃ for 1-10 h to obtain the fixed layer; the fixed layer reacts with potassium ferricyanide for 1-10 hours under the conditions that the pH value is 1-3 and the temperature is 20-60 ℃ to obtain a sensing film fixed with Prussian blue;
(2) Mixing the sensing film 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 film.
30. A biosensing material, characterized in that it comprises a biosensing film according to any one of claims 1-6.
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