WO2023246661A1 - 天然高分子化合物在生物传感器中的应用 - Google Patents

天然高分子化合物在生物传感器中的应用 Download PDF

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WO2023246661A1
WO2023246661A1 PCT/CN2023/100831 CN2023100831W WO2023246661A1 WO 2023246661 A1 WO2023246661 A1 WO 2023246661A1 CN 2023100831 W CN2023100831 W CN 2023100831W WO 2023246661 A1 WO2023246661 A1 WO 2023246661A1
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natural polymer
polymer compound
biosensor
oxidized
film layer
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PCT/CN2023/100831
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English (en)
French (fr)
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刘良岭
林静
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上海联影微电子科技有限公司
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Priority to EP23821472.0A priority Critical patent/EP4343316A1/en
Publication of WO2023246661A1 publication Critical patent/WO2023246661A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction

Definitions

  • This specification relates to the technical field, and in particular to the application of a natural polymer compound in a biosensor.
  • Biosensors are usually used to measure substances in living organisms, and their applications are becoming more and more widespread. For example, biosensors can be used to measure blood sugar in the body. However, many implantable biosensors are susceptible to immune responses in the body and tend to have a short lifespan.
  • some biotoxic cross-linking agents such as glutaraldehyde, photoinitiators or acrylics, are used in the existing sensitive element fixation process in biosensors, which not only affects the activity and stability of the sensitive element, but also affects the activity and stability of the sensitive element. The residual cross-linking agent will also strengthen the body's rejection reaction, thereby reducing the service life, and is not conducive to the safety of mass production.
  • the biosensor may include a working electrode including an enzyme membrane layer.
  • the enzyme film layer includes sensitive elements fixed with an oxidized natural polymer compound as a cross-linking agent, wherein the oxidized natural polymer compound is obtained by oxidation treatment of the natural polymer compound.
  • natural polymer compounds include cellulose and its derivatives, sodium alginate, chitosan and its derivatives, hyaluronic acid, konjac mannan, starch and its derivatives, pectin and carrageenan at least one of them.
  • the oxidized natural polymer compound is a dialdehyde-formed natural polymer compound.
  • the oxidation degree of the oxidized natural polymer compound is 5% to 85%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 30% to 60%.
  • the sensitive motif includes one of glucose oxidase, lactate oxidase, uricase, or alcohol oxidase.
  • the enzyme film layer further includes an enzyme stabilizer.
  • the thickness of the enzyme film layer ranges from 0.1 ⁇ m to 20 ⁇ m.
  • the thickness of the enzyme film layer ranges from 1 ⁇ m to 5 ⁇ m.
  • the biosensor further includes at least one of a flexible substrate, a reference electrode, and a counter electrode.
  • the thickness of the flexible substrate ranges from 10 ⁇ m to 300 ⁇ m.
  • the working electrode further includes at least one of a metal layer and a protective layer.
  • the metal layer of the working electrode is a first metal layer, a second metal layer and a third metal layer in order from the inside to the outside; wherein the first metal layer is Cr, Ti, Ni- At least one of Cr; the second metal layer is at least one of gold, copper, and silver; and the third metal layer is at least one of platinum, platinum-iridium, and platinum-carbon.
  • the protective layer includes at least one of polyurethane, polyethylene glycol, cellulose acetate, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene.
  • One embodiment of this specification provides a method for preparing a biosensor.
  • the method includes: oxidizing natural polymer compounds to obtain oxidized natural polymer compounds; and using the oxidized natural polymer compounds as cross-linking agents to fix sensitive motifs on the biological in the working electrode of the sensor.
  • oxidizing natural polymer compounds to obtain oxidized natural polymer compounds specifically includes the following steps: dissolving the natural polymer compound in a first solvent to obtain a natural polymer compound solution; dissolving the oxidant In the second solvent, an oxidizing solution is obtained; and the natural polymer compound solution and the oxidizing solution are mixed, and the oxidized natural polymer compound is obtained after an oxidation reaction.
  • the oxidation degree of the oxidized natural polymer compound is 5% to 85%.
  • the oxidation degree of the oxidized natural polymer compound is 30% to 60%.
  • the working electrode includes an enzyme film layer, and the enzyme film layer includes the sensitive element immobilized with the oxidized natural polymer compound.
  • the above method further includes a method of preparing the enzyme film layer.
  • the preparation method of the enzyme film layer includes the following steps: mixing the enzyme stabilizer, the sensitive element and the oxidized natural polymer compound in a third solvent to obtain a mixed solution; and The mixed solution is coated on the metal layer, and the enzyme film layer is obtained after cross-linking reaction.
  • the concentration of the sensitive motif is 0.5% ⁇ 20%; the concentration of the oxidized natural polymer compound is 0.1% ⁇ 20%; the concentration of the enzyme stabilizer It is 0.5% to 20%; the temperature of the cross-linking reaction is 0°C to 37°C.
  • the concentration of the sensitive motif is 2% to 10%; the concentration of the oxidized natural polymer compound is 5% to 15%; and the concentration of the enzyme stabilizer is It is 0.5% to 5%; the temperature of the cross-linking reaction is 5°C to 15°C.
  • One embodiment of this specification also provides the application of the biosensor in biosensing.
  • the biosensor is used to detect at least one of glucose, uric acid, lactic acid or alcohols.
  • Figure 1 is a schematic plan view of a flexible electrode in a biosensor according to some embodiments of this specification
  • Figure 2 is a schematic cross-sectional view of a working electrode in a flexible electrode according to some embodiments of this specification
  • Figure 3A is a graph showing sensitivity test results of a biosensor according to some embodiments of this specification.
  • Figure 3B is a diagram of stability test results of a biosensor according to some embodiments of this specification.
  • Figure 4A is a graph of sensitivity test results of yet another biosensor according to some embodiments of this specification.
  • Figure 4B is a diagram of stability test results of yet another biosensor according to some embodiments of this specification.
  • Figure 5A is a graph of sensitivity test results of yet another biosensor according to some embodiments of this specification.
  • Figure 5B is a graph showing stability test results of yet another biosensor according to some embodiments of this specification.
  • Figure 6 is a chart showing the cytotoxicity test results of the working electrode according to some embodiments of this specification.
  • system means of distinguishing between different components, elements, parts, portions or assemblies at different levels.
  • said words may be replaced by other expressions if they serve the same purpose.
  • a biosensor is a device that can convert biological signals into readable electrical signals. It is used to detect and monitor chemical substances, bioactive molecules, metabolic activities of cells and other biological processes in organisms. It is widely used in medical and health care, Environmental monitoring and food safety and other fields.
  • the biosensor in this specification may include a biometric module, a signal conversion module, an energy supply module, a data processing module and an output module.
  • Biorecognition modules usually include sensitive primitives such as antibodies, enzymes, nucleic acids, and cells. During the working process of the biosensor, the sensitive primitives in the biorecognition module specifically recognize the molecules to be measured, resulting in some physical or chemical events, resulting in changes in chemical signals.
  • the molecules to be tested may include biomolecules, metabolites, drugs, or toxins.
  • biomolecules may include proteins, nucleic acids, enzymes, cytokines, and the like. Metabolites can include glucose, uric acid, lactic acid, ethanol, etc.
  • the signal converter in the signal conversion module can convert chemical signals into readable electrical signals.
  • Signal converters include electrochemical converters, optical converters, piezoelectric converters and thermal converters.
  • the basic working principle of the electrochemical converter is to generate current or potential difference through the electrochemical reaction between the biometric module and the electrode. This current or potential difference can be measured and recorded by the detection system.
  • the energy supply module is a module that provides energy for the biosensing process.
  • Energy supply methods include pure chemical energy (such as oxygen), solar energy, motion energy or wireless electromagnetic radiation.
  • the data processing module can further process the electrical signals in the signal conversion module into signals that are easier for users to understand.
  • the data processing module may include electrical signal acquisition, electrical signal processing and other steps.
  • the output module can present the collection results processed by the data processing module to the user.
  • users include but are not limited to personnel in medical and health institutions, personal health managers, athletes, sports enthusiasts, personnel in environmental monitoring agencies, and food or beverage production or quality supervisory personnel.
  • the biosensor includes a working electrode including an enzyme membrane layer.
  • the enzyme film layer includes sensitive elements fixed with an oxidized natural polymer compound as a cross-linking agent, wherein the oxidized natural polymer compound is obtained by oxidation treatment of a natural polymer compound.
  • the biosensor further includes at least one of a flexible substrate, a reference electrode, and a counter electrode.
  • the thickness of the flexible substrate ranges from 10 ⁇ m to 300 ⁇ m.
  • the flexible substrate is made of at least one of polyimide and polyethylene terephthalate.
  • the working electrode further includes at least one of a metal layer and a protective layer.
  • the metal layer of the working electrode is a first metal layer, a second metal layer and a third metal layer in order from the inside to the outside; wherein the first metal layer is Cr, Ti, Ni- At least one of Cr; the second metal layer It is at least one of gold, copper, and silver; the third metal layer is at least one of platinum, platinum-iridium, and platinum-carbon.
  • the protective layer includes at least one of polyurethane, polyethylene glycol, cellulose acetate, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene.
  • the biosensor includes a flexible electrode 100.
  • the flexible electrode 100 includes a flexible substrate 10 and a working electrode 20 , a reference electrode 30 and a counter electrode 40 located on the same surface of the flexible substrate 10 .
  • a metal layer can be covered on the flexible substrate 10 through micro-nano processing, and the metal layer can be patterned to form the working electrode 20 , the reference electrode 30 and the Counter electrode 40 is a three-electrode system.
  • Micro-nano processing refers to a series of semiconductor process-based technologies such as photolithography, development, magnetron sputtering, wet etching, plasma cleaning and laser cutting. Preparing flexible electrodes through micro-nano processing technology can improve processing accuracy.
  • the working electrode 20 includes a metal layer 201 , an enzyme film layer 202 and a protective layer 203 that are stacked in sequence.
  • the metal layer 201 is located between the flexible substrate 10 and the protective layer 203 .
  • the metal layer 201 includes a first metal part (not shown in the figure), a second metal part (not shown in the figure) and a third metal part (not shown in the figure) that are stacked in sequence. ).
  • the material of the first metal part includes chromium, titanium or nickel-chromium alloy
  • the material of the second metal part includes gold, copper or silver
  • the material of the third metal part includes platinum, platinum, etc. Iridium alloy or platinum carbon mixture.
  • the second metal part has good electrical conductivity, which improves the electron transmission efficiency of the biosensor
  • the third metal part has a catalytic effect on hydrogen peroxide, reducing the impact of hydrogen peroxide generated during the enzyme catalysis on the catalytic reaction. Impact.
  • the thickness of the enzyme film layer ranges from 0.1 ⁇ m to 20 ⁇ m. Preferably, the thickness of the enzyme film layer ranges from 1 ⁇ m to 5 ⁇ m.
  • a spin coating process can be used to control the thickness of the enzyme film layer by controlling the rotation speed of the spin coating machine. In some embodiments, the amount of cross-linking agent and cross-linking temperature can be adjusted to achieve effective control of the thickness of the enzyme film layer.
  • Natural polymer compounds are compounds that can be produced by organisms themselves through biochemical effects or photosynthesis. Compared with other synthetic organic compounds, natural polymer compounds are more biocompatible because they are derived from living organisms. After entering the human body, the human body will greatly reduce the occurrence of immune rejection reactions. Natural polymer compounds contain multiple repeating structural units. For example, sodium alginate is composed of ⁇ -D-mannuronic acid (M) and ⁇ -L-glucuronic acid (G) residues connected through 1,4-glycosidic bonds, which are repeatedly arranged into MM fragments, GG fragments or Natural polymer compound of MG fragment.
  • M ⁇ -D-mannuronic acid
  • G ⁇ -L-glucuronic acid
  • the natural polymer compound is a natural polysaccharide polymer compound.
  • Polysaccharide in this article refers to a natural polymer compound composed of multiple monosaccharide molecules combined through glycosidic bonds through dehydration and condensation.
  • the polysaccharide polymer compounds include plant polysaccharides (such as cellulose, hemicellulose, starch, pectin), animal polysaccharides (such as chitin, chitosan, heparin, chondroitin sulfate), Agar polysaccharides (such as agar, alginic acid, carrageenan), fungal polysaccharides (such as D-glucan, D-galactan, mannan), microbial polysaccharides (such as dextran, xanthan gum, gelatin lactose, pullulose).
  • plant polysaccharides such as cellulose, hemicellulose, starch, pectin
  • animal polysaccharides such as chitin, chitosan, heparin, chondroitin sulfate
  • the natural polymer compound contains a hydroxyl group.
  • the natural polymer compounds include cellulose and its derivatives, sodium alginate, chitosan and its derivatives, hyaluronic acid, konjac mannan, starch and its derivatives, pectin, and At least one of the carrageenans.
  • the natural polymer compound has high mechanical strength, and after being used as a sensitive unit fixed by a cross-linking agent, the working electrode has better stability.
  • the natural polymer compound includes sodium alginate or konjac mannan.
  • the natural polymer compound can be oxidized by an oxidizing agent to obtain the oxidized natural polymer compound.
  • Oxidizing agents include, but are not limited to, metal oxides and their salts (such as chromium reagent, potassium permanganate, manganese dioxide), nitric acid, periodic acid, periodate, or dimethyl sulfoxide.
  • the hydroxyl groups in the oxidized natural polymer compound are at least partially oxidized to form aldehyde groups.
  • At least partially oxidized in this article means that at least one hydroxyl group of the natural polymer compound is oxidized.
  • Part may refer to the proportion of hydroxyl groups.
  • “part” can be one-half, one-fifth, or one-tenth, which respectively represent one-half, one-fifth, or one-fifth of the total number of hydroxyl groups in the natural polymer compound. Or a tenth.
  • the oxidation degree of the natural polymer compound can be controlled by controlling the concentration or type of the oxidizing agent.
  • the oxidized natural polymer compound is a dialdehyde-formed natural polymer compound.
  • Dialdehydeization refers to the oxidation of two hydroxyl groups on the structural units of natural polymer compounds into aldehyde groups.
  • dialdehyde-based cellulose can be obtained by selective oxidation of the hydroxyl groups on C2-C3 of the glucose units of cellulose by periodate, and the hydroxyl groups on C2-C3 are oxidized into aldehyde groups.
  • At least one of sodium alginate, chitosan and its derivatives, hyaluronic acid, konjac mannan, starch and its derivatives, pectin and carrageenan can be mixed with an oxidizing agent, Obtained dialdehyde-based sodium alginate, dialdehyde-based chitosan and its derivatives, dialdehyde-based hyaluronic acid, dialdehyde-based konjac mannan, dialdehyde-based starch and its derivatives, dialdehyde-based pectin and dialdehyde-based pectin. At least one kind of aldehyde carrageenan.
  • the oxidation degree of the oxidized natural polymer compound is 5% to 85%.
  • the degree of oxidation refers to the proportion of hydroxyl groups in natural polymer compounds that are oxidized to aldehyde groups.
  • the aldehyde group content can be used to express the oxidation degree of oxidized natural polymer compounds. For example, if the aldehyde group content is 6%, the oxygen of the natural polymer compound will be oxidized.
  • the chemical degree is 6%.
  • the chemical titration method can be used to measure the aldehyde group content of the oxidized natural polymer compound. The method for measuring the degree of oxidation is not specifically limited in this article.
  • the oxidation degree of the oxidized natural polymer compound is 5%-60%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 20%-70%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 30%-80%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 5%, 20%, 30%, 40%, 50%, 60% or 85%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 30% to 60%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 35%, 45%, or 55%.
  • Sensitive motifs are bioactive units that selectively act on target substances (eg, substrates of enzymes) in the enzyme membrane layer.
  • the sensitive moiety may be a compound bearing a primary amino group.
  • the sensitive motif includes one of glucose oxidase, lactate oxidase, uricase, or alcohol oxidase.
  • the sensitive motif is glucose oxidase.
  • the enzyme film layer further includes an enzyme stabilizer.
  • Enzyme stabilizer is a substance used to enhance the stability and activity of enzymes. It can protect enzymes from environmental factors (such as temperature, pH, oxygen, etc.) and extend the service life and activity of enzymes. Enzyme stabilizers include, but are not limited to, polyethylene glycol (PEG), proteins, peptides, amino acids, cholesterol, etc.
  • the thickness of the enzyme film layer ranges from 0.1 ⁇ m to 20 ⁇ m. In some embodiments, the thickness of the enzyme film layer ranges from 0.1 ⁇ m to 10 ⁇ m. In some embodiments, the thickness of the enzyme film layer ranges from 0.1 ⁇ m to 5 ⁇ m. In some embodiments, the thickness of the enzyme film layer ranges from 1 ⁇ m to 5 ⁇ m. In some embodiments, the thickness of the enzyme film layer may be 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, or 5 ⁇ m.
  • an enzyme membrane containing glucose oxidase can be used as the sensor of the biorecognition module, and an oxygen electrode can be used as the energy supply module to form a glucose oxidase biosensor.
  • an oxygen electrode can be used as the energy supply module to form a glucose oxidase biosensor.
  • oxidized natural polymer compounds are obtained by oxidizing natural polymer chemicals, and the oxidized natural polymer compounds are used as cross-linking agents to fix sensitive elements (such as glucose oxidase) on the biosensor. in the working electrode to ensure the activity and stability of sensitive elements and avoid the biological toxicity of traditional chemical cross-linking agents. properties, improving the biocompatibility of the working electrode.
  • sensitive elements such as glucose oxidase
  • One of the embodiments of this specification provides a method for preparing a biosensor.
  • the method includes: oxidizing a natural polymer compound to obtain an oxidized natural polymer compound; and using the oxidized natural polymer compound as a cross-linking agent.
  • the sensitive element is fixed in the working electrode.
  • the natural polymer compound is subjected to oxidation treatment, and obtaining the oxidized natural polymer compound specifically includes the following steps:
  • Step S11 Dissolve the natural polymer compound in a first solvent to obtain a natural polymer compound solution.
  • the natural polymer compounds include cellulose and its derivatives, sodium alginate, chitosan and its derivatives, hyaluronic acid, konjac mannan, starch and its derivatives, pectin, and At least one of the carrageenans.
  • the first solvent refers to a solvent that can dissolve natural polymer compounds.
  • the first solvent may be an organic solvent such as ethanol, or an acidic or alkaline solvent.
  • the first solvent is water.
  • Step S12 Dissolve the oxidizing agent in the second solvent to obtain an oxidizing solution.
  • the second solvent refers to a solvent capable of dissolving the oxidizing agent.
  • the second solvent includes water.
  • the oxidizing agent may be sodium periodate. Specifically, sodium periodate is dissolved in water to obtain an oxidizing solution.
  • Step S13 Mix the natural polymer compound solution and the oxidizing solution to obtain the oxidized natural polymer compound after the oxidation reaction.
  • the oxidized natural polymer compound may be a dialdehyde-formed natural polymer compound.
  • the oxidizing agent such as sodium periodate
  • a terminator may be added to terminate the oxidation reaction.
  • the terminator includes ethylene glycol.
  • oxidized natural polymer compounds with different oxidation degrees can be obtained by controlling the ratio or mixing time between the oxidizing agent and the natural polymer compound.
  • the oxidized natural polymer compound is obtained by oxidizing the natural polymer compound solution and the oxidizing solution for 0.5 to 48 hours. In some embodiments, the oxidized natural polymer compound is obtained by oxidizing the natural polymer compound solution and the oxidizing solution for 8 to 24 hours. In some embodiments, the oxidized natural polymer compound is obtained after oxidizing the natural polymer compound solution and the oxidizing solution for 1 hour, 4 hours, 10 hours, 12 hours, 15 hours, or 24 hours.
  • the initial reaction concentration of the polymer compound in the mixed solution is between 0.1% and 20%. In some embodiments, the polymer compound is in the mixed liquid The initial reaction concentration in is 0.1%, 0.5%, 5%, 10%, or 20%. In some embodiments, the initial reaction concentration of the polymer compound in the mixed solution is between 1% and 5%. In some embodiments, the initial reaction concentration of the polymer compound in the mixed solution is 1% or 3%.
  • the initial reaction concentration of the oxidant in the mixed solution is between 0.1% and 40%. In some embodiments, the initial reaction concentration of the oxidant in the mixed solution is 0.1%, 5%, 10%, 20% or 40%. In some embodiments, the initial reaction concentration of the oxidant in the mixed solution is between 1% and 10%. In some embodiments, the initial reaction concentration of the oxidant in the mixed solution is 0.3%, 0.5%, 1%, 3%, 8%, or 10%.
  • the oxidation degree of the oxidized natural polymer compound is 5% to 85%.
  • the oxidation degree of the oxidized natural polymer compound is 5%-60%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 20%-70%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 30%-80%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 5%, 20%, 30%, 40%, 50%, 60% or 85%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 30% to 60%. In some embodiments, the oxidation degree of the oxidized natural polymer compound is 35%, 45%, or 55%.
  • the embodiments of this specification control the oxidation degree of the final oxidized natural polymer compound by adjusting the initial reaction concentration of the oxidant and the natural polymer compound in the mixed solution and the time of the oxidation reaction, thereby controlling the number of sensitive primitives to ensure that the sensor is properly transmitted. sensitivity range.
  • the preparation method of the biosensor includes: using the oxidized natural polymer compound as a cross-linking agent for fixing sensitive elements in the working electrode.
  • the working electrode includes an enzyme film layer, and the enzyme film layer includes the sensitive element immobilized with the oxidized natural polymer compound.
  • the oxidized natural polymer compound is obtained by oxidizing the natural polymer compound.
  • the natural polymer compounds include cellulose and its derivatives, sodium alginate, chitosan and its derivatives, hyaluronic acid, konjac mannan, starch and its derivatives, pectin, and At least one of the carrageenans.
  • the sensitive moiety may be a compound bearing a primary amino group.
  • the sensitive motif includes one of glucose oxidase, lactate oxidase, uricase, or alcohol oxidase.
  • the method of preparing the biosensor further includes a method of preparing the enzyme film layer.
  • the preparation method of the enzyme film layer includes: mixing the enzyme stabilizer, the sensitive element and the oxidized natural polymer compound in a third solvent to obtain a mixed solution; and mixing the mixed solution. The solution is coated on the metal layer, and the cross-linking reaction After the reaction, the enzyme film layer is obtained.
  • the third solvent includes water.
  • the sensitive motif includes glucose oxidase.
  • the enzyme stabilizer includes serum proteins (eg, bovine serum albumin), serum enzymes, or other proteins with enzyme stabilizing effects.
  • the cross-linking reaction includes a Schiff base reaction between a primary amino group in glucose oxidase and an aldehyde group in an oxidized natural polymer compound.
  • the concentration of the sensitive motif is 0.5% to 20%; the concentration of the oxidized natural polymer compound is 0.1% to 20%; and the concentration of the enzyme stabilizer is 0.5% ⁇ 20%. In some embodiments, in the mixed solution, the concentration of the sensitive motif is 5% to 20%; the concentration of the oxidized natural polymer compound is 1% to 20%; and the concentration of the enzyme stabilizer is 5% ⁇ 20%. In some embodiments, in the mixed solution, the concentration of the sensitive motif is 5% to 10%; the concentration of the oxidized natural polymer compound is 10% to 20%; and the concentration of the enzyme stabilizer is 5%. ⁇ 10%. In some embodiments, in the mixed solution, the concentration of the sensitive motif is 2% to 10%; the concentration of the oxidized natural polymer compound is 5% to 15%; and the concentration of the enzyme stabilizer is 0.5% to 5%.
  • the temperature of the cross-linking reaction is 0°C to 37°C. In some embodiments, the temperature of the cross-linking reaction is 5°C to 15°C. In some embodiments, the temperature of the cross-linking reaction is 0°C to 10°C. In some embodiments, the temperature of the cross-linking reaction is 4°C.
  • the preparation method of the enzyme film layer 202 includes the following steps:
  • Step S21 Mix serum protein, the glucose oxidase and the oxidized natural polymer compound in a solvent to obtain a glucose oxidase mixed solution.
  • the solvent is water.
  • different proportions of the serum protein and the glucose oxidase are formulated into an aqueous solution of a certain concentration, and then the oxidized natural polymer compound is configured into an aqueous solution of a certain concentration, and the two are mixed to obtain the glucose oxidized enzyme. enzyme mixed solution.
  • the concentration of the serum protein is 0.5% to 20%
  • the concentration of the glucose oxidase is 0.5% to 20%
  • the oxidized natural polymer compound The concentration is 0.1% to 20%.
  • Step S22 The glucose oxidase mixed solution is coated on the metal layer, and the enzyme film layer 202 is obtained after a cross-linking reaction.
  • the glucose oxidase mixed solution obtained by mixing is immediately coated on the metal layer, and cross-linked at a certain temperature for a certain period of time, so that the primary amino groups in the glucose oxidase and the oxidized natural polymer in compounds The aldehyde group undergoes Schiff base reaction to form the stable enzyme film layer 202. It can be understood that the cross-linking time required for cross-linking at different temperatures is also different.
  • the temperature at which the glucose oxidase mixed solution performs the cross-linking reaction on the metal layer is 0°C to 37°C. In some embodiments, the temperature at which the glucose oxidase mixed solution performs a cross-linking reaction on the metal layer is 4°C.
  • polymer compounds are often used in the field of materials, specifically for the preparation of hydrogels, human tissue scaffolds, etc.
  • the thickness of the enzyme film layer may be increased.
  • cross-linking is not specified in some embodiments of this specification.
  • the dosage and temperature of the agent are controlled by process parameters, thereby achieving effective control of the thickness of the enzyme film layer. This ensures that the enzyme film layer is neither too thick and easy to fall off, affecting stability, nor too thin, affecting sensing performance.
  • the material of the protective layer 203 includes at least one of polyurethane, polyethylene glycol, cellulose acetate, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene.
  • the protective layer 203 can be prepared by dip coating. Specifically, the material of the protective layer (i.e., at least one of polyurethane, polyethylene glycol, cellulose acetate, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene) is dissolved in a solvent to obtain a protective layer solution, and then The enzyme film layer 202 is placed in the protective layer solution, taken out and dried to obtain the protective layer.
  • the material of the protective layer i.e., at least one of polyurethane, polyethylene glycol, cellulose acetate, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene
  • the enzyme film layer 202 is placed in the protective layer solution, taken out and dried to obtain the protective layer.
  • the solvent of the protective layer includes at least one of tetrahydrofuran, N,N-dimethylformamide (DMF), cyclohexane, and acetone.
  • the material of the protective layer ie, at least one of polyurethane, polyethylene glycol, cellulose acetate, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene
  • the concentration is 0.5% to 10%.
  • biosensors may include immunosensors, enzyme sensors, nucleic acid sensors, and cell sensors.
  • the biosensor is used to detect at least one of glucose, uric acid, lactic acid or alcohols.
  • the biosensor is a glucose biosensor and the sensitive element includes glucose oxidase.
  • the first step dissolve 3g of sodium alginate in 150ml of distilled water to obtain a sodium alginate aqueous solution.
  • the third step add the sodium periodate aqueous solution dropwise to the sodium alginate aqueous solution, and react in the dark at room temperature for 24 hours. Then add 5 ml of ethylene glycol and stir for 1 hour to terminate the reaction. Purify by dialysis and dry in an oven. Finally, oxidized sodium alginate is obtained.
  • the fourth step is to dissolve oxidized sodium alginate in distilled water to prepare an oxidized sodium alginate aqueous solution with a concentration of 3%, add glucose oxidase to it to make the concentration 30 mg/ml, and then add bovine serum albumin to it , to a concentration of 20 mg/ml, stir and dissolve to obtain a pre-crosslinked glucose oxidase mixed solution.
  • the fifth step is to apply the above pre-crosslinked glucose oxidase mixed solution on the metal layer, cross-link it in a refrigerator at 4°C for 24 hours, and then clean the uncross-linked glucose oxidase with distilled water to obtain an enzyme film layer. .
  • the sixth step is to put the enzyme film layer into a polyurethane solution with a concentration of 5%, take it out and dry it at room temperature to form a protective layer, thereby obtaining a working electrode.
  • the third step add the sodium periodate aqueous solution dropwise to the carboxymethyl cellulose aqueous solution, and react in the dark at 4°C for 24 hours. Then add 5 ml of ethylene glycol and stir for 1 hour to terminate the reaction. Then filter and place in an oven. After medium drying, oxidized carboxymethyl cellulose is obtained.
  • the fourth step is to dissolve oxidized carboxymethyl cellulose in distilled water to prepare an oxidized carboxymethyl cellulose aqueous solution with a concentration of 5%, add glucose oxidase to it to make the concentration 20 mg/ml, and then add glucose oxidase to it. Add bovine serum albumin to a concentration of 15 mg/ml, stir and dissolve, and obtain a pre-crosslinked glucose oxidase mixed solution.
  • the fifth step is to apply the above pre-crosslinked glucose oxidase mixed solution on the metal layer, cross-link it in a refrigerator at 4°C for 12 hours, and then clean the uncross-linked glucose oxidase with distilled water to obtain an enzyme film layer. .
  • the sixth step is to put the enzyme film layer into a polyurethane solution with a concentration of 5%, take it out and dry it at room temperature to form a protective layer, thereby obtaining a working electrode.
  • the first step dissolve 1g of pectin in 100ml of distilled water to obtain a pectin aqueous solution.
  • the third step add the sodium periodate aqueous solution dropwise to the pectin aqueous solution, and react in the dark at 4°C for 12 hours. Then add 5 ml of ethylene glycol and stir for 1 hour to terminate the reaction. Then filter and dry in an oven. Oxidized pectin is obtained.
  • the fourth step is to dissolve the oxidized pectin in distilled water to prepare an oxidized pectin aqueous solution with a concentration of 1%, add glucose oxidase to it to make the concentration 10 mg/ml, and then add bovine serum albumin to it to make the solution The concentration is 8 mg/ml. After stirring and dissolving, a pre-crosslinked glucose oxidase mixed solution is obtained.
  • the fifth step is to apply the above pre-crosslinked glucose oxidase mixed solution on the metal layer, cross-link it in a refrigerator at 4°C for 12 hours, and then clean the uncross-linked glucose oxidase with distilled water to obtain an enzyme film layer. .
  • the sixth step is to put the enzyme film layer into a polyurethane solution with a concentration of 5%, take it out and dry it at room temperature to form a protective layer, thereby obtaining a working electrode.
  • the working electrodes prepared in Examples 1 to 3 and Comparative Example 1 were used to prepare glucose biosensors respectively.
  • the preparation method of the glucose biosensor in Comparative Example 1 is as follows:
  • polyimide (PI) with a thickness of 120 ⁇ m is selected as a flexible substrate.
  • the glass or silicon wafer support substrate and PI are cleaned and then dried with a nitrogen gun.
  • polydimethylsiloxane (PDMS) is spin-coated onto the support substrate and cured by heating.
  • the cleaned PI is adsorbed using a laminating machine and a support substrate coated with PDMS.
  • the fourth step is to use a magnetron sputtering instrument to sputter a layer of adhesive layer chromium (Cr) on the PI flexible substrate, then sputter the conductive layer gold (Au), then spin-coat a layer of photoresist, and then use an exposure machine
  • the patterning process is carried out and finally the development reaction is carried out.
  • Wet etch the conductive layer Au then clean it with plenty of water and blow dry with nitrogen.
  • the adhesive layer Cr is etched until the non-pattern area is completely exposed in PI color (reddish brown), cleaned with plenty of water and blown dry with nitrogen. Finally, peel off the photoresist and check whether the wires are connected.
  • the PI flexible substrate is deposited with parylene, then spin-coated with photoresist, developed for patterning, and then subjected to plasma treatment to clean the developed part of the parylene deposit, and finally the photoresist is Peel off and check the lead area for insulation.
  • the sixth step is to prepare the working electrode and the counter electrode: first spin-coat a layer of photoresist on the PI flexible substrate of the working electrode and the counter electrode, then use an exposure machine to perform the patterning process, and finally perform a development reaction, wash with water and use nitrogen Blow dry.
  • Use a magnetron sputtering instrument to sputter an adhesive layer Cr on the PI flexible substrate, then sputter the conductive layer platinum (Pt), and soak it in acetone with ultrasound until the working electrode (WE) and counter electrode (CE) patterns are complete. Lift it clean, and then use clean acetone and wine respectively. Rinse with semen and water and blow dry with a nitrogen gun.
  • Use a multimeter to test the continuity of WE, CE and the corresponding component holes (PAD), and the insulation of different wires. Obtain working electrode and counter electrode.
  • the seventh step is to prepare a working electrode containing an enzyme film layer: dissolve glucose oxidase and bovine serum albumin in phosphate buffer saline (PBS), and then add 2.5% glutaraldehyde solution to it to obtain pre-cross-linked glucose oxidase. enzyme mixed solution.
  • PBS phosphate buffer saline
  • the above pre-crosslinked glucose oxidase mixed solution was applied to the metal layer of the working electrode, and cross-linked in a refrigerator at 4°C for 24 hours.
  • the uncross-linked glucose oxidase was then washed with distilled water to obtain an enzyme film layer.
  • Put the working electrode containing the enzyme film layer into a polyurethane solution with a concentration of 5% take it out and dry it at room temperature to form a protective layer, thereby obtaining a dry working electrode containing the enzyme film layer.
  • the eighth step is to prepare the reference electrode: electroplating the silver layer, using a pulse current source device for galvanostatic plating.
  • Chlorination treatment first treat with argon plasma for 5 minutes, then treat with ferric chloride solution, finally rinse with water and blow dry with nitrogen to obtain the reference electrode.
  • the working electrodes prepared in Examples 1 to 3 were used to prepare glucose biosensors according to the preparation method of Comparative Example 1.
  • glucose biosensors were prepared from the working electrode in Example 1 according to the method of Comparative Example 1, and sensitivity tests were performed on these five batches of sensors at glucose concentrations of 0 to 30 mM.
  • the sensitivity test results of the biosensor prepared by the working electrode in Example 1 are shown in Figure 3A.
  • the test results show that the five batches of glucose biosensors prepared by the working electrode in Example 1 have a glucose concentration in the range of 0 to 30mM. Linearity can be achieved within the test, and the batch-to-batch variability of five batches of glucose biosensors is less than 5%.
  • a sensitivity test was performed on the glucose biosensor prepared from the working electrode in Example 2-3. Specifically, a glucose biosensor was prepared from the working electrode in Example 2-3 according to the method of Comparative Example 1, and the sensitivity test of the sensor was performed at a glucose concentration of 0 to 20 mM.
  • the sensitivity test results of the biosensor prepared by the working electrode in Example 2 are shown in Figure 4A.
  • the test results show that the glucose biosensor prepared by the working electrode in Example 2 has uniform sensitivity in the range of 0 to 20 mM glucose concentration. Can achieve linearity.
  • the sensitivity test results of the biosensor prepared by the working electrode in Example 3 are shown in Figure 5A.
  • the test results show that the glucose biosensor prepared by the working electrode in Example 3 has uniform sensitivity in the range of 0 to 20 mM glucose concentration. Can achieve linearity.
  • the sensitivity test results of the biosensor prepared by the working electrode in Example 1 are shown in Figure 3B.
  • the test results show that the sensitivity change of the glucose biosensor prepared from the working electrode in Example 1 is less than 2% within 30 days. This shows that the glucose biosensor prepared by the present invention has long-term stability.
  • the sensitivity test results of the biosensor prepared by the working electrode in Example 2 are shown in Figure 4B.
  • the test results show that the sensitivity of the glucose biosensor prepared from the working electrode in Example 2 changes below 10% within 30 days. This shows that the glucose biosensor prepared by the present invention has long-term stability.
  • the sensitivity test results of the biosensor prepared by the working electrode in Example 3 are shown in Figure 5B.
  • the test results show that the sensitivity change of the glucose biosensor prepared from the working electrode in Example 3 is less than 5% within 30 days. This shows that the glucose biosensor prepared by the present invention has long-term stability.
  • the working electrode cytotoxicity test method is: soak the working electrodes prepared in Examples 1 to 3 and Comparative Example 1 in PBS for extraction to obtain an extract, and conduct a cytotoxicity test on the obtained extract. .
  • Figure 6 is a chart showing the cytotoxicity test results of the working electrode according to some embodiments of this specification.
  • the beneficial effects that may be brought about by the embodiments of this specification include but are not limited to: (1) oxidizing natural polymer chemicals to obtain oxidized natural polymer compounds, and using the oxidized natural polymer compounds as cross-linking agents to bind sensitive elements Fixed in the working electrode of the biosensor, it avoids the biological toxicity of traditional chemical cross-linking agents and improves the biocompatibility of the working electrode of the biosensor; (2) Preparation of the oxidation reaction and enzyme film layer by adjusting the participation of natural polymer compounds
  • the process parameters in the process optimize and improve the controllability of the environment and process of chemical bonding of sensitive primitives, which is conducive to maintaining the quantity and activity of sensitive primitives to ensure the activity and stability of sensitive primitives, thereby improving biological efficiency. Sensor consistency and long-term stability.
  • the possible beneficial effects may be any one or a combination of the above, or any other possible beneficial effects.
  • numbers are used to describe the quantities of components and properties. It should be understood that such numbers used to describe the embodiments are modified by the modifiers "about”, “approximately” or “substantially” in some examples. Grooming. Unless otherwise stated, “about,” “approximately,” or “substantially” means that the stated number is allowed to vary by ⁇ 20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximations that may vary depending on the desired features of the individual embodiment. In some embodiments, numerical parameters should account for the specified number of significant digits and use general digit preservation methods. Although the numerical ranges and parameters used to identify the breadth of ranges in some embodiments of this specification are approximations, in specific embodiments, such numerical values are set as accurately as is feasible.

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Abstract

本说明书实施例提供一种生物传感器，所述生物传感器包括工作电极，所述工作电极包括酶膜层，所述酶膜层中包括用氧化天然高分子化合物作为交联剂固定的敏感基元，其中，所述氧化天然高分子化合物是由天然高分子化合物经氧化处理得到的。

Description

天然高分子化合物在生物传感器中的应用
优先权信息
本发明要求2022年06月20日提交的申请号为202210698681.8的中国专利申请的优先权，其全部内容通过引用并入本文。
技术领域
本说明书涉及技术领域，尤其涉及一种天然高分子化合物在生物传感器中的应用。
背景技术
生物传感器通常用于生物体内物质的测定，其应用越来越广泛。例如，生物传感器可用于体内血糖的测定。然而许多可植入的生物传感器都容易在体内受到免疫反应，并且使用寿命往往较短。此外，现有的生物传感器中的敏感基元固定工艺都会应用一些有生物毒性的交联剂，例如戊二醛、光引发剂或丙烯酸类等，不仅会影响敏感基元的活性和稳定性，残留的交联剂也会加强人体的排异反应，从而降低使用寿命，同时也不利于批量的生产安全。
发明内容
本说明书实施例之一提供一种生物传感器。生物传感器可以包括工作电极，所述工作电极包括酶膜层。在一些实施例中，酶膜层中包括用氧化天然高分子化合物作为交联剂固定的敏感基元，其中，氧化天然高分子化合物是由天然高分子化合物经氧化处理得到的。
在一些实施例中，天然高分子化合物包括纤维素及其衍生物、海藻酸钠、壳聚糖及其衍生物、透明质酸、魔芋甘露聚糖、淀粉及其衍生物、果胶和卡拉胶中的至少一种。
在一些实施例中，所述氧化天然高分子化合物为双醛化天然高分子化合物。
在一些实施例中，所述氧化天然高分子化合物的氧化度为5％～85％，在一些实施例中，所述氧化天然高分子化合物的氧化度为30％～60％。
在一些实施例中，所述敏感基元包括葡萄糖氧化酶、乳酸氧化酶、尿酸酶、或醇氧化酶中的一种。
在一些实施例中，所述酶膜层还包括酶稳定剂。
在一些实施例中，所述酶膜层的厚度范围在0.1μm-20μm之间。
在一些实施例中，所述酶膜层的厚度范围在1μm-5μm之间。
在一些实施例中，所述生物传感器还包括柔性基底、参比电极、对电极中的至少一种。
在一些实施例中，所述柔性基底的厚度范围在10μm～300μm之间。
在一些实施例中，所述工作电极还包括金属层、保护层中的至少一种。
在一些实施例中，所述工作电极的所述金属层由内到外依次为第一金属层、第二金属层和第三金属层；其中所述第一金属层为Cr、Ti、Ni-Cr中的至少一种；所述第二金属层为金、铜、银中的至少一种；所述第三金属层为铂、铂铱、铂碳中的至少一种。
在一些实施例中，所述保护层包括聚氨酯、聚乙二醇、醋酸纤维素、聚乙烯醇、聚乙烯吡咯烷酮、聚乙烯中的至少一种。
本说明书实施例之一提供一种生物传感器的制备方法。在一些实施例中，所述方法包括：对天然高分子化合物进行氧化处理，得到氧化天然高分子化合物；以及将所述氧化天然高分子化合物作为交联剂，将敏感基元固定在所述生物传感器的工作电极中。
在一些实施例中，对天然高分子化合物进行氧化处理，得到氧化天然高分子化合物具体包括以下步骤：将所述天然高分子化合物溶于第一溶剂中，得到天然高分子化合物溶液；将氧化剂溶于第二溶剂中，得到氧化性溶液；以及将所述天然高分子化合物溶液和所述氧化性溶液混合，氧化反应后得到所述氧化天然高分子化合物。
在一些实施例中，所述氧化天然高分子化合物的氧化度为5％～85％。
在一些实施例中，所述氧化天然高分子化合物的氧化度为30％～60％。
在一些实施例中，所述工作电极包括酶膜层，所述酶膜层中包括用所述氧化天然高分子化合物固定的所述敏感基元。
在一些实施例中，上述方法进一步包括制备所述酶膜层的方法。在一些实施例中，所述酶膜层的制备方法包括以下步骤：将酶稳定剂、所述敏感基元和所述氧化天然高分子化合物于第三溶剂中混合，得到混合溶液；以及将所述混合溶液涂覆在金属层上，交联反应后得到所述酶膜层。
在一些实施例中，在所述混合溶液中，所述敏感基元的浓度为0.5％～20％；所述氧化天然高分子化合物的浓度为0.1％～20％；所述酶稳定剂的浓度为0.5％～20％；所述交联反应的温度为0℃～37℃。
在一些实施例中，在所述混合溶液中，所述敏感基元的浓度为2％～10％；所述氧化天然高分子化合物的浓度为5％～15％；所述酶稳定剂的浓度为0.5％～5％；所述交联反应的温度为5℃～15℃。
本说明书实施例之一还提供所述的生物传感器在生物传感中的应用。
在一些实施例中，所述生物传感器用于检测葡萄糖、尿酸、乳酸或醇类物质中的至少一种。
附图说明
本说明书将以示例性实施例的方式进一步说明，这些示例性实施例将通过附图进行详细描述。这些实施例并非限制性的，在这些实施例中，相同的编号表示相同的结构，其中：
图1是根据本说明书一些实施例所示的生物传感器中的柔性电极的平面示意图；
图2是根据本说明书一些实施例所示的柔性电极中的工作电极的截面示意图；
图3A是根据本说明书一些实施例所示的生物传感器的灵敏度测试结果图；
图3B是根据本说明书一些实施例所示的生物传感器的稳定性测试结果图；
图4A是根据本说明书一些实施例所示的又一生物传感器的灵敏度测试结果图；
图4B是根据本说明书一些实施例所示的又一生物传感器的稳定性测试结果图；
图5A是根据本说明书一些实施例所示的又一生物传感器的灵敏度测试结果图；
图5B是根据本说明书一些实施例所示的又一生物传感器的稳定性测试结果图；
图6根据本说明书一些实施例所示的工作电极的细胞毒性测试结果图。
附图标记：100-柔性电极；10-柔性基底；20-工作电极；201-金属层；202-酶膜层；203-保护层；30-参比电极；40-对电极。
具体实施方式
为了更清楚地说明本说明书实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单的介绍。显而易见地，下面描述中的附图仅仅是本说明书的一些示例或实施例，对于本领域的普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图将本说明书应用于其它类似情景。除非从语言环境中显而易见或另做说明，图中相同标号代表相同结构或操作。
应当理解，本文使用的“系统”、“装置”、“单元”和/或“模块”是用于区分不同级别的不同组件、元件、部件、部分或装配的一种方法。然而，如果其他词语可实现相同的目的，则可通过其他表达来替换所述词语。
如本说明书和权利要求书中所示，除非上下文明确提示例外情形，“一”、“一个”、“一种”和/或“该”等词并非特指单数，也可包括复数。一般说来，术语“包括”与“包含”仅提示包括已明确标识的步骤和元素，而这些步骤和元素不构成一个排它性的罗列，方法或者设备也可能包含其它的步骤或元素。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的 所列项目的任意的和所有的组合。
生物传感器是一种能够将生物学信号转化为可读取的电学信号的装置，用于检测和监测生物体内的化学物质、生物活性分子、细胞的代谢活动等生物过程，广泛应用于医疗卫生、环境监测和食品安全等领域。在一些实施例中，本说明书中的生物传感器可以包括生物识别模块、信号转换模块、能源供应模块、数据处理模块和输出模块。
生物识别模块通常包括抗体、酶、核酸和细胞等敏感基元。在生物传感器工作过程中，生物识别模块中的敏感基元与待测分子发生特异性识别，产生一些物理或化学事件，导致化学信号变化。待测分子可以包括生物分子、代谢产物、药物或毒素。例如，生物分子可以包括蛋白质、核酸、酶、细胞因子等。代谢产物可以包括葡萄糖、尿酸、乳酸、乙醇等。
信号转换模块中的信号转换器可以将化学信号转换为可读取的电学信号。信号转换器包括电化学转换器、光学转换器、压电转换器和热敏转换器。其中，电化学转换器的基本工作原理是通过生物识别模块和电极之间的电化学反应产生电流或电势差。该电流或电势差可被检测系统测量和记录。
能源供应模块是为生物传感过程提供能量的模块。能源供应方式包括采用纯化学能源(例如氧)、太阳能、运动能或无线电磁辐射。
数据处理模块可以将信号转换模块中的电信号进一步处理为用户更容易理解的信号。数据处理模块可以包括电信号采集、电信号处理等步骤。
输出模块可以将数据处理模块处理后的采集结果呈现给用户。其中，用户包括但不限于医疗和健康机构的人员、个人健康管理者、运动员、运动爱好者、环境监测机构人员、食品或饮料的生产或质量监督人员。
本说明书实施例之一提供一种生物传感器。在一些实施例中，所述生物传感器包括工作电极，所述工作电极包括酶膜层。其中，所述酶膜层中包括用氧化天然高分子化合物作为交联剂固定的敏感基元，其中，所述氧化天然高分子化合物是由天然高分子化合物经氧化处理得到的。
在一些实施例中，所述生物传感器还包括柔性基底、参比电极、对电极中的至少一种。在一些实施例中，所述柔性基底的厚度范围在10μm～300μm之间。在一些实施例中，所述柔性基底的材质包括聚酰亚胺以及聚对苯二甲酸乙二醇酯中的至少一种。
在一些实施例中，所述工作电极还包括金属层、保护层中的至少一种。
在一些实施例中，所述工作电极的所述金属层由内到外依次为第一金属层、第二金属层和第三金属层；其中所述第一金属层为Cr、Ti、Ni-Cr中的至少一种；所述第二金属层 为金、铜、银中的至少一种；所述第三金属层为铂、铂铱、铂碳中的至少一种。
在一些实施例中，所述保护层包括聚氨酯、聚乙二醇、醋酸纤维素、聚乙烯醇、聚乙烯吡咯烷酮、聚乙烯中的至少一种。
示例性地，如图1和图2所示，所述生物传感器包括柔性电极100。在一些实施例中，所述柔性电极100包括柔性基底10以及位于所述柔性基底10同一表面上的工作电极20、参比电极30和对电极40。
在一些实施例中，可在所述柔性基底10上通过微纳加工的方式覆盖金属层，并将所述金属层图案化，以形成所述工作电极20、所述参比电极30和所述对电极40的三电极体系。
微纳加工是指光刻、显影、磁控溅射、湿法刻蚀、等离子清洗以及激光切割等一系列基于半导体工艺的技术。通过微纳加工工艺制备柔性电极，可以提高加工精度。
请参阅图2，在一些实施例中，所述工作电极20包括依次层叠设置的金属层201、酶膜层202以及保护层203。其中，所述金属层201位于所述柔性基底10和所述保护层203之间。
在一些实施例中，所述金属层201包括依次层叠设置的第一金属部(图中未示出)、第二金属部(图中未示出)和第三金属部(图中未示出)。在一些实施例中，所述第一金属部的材质包括铬、钛或镍铬合金，所述第二金属部的材质包括金、铜或银，所述第三金属部的材质包括铂、铂铱合金或铂碳混合物。其中，所述第二金属部具有较好的导电性，提高生物传感器的电子传输效率，所述第三金属部对过氧化氢具有催化作用，减少酶催化过程中产生的过氧化氢对催化反应的影响。
在一些实施例中，所述酶膜层的厚度范围在0.1μm-20μm之间。优选地，所述酶膜层的厚度范围在1μm-5μm之间。在一些实施例中，可以利用旋涂工艺，通过控制旋涂机的旋转速度来控制酶膜层的厚度。在一些实施例中，可以对交联剂的用量、交联温度进行调整，以实现对酶膜层的厚度的有效控制。
本文所指的“天然高分子化合物”是生物体本身能够通过生化作用或光合作用产生的化合物。相对于其他合成有机物来说，天然高分子化合物由于来源于生物体内，因此更具有生物相容性，进入人体后人体会大大降低免疫排斥反应的发生。天然高分子化合物含有多个重复结构单元。例如，海藻酸钠是由β-D-甘露糖醛酸(M)和α-L-葡糖醛酸(G)残基通过1,4-糖苷键连接，重复排列成MM片段、GG片段或MG片段的天然高分子化合物。
在一些实施例中，所述天然高分子化合物为天然多糖类高分子化合物。
本文中的“多糖”是指由多个单糖分子经失水缩聚，通过糖苷键结合而成的天然高分子化合物。在一些实施例中，所述多糖类高分子化合物包括植物多糖(如纤维素、半纤维素、淀粉、果胶)，动物多糖(如甲壳素、壳聚糖、肝素、硫酸软骨素)，琼脂多糖(如琼脂、海藻酸、角叉藻聚糖)，菌类多糖(如D-葡聚糖、D-半乳聚糖、甘露聚糖)，微生物多糖(如右旋糖酐、黄原胶、凝乳糖、出芽短梗孢糖)。
在一些实施例中，所述天然高分子化合物含有羟基基团。
在一些实施例中，所述天然高分子化合物包括纤维素及其衍生物、海藻酸钠、壳聚糖及其衍生物、透明质酸、魔芋甘露聚糖、淀粉及其衍生物、果胶和卡拉胶中的至少一种。
在一些实施例中，所述天然高分子化合物的机械强度较高，作为交联剂固定的敏感基元后，工作电极的稳定性较好。在一些实施例中，所述天然高分子化合物包括海藻酸钠或魔芋甘露聚糖。
在一些实施例中，可以通过氧化剂对天然高分子化合物进行氧化处理，获得氧化天然高分子化合物。氧化剂包括但不限于金属氧化物及其盐(如铬试剂、高锰酸钾、二氧化锰)、硝酸、过碘酸、高碘酸盐、或二甲亚砜。
在一些实施例中，所述氧化天然高分子化合物中的羟基基团至少部分被氧化，形成醛基。
本文中的“至少部分被氧化”是指天然高分子化合物至少有一个羟基被氧化。“部分”可以指羟基的比例。例如，“部分”可以为二分之一、五分之一、或十分之一，分别代表天然高分子化合物被氧化的羟基的数量占总羟基数量的二分之一、五分之一、或十分之一。在一些实施例中，可以通过控制氧化剂的浓度或种类来控制天然高分子化合物的氧化度。
在一些实施例中，所述氧化天然高分子化合物为双醛化天然高分子化合物。
本文所用的“双醛化”是指天然高分子化合物的结构单元上的两个羟基被氧化为醛基。例如，通过高碘酸盐对纤维素葡萄糖单元C2-C3上的羟基选择性氧化，C2-C3上的羟基被氧化成醛基，可获得双醛基纤维素。在一些实施例中，可以将海藻酸钠、壳聚糖及其衍生物、透明质酸、魔芋甘露聚糖、淀粉及其衍生物、果胶和卡拉胶中的至少一种与氧化剂进行混合，得到双醛基海藻酸钠、双醛基壳聚糖及其衍生物、双醛基透明质酸、双醛基魔芋甘露聚糖、双醛基淀粉及其衍生物、双醛基果胶和双醛基卡拉胶中的至少一种。
在一些实施例中，所述氧化天然高分子化合物的氧化度为5％～85％。
氧化度是指天然高分子化合物中的羟基被氧化成醛基的比例。可以用醛基含量来表示氧化天然高分子化合物的氧化度。例如，醛基含量为6％，则氧化天然高分子化合物的氧 化度为6％。可以采用例如化学滴定法测定氧化天然高分子化合物的醛基含量，本文对测定氧化度的方法不做具体限定。
在一些实施例中，所述氧化天然高分子化合物的氧化度为5％-60％。在一些实施例中，所述氧化天然高分子化合物的氧化度为20％-70％。在一些实施例中，所述氧化天然高分子化合物的氧化度为30％-80％。在一些实施例中，所述氧化天然高分子化合物的氧化度为5％、20％、30％、40％、50％、60％或85％。在一些实施例中，所述氧化天然高分子化合物的氧化度为30％～60％。在一些实施例中，所述氧化天然高分子化合物的氧化度为35％、45％、或55％。
敏感基元是在酶膜层中对目标物(例如，酶的底物)进行选择性作用的生物活性单元。
在一些实施例中，所述敏感基元可以是带有伯氨基的化合物。在一些实施例中，所述敏感基元包括葡萄糖氧化酶、乳酸氧化酶、尿酸酶、或醇氧化酶中的一种。在一些实施例中，所述敏感基元为葡萄糖氧化酶。
在一些实施例中，所述酶膜层还包括酶稳定剂。酶稳定剂是一种用于增强酶的稳定性和活性的物质，能够保护酶免受环境因素(如温度、pH、氧气等)的影响，延长酶的使用寿命和活性。酶稳定剂包括但不限于聚乙二醇(PEG)、蛋白质、多肽、氨基酸、胆固醇等。
在一些实施例中，所述酶膜层的厚度范围在0.1μm-20μm之间。在一些实施例中，所述酶膜层的厚度范围在0.1μm-10μm之间。在一些实施例中，所述酶膜层的厚度范围在0.1μm-5μm之间。在一些实施例中，所述酶膜层的厚度范围在1μm-5μm之间。在一些实施例中，所述酶膜层的厚度可以为1μm、2μm、3μm、4μm、5μm。
在一些实施例中，可以用含有葡萄糖氧化酶的酶膜作生物识别模块的感受器，以氧电极作为能源供应模块，构成葡萄糖氧化酶生物传感器。当该生物传感器放入待测溶液中时，待测溶液内的溶解氧和待测葡萄糖同时渗入酶膜，在氧存在下，葡萄糖被葡萄糖氧化酶催化氧化成为葡萄糖酸，同时消耗氧而生成过氧化氢。此时溶液中氧浓度的下降，信号转换模块将此变化转变为电信号，经过数据处理模块处理后，输出模块输出为待测溶液中葡萄糖的浓度值。
本说明书实施例通过对天然高分子化学物进行氧化处理，得到氧化天然高分子化合物，并采用所述氧化天然高分子化合物作为交联剂将敏感基元(如葡萄糖氧化酶)固定在生物传感器的工作电极中，以保证敏感基元的活性和稳定性，避免了传统化学交联剂的生物毒 性，提高了工作电极的生物相容性。
本说明书实施例之一提供一种生物传感器的制备方法，所述方法包括：对天然高分子化合物进行氧化处理，得到氧化天然高分子化合物以及将所述氧化天然高分子化合物作为交联剂，用于将敏感基元固定在所述工作电极中。
在一些实施例中，对所述天然高分子化合物进行氧化处理，得到所述氧化天然高分子化合物具体包括以下步骤：
步骤S11，将所述天然高分子化合物溶于第一溶剂中，得到天然高分子化合物溶液。
在一些实施例中，所述天然高分子化合物包括纤维素及其衍生物、海藻酸钠、壳聚糖及其衍生物、透明质酸、魔芋甘露聚糖、淀粉及其衍生物、果胶和卡拉胶中的至少一种。第一溶剂是指能够溶解天然高分子化合物的溶剂。在一些实施例中，所述第一溶剂可以为乙醇等有机溶剂，或酸性、碱性溶剂。在一些实施例中，所述第一溶剂为水。
步骤S12，将氧化剂溶于第二溶剂中，得到氧化性溶液。第二溶剂是指能够溶解氧化剂的溶剂。在一些实施例中，所述第二溶剂包括水。
在一些实施例中，所述氧化剂可以为高碘酸钠。具体地，将高碘酸钠溶于水中，得氧化性溶液。
步骤S13，将所述天然高分子化合物溶液和所述氧化性溶液混合，氧化反应后得到所述氧化天然高分子化合物。
在一些实施例中，所述氧化天然高分子化合物可为双醛化天然高分子化合物。具体地，所述氧化剂(如高碘酸钠)将所述天然高分子化合物中的糖环氧化，以得到包含两个醛基的氧化高分子化合物。
在一些实施例中，可以加入终止剂以终止氧化反应。在一些实施例中，终止剂包括乙二醇。
在一些实施例中，可以通过控制氧化剂和天然高分子化合物之间的比例或混合时间得到不同氧化度的氧化天然高分子化合物。
在一些实施例中，将天然高分子化合物溶液和所述氧化性溶液氧化反应0.5h-48h后得到氧化天然高分子化合物。在一些实施例中，将天然高分子化合物溶液和所述氧化性溶液氧化反应8h-24h后得到氧化天然高分子化合物。在一些实施例中，将天然高分子化合物溶液和所述氧化性溶液氧化反应1h、4h、10h、12h、15h、或24h后得到氧化天然高分子化合物。
在一些实施例中，天然高分子化合物溶液和所述氧化性溶液混合后，高分子化合物在混合液中的初始反应浓度介于0.1％-20％之间。在一些实施例中，高分子化合物在混合液 中的初始反应浓度为0.1％、0.5％、5％、10％、或20％。在一些实施例中，高分子化合物在混合液中的初始反应浓度介于1％-5％之间。在一些实施例中，高分子化合物在混合液中的初始反应浓度为1％或3％。
在一些实施例中，天然高分子化合物溶液和所述氧化性溶液混合后，氧化剂在混合液中的初始反应浓度介于0.1％-40％之间。在一些实施例中，氧化剂在混合液中的初始反应浓度为0.1％、5％、10％、20％或40％。在一些实施例中，氧化剂在混合液中的初始反应浓度介于1％-10％之间。在一些实施例中，氧化剂在混合液中的初始反应浓度为0.3％、0.5％、1％、3％、8％、或10％。
在一些实施例中，所述氧化天然高分子化合物的氧化度为5％～85％。
在一些实施例中，所述氧化天然高分子化合物的氧化度为5％-60％。在一些实施例中，所述氧化天然高分子化合物的氧化度为20％-70％。在一些实施例中，所述氧化天然高分子化合物的氧化度为30％-80％。在一些实施例中，所述氧化天然高分子化合物的氧化度为5％、20％、30％、40％、50％、60％或85％。在一些实施例中，所述氧化天然高分子化合物的氧化度为30％～60％。在一些实施例中，所述氧化天然高分子化合物的氧化度为35％、45％、或55％。
本说明书实施例通过调整氧化剂和天然高分子化合物在混合液中的初始反应浓度、氧化反应的时间来控制最终氧化天然高分子化合物的氧化度，进而控制敏感基元的数量，保证传感器在合适传感灵敏度区间。
在一些实施例中，所述生物传感器的制备方法包括：将所述氧化天然高分子化合物作为交联剂，用于将敏感基元固定在所述工作电极中。
在一些实施例中，所述工作电极包括酶膜层，所述酶膜层中包括用所述氧化天然高分子化合物固定的所述敏感基元。
在一些实施例中，所述氧化天然高分子化合物通过将天然高分子化合物氧化后的得到。在一些实施例中，所述天然高分子化合物包括纤维素及其衍生物、海藻酸钠、壳聚糖及其衍生物、透明质酸、魔芋甘露聚糖、淀粉及其衍生物、果胶和卡拉胶中的至少一种。
在一些实施例中，所述敏感基元可以是带有伯氨基的化合物。在一些实施例中，所述敏感基元包括葡萄糖氧化酶、乳酸氧化酶、尿酸酶、或醇氧化酶中的一种。
在一些实施例中，所述生物传感器的制备方法还包括制备所述酶膜层的方法。在一些实施例中，所述酶膜层的制备方法包括：将酶稳定剂、所述敏感基元和所述氧化天然高分子化合物于第三溶剂中混合，得到混合溶液；以及将所述混合溶液涂覆在金属层上，交联反 应后得到所述酶膜层。在一些实施例中，所述第三溶剂包括水。
在一些实施例中，所述敏感基元包括葡萄糖氧化酶。
在一些实施例中，所述酶稳定剂包括血清蛋白(例如，牛血清蛋白)、血清酶、或其他具有酶稳定作用的蛋白质。
在一些实施例中，所述交联反应包括葡萄糖氧化酶中的伯氨基与氧化天然高分子化合物中的醛基发生希夫碱反应。
在一些实施例中，混合溶液中，所述敏感基元的浓度为0.5％～20％；所述氧化天然高分子化合物的浓度为0.1％～20％；所述酶稳定剂的浓度为0.5％～20％。在一些实施例中，混合溶液中，所述敏感基元的浓度为5％～20％；所述氧化天然高分子化合物的浓度为1％～20％；所述酶稳定剂的浓度为5％～20％。在一些实施例中，混合溶液中，所述敏感基元的浓度为5％～10％；所述氧化天然高分子化合物的浓度为10％～20％；所述酶稳定剂的浓度为5％～10％。在一些实施例中，在所述混合溶液中，所述敏感基元的浓度为2％～10％；所述氧化天然高分子化合物的浓度为5％～15％；所述酶稳定剂的浓度为0.5％～5％。
在一些实施例中，所述交联反应的温度为0℃～37℃。在一些实施例中，所述交联反应的温度为5℃～15℃。在一些实施例中，所述交联反应的温度为0℃～10℃。在一些实施例中，所述交联反应的温度为4℃。
现以所述敏感基元为葡萄糖氧化酶、酶稳定剂为血清蛋白为例进行酶膜层的制备说明。所述酶膜层202的制备方法包括以下步骤：
步骤S21，将血清蛋白、所述葡萄糖氧化酶和所述氧化天然高分子化合物于溶剂中混合，得到葡萄糖氧化酶混合溶液。在一些实施例中，所述溶剂为水。
具体地，将不同比例的所述血清蛋白和所述葡萄糖氧化酶配制成一定浓度的水溶液，然后将所述氧化天然高分子化合物配置成一定浓度的水溶液，将两者混合后得到所述葡萄糖氧化酶混合溶液。
在一些实施例中，在所述葡萄糖氧化酶混合溶液中，所述血清蛋白的浓度为0.5％～20％，所述葡萄糖氧化酶的浓度为0.5％～20％，所述氧化天然高分子化合物的浓度为0.1％～20％。
步骤S22，将所述葡萄糖氧化酶混合溶液涂覆在所述金属层上，交联反应后得到所述酶膜层202。
具体地，将混合得到的所述葡萄糖氧化酶混合溶液立即涂敷在所述金属层上，在一定温度下交联一定时间，使所述葡萄糖氧化酶中的伯氨基与所述氧化天然高分子化合物中的 醛基发生希夫碱反应，以形成稳定的所述酶膜层202。可以理解，在不同温度下交联所需要的交联时间也不同。
在一些实施例中，所述葡萄糖氧化酶混合溶液在所述金属层上进行交联反应的温度为0℃～37℃。在一些实施例中，所述葡萄糖氧化酶混合溶液在所述金属层上进行交联反应的温度为4℃。
由于天然高分子的生物相容性比较好，因此高分子化合物在常常被用于在材料领域，具体的，用于水凝胶、人体组织支架等的制备。然而，天然高分子化合物在生物传感器方面应用时，由于高分子本身的性质，可能会增加酶膜层厚度，考虑到需要对酶膜层进行厚度控制，因此，本说明书一些实施例中对交联剂的用量、温度进行了工艺参数的控制，进而实现了对酶膜层的厚度的有效控制。使得酶膜层既不至于过厚易脱落影响稳定性，又不至于过薄影响传感性能。
在一些实施例中，所述保护层203的材质包括聚氨酯、聚乙二醇、醋酸纤维素、聚乙烯醇、聚乙烯吡咯烷酮以及聚乙烯中的至少一种。
其中，所述保护层203可采用浸涂法制备。具体地，将所述保护层的材质(即聚氨酯、聚乙二醇、醋酸纤维素、聚乙烯醇、聚乙烯吡咯烷酮以及聚乙烯中的至少一种)溶于溶剂中，得到保护层溶液，再将所述酶膜层202放入在所述保护层溶液中，取出后干燥，即得到所述保护层。
在一些实施例中，所述保护层的溶剂包括四氢呋喃、N,N-二甲基甲酰胺(DMF)、环己烷以及丙酮中的至少一种。
在一些实施例中，在所述保护层溶液中，所述保护层的材质(即聚氨酯、聚乙二醇、醋酸纤维素、聚乙烯醇、聚乙烯吡咯烷酮以及聚乙烯中的至少一种)的浓度为0.5％～10％。
本说明书实施例之一还提供了一种生物传感器在生物传感中的应用。在一些实施例中，所述生物传感器可以包括：免疫传感器、酶传感器、核酸传感器和细胞传感器。
在一些实施例中，所述生物传感器用于检测葡萄糖、尿酸、乳酸或醇类物质中的至少一种。
在一些实施例中，所述生物传感器为葡萄糖生物传感器，所述敏感基元包括葡萄糖氧化酶。
以下实施例是对与上述一些实施例相关的实施例的一些更具体的说明。这些实施例中的部分内容也可以与其他实施例中的相应内容替换或组合，从而形成新的实施例。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特 殊说明，均为自常规生化试剂公司购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。应当理解，以下实施例是为了更好地解释本发明，并不旨在限制本发明。
实施例1
第一步，将3g海藻酸钠溶于150ml蒸馏水中，得到海藻酸钠水溶液。
第二步，将2g高碘酸钠溶于50ml蒸馏水中，得到高碘酸钠水溶液。
第三步，将高碘酸钠水溶液滴加在海藻酸钠水溶液中，并在室温下避光反应24h，然后加入5ml乙二醇搅拌1h以终止反应，再通过透析纯化，并在烘箱中干燥后得到氧化海藻酸钠。
第四步，将氧化海藻酸钠溶解在蒸馏水中，配制成浓度为3％的氧化海藻酸钠水溶液，并向其中加入葡萄糖氧化酶，使其浓度为30mg/ml，再向其中加入牛血清蛋白，使其浓度为20mg/ml，搅拌溶解后得到预交联的葡萄糖氧化酶混合溶液。
第五步，将上述预交联的葡萄糖氧化酶混合溶液涂敷在金属层上，并在4℃冰箱交联24h，再用蒸馏水清洗未交联的所述葡萄糖氧化酶，以得到酶膜层。
第六步，将酶膜层放入在浓度为5％的聚氨酯溶液中，取出后经室温干燥，形成一层保护层，从而得到工作电极。
实施例2
第一步，将5g羧甲基纤维素溶于500ml蒸馏水中，得到羧甲基纤维素水溶液。
第二步，将4.5g高碘酸钠溶于50ml蒸馏水中，得到高碘酸钠水溶液。
第三步，将高碘酸钠水溶液滴加在羧甲基纤维素水溶液中，并在4℃下避光反应24h，然后加入5ml乙二醇搅拌1h以终止反应，再通过过滤，并在烘箱中干燥后得到氧化羧甲基纤维素。
第四步，将氧化羧甲基纤维素溶解在蒸馏水中，配制成浓度为5％的氧化羧甲基纤维素水溶液，并向其中加入葡萄糖氧化酶，使其浓度为20mg/ml，再向其中加入牛血清蛋白，使其浓度为15mg/ml，搅拌溶解后得到预交联的葡萄糖氧化酶混合溶液。
第五步，将上述预交联的葡萄糖氧化酶混合溶液涂敷在金属层上，并在4℃冰箱交联12h，再用蒸馏水清洗未交联的所述葡萄糖氧化酶，以得到酶膜层。
第六步，将酶膜层放入在浓度为5％的聚氨酯溶液中，取出后经室温干燥，形成一层保护层，从而得到工作电极。
实施例3
第一步，将1g果胶溶于100ml蒸馏水中，得到果胶水溶液。
第二步，将1g高碘酸钠溶于50ml蒸馏水中，得到高碘酸钠水溶液。
第三步，将高碘酸钠水溶液滴加在果胶水溶液中，并在4℃下避光反应12h，然后加入5ml乙二醇搅拌1h以终止反应，再通过过滤，并在烘箱中干燥后得到氧化果胶。
第四步，将氧化果胶溶解在蒸馏水中，配制成浓度为1％的氧化果胶水溶液，并向其中加入葡萄糖氧化酶，使其浓度为10mg/ml，再向其中加入牛血清蛋白，使其浓度为8mg/ml，搅拌溶解后得到预交联的葡萄糖氧化酶混合溶液。
第五步，将上述预交联的葡萄糖氧化酶混合溶液涂敷在金属层上，并在4℃冰箱交联12h，再用蒸馏水清洗未交联的所述葡萄糖氧化酶，以得到酶膜层。
第六步，将酶膜层放入在浓度为5％的聚氨酯溶液中，取出后经室温干燥，形成一层保护层，从而得到工作电极。
对比例1
将实施例1～3以及对比例1中制备得到的工作电极分别用于制备葡萄糖生物传感器。其中，对比例1中葡萄糖生物传感器的制备方法如下：
第一步，选取厚度为120μm的聚酰亚胺(PI)作为柔性基底将玻璃或硅片支撑衬底和PI清洗后用氮气枪吹干。
第二步，将聚二甲基硅氧烷(PDMS)旋涂到支撑衬底上，并加热固化。
第三步，将清洗好的PI用贴膜机和涂有PDMS的支撑衬底吸附。
第四步，采用磁控溅射仪在PI柔性基底上溅射一层粘接层铬(Cr)，再溅射导电层金(Au)，然后旋涂一层光刻胶，接着采用曝光机进行图案化过程，最后进行显影反应。对导电层Au进行湿法刻蚀，然后用大量水清洗干净并用氮气吹干。然后进行粘接层Cr刻蚀，直到非图案区域完全裸露出PI颜色(红棕色)，用大量水清洗干净并用氮气吹干。最后将光刻胶剥离，并检查导线是否导通。
第五步，对PI柔性基底进行聚对二甲苯沉积，然后旋涂光刻胶，显影进行图案化，再进行等离子处理，以对显影部分的聚对二甲苯沉积进行清洗，最后将光刻胶剥离，并检查引线区域是否绝缘。
第六步，制备工作电极和对电极：先在工作电极和对电极的PI柔性基底上分别旋涂一层光刻胶，接着采用曝光机进行图案化过程，最后进行显影反应，水洗后用氮气吹干。采用磁控溅射仪在PI柔性基底上溅射一层粘接层Cr，再溅射导电层铂(Pt)，并在丙酮浸泡超声，直到工作电极(WE)、对电极(CE)图案完全抬离干净，再分别用干净的丙酮、酒 精、水冲洗干净并用氮气枪吹干，用万用表测试WE、CE与相对应的元件孔(PAD)的导通性，不同导线的绝缘性。得到工作电极和对电极。
第七步，制备包含酶膜层的工作电极：将葡萄糖氧化酶和牛血清蛋白溶解于磷酸盐缓冲液(PBS)中，然后向其中添加2.5％的戊二醛溶液，得到预交联的葡萄糖氧化酶混合溶液。将上述预交联的葡萄糖氧化酶混合溶液涂敷在工作电极的金属层上，并在4℃冰箱交联24h，再用蒸馏水清洗未交联的所述葡萄糖氧化酶，以得到酶膜层。将包含酶膜层的工作电极放入在浓度为5％的聚氨酯溶液中，取出后经室温干燥，形成一层保护层，从而得到干燥的包含酶膜层工作电极。
第八步，制备参比电极制备：电镀银层，采用脉冲电流源装置进行恒电流方式电镀。氯化处理：先用氩气等离子处理5分钟，然后用氯化铁溶液处理，最后用水冲洗干净并用氮气吹干，得到参比电极。
按照对比例1的制备方法将实施例1～3中制备得到的工作电极分别用于制备葡萄糖生物传感器。
实施例4生物传感器的灵敏度和稳定性测试
(一)对由实施例1-3中的工作电极制备得到的葡萄糖生物传感器进行灵敏度测试。
具体地，由实施例1中的工作电极按照对比例1的方法制备5批葡萄糖生物传感器，并对这5批传感器在0～30mM的葡萄糖浓度下进行灵敏度测试。
实施例1中工作电极所制备的生物传感器的灵敏度测试结果如图3A所示，测试结果表明，由实施例1中的工作电极制备得到的5批葡萄糖生物传感器在0～30mM的葡萄糖浓度的范围内均能实现线性，且5批葡萄糖生物传感器的批次间的差异性在5％以下。
对由实施例2-3中的工作电极制备得到的葡萄糖生物传感器进行灵敏度测试。具体地，由实施例2-3中的工作电极按照对比例1的方法制备葡萄糖生物传感器，并对传感器在0～20mM的葡萄糖的浓度下进行灵敏度测试。
实施例2中工作电极所制备的生物传感器的灵敏度测试结果如图4A所示，测试结果表明，由实施例2中的工作电极制备得到的葡萄糖生物传感器在0～20mM的葡萄糖浓度的范围内均能实现线性。
实施例3中工作电极所制备的生物传感器的灵敏度测试结果如图5A所示，测试结果表明，由实施例3中的工作电极制备得到的葡萄糖生物传感器在0～20mM的葡萄糖浓度的范围内均能实现线性。
(二)对由实施例1-3中的工作电极制备得到的葡萄糖生物传感器进行长期稳定性测 试。
实施例1中工作电极所制备的生物传感器的灵敏度测试结果如图3B所示。测试结果表明，由实施例1中的工作电极制备得到的葡萄糖生物传感器在30天内灵敏度变化在2％以下。这表明本发明制备的葡萄糖生物传感器具有长期的稳定性。
实施例2中工作电极所制备的生物传感器的灵敏度测试结果如图4B所示。测试结果表明，由实施例2中的工作电极制备得到的葡萄糖生物传感器在30天内灵敏度变化在10％以下。这表明本发明制备的葡萄糖生物传感器具有长期的稳定性。
实施例3中工作电极所制备的生物传感器的灵敏度测试结果如图5B所示。测试结果表明，由实施例3中的工作电极制备得到的葡萄糖生物传感器在30天内灵敏度变化在5％以下。这表明本发明制备的葡萄糖生物传感器具有长期的稳定性。
由以上测试结果可知，由氧化后的海藻酸钠作为酶膜层的交联剂应用到生物传感器中与敏感基元进行交联时，制备得到的传感器的稳定性最佳。这可能是由于海藻酸钠本身的分子结构使得最终的交联效果最佳，在保证敏感基元数量的同时，酶膜层中的敏感基元也能够更稳定地被固定在工作电极中。
实施例5工作电极的细胞毒性测试
分别对实施例1～3以及对比例1中制备得到的工作电极进行细胞毒性测试。
工作电极细胞毒性测试方法为：分别将实施例1～3以及对比例1中制备得到的工作电极浸泡在PBS中进行浸提，以得到浸提液，并将所得的浸提液进行细胞毒性试验。
图6根据本说明书一些实施例所示的工作电极的细胞毒性测试结果图。
请参阅图6，测试结果表明细胞在实施例1～3制备得到的工作电极的浸提液中存活率较高，但细胞在对比例1制备得到的工作电极的浸提液中的存活率较低。这说明氧化天然高分子化合物的毒性低于戊二醛，从而说明本发明采用氧化天然高分子化合物作为交联剂明显优于现有技术采用戊二醛作为交联剂。
本说明书实施例可能带来的有益效果包括但不限于：(1)通过对天然高分子化学物进行氧化处理，得到氧化天然高分子化合物，采用氧化天然高分子化合物作为交联剂将敏感基元固定在生物传感器的工作电极中，避免了传统化学交联剂的生物毒性，提高了生物传感器工作电极的生物相容性；(2)通过调整天然高分子化合物参与的氧化反应与酶膜层制备过程中的工艺参数，优化和提高了敏感基元化学键合的环境和过程的可控性，有利于保持敏感基元的数量和活性，以保证敏感基元的活性和稳定性，进而提高了生物传感器的一致性和长期稳定性。
需要说明的是，不同实施例可能产生的有益效果不同，在不同的实施例里，可能产生的有益效果可以是以上任意一种或几种的组合，也可以是其他任何可能获得的有益效果。
上文已对基本概念做了描述，显然，对于本领域技术人员来说，上述详细披露仅仅作为示例，而并不构成对本说明书的限定。虽然此处并没有明确说明，本领域技术人员可能会对本说明书进行各种修改、改进和修正。该类修改、改进和修正在本说明书中被建议，所以该类修改、改进、修正仍属于本说明书示范实施例的精神和范围。
同时，本说明书使用了特定词语来描述本说明书的实施例。如“一个实施例”、“一些实施例”、和/或“一些实施例”意指与本说明书至少一个实施例相关的某一特征、结构或特点。因此，应强调并注意的是，本说明书中在不同位置两次或多次提及的“一些实施例”或“一个实施例”或“一个替代性实施例”并不一定是指同一些实施例。此外，本说明书的一个或多个实施例中的某些特征、结构或特点可以进行适当的组合。
此外，除非权利要求中明确说明，本说明书所述处理元素和序列的顺序、数字字母的使用、或其他名称的使用，并非用于限定本说明书流程和方法的顺序。尽管上述披露中通过各种示例讨论了一些目前认为有用的发明实施例，但应当理解的是，该类细节仅起到说明的目的，附加的权利要求并不仅限于披露的实施例，相反，权利要求旨在覆盖所有符合本说明书实施例实质和范围的修正和等价组合。例如，虽然以上所描述的系统组件可以通过硬件设备实现，但是也可以只通过软件的解决方案得以实现，如在现有的服务器或移动设备上安装所描述的系统。
同理，应当注意的是，为了简化本说明书披露的表述，从而帮助对一个或多个发明实施例的理解，前文对本说明书实施例的描述中，有时会将多种特征归并至一个实施例、附图或对其的描述中。但是，这种披露方法并不意味着本说明书对象所需要的特征比权利要求中提及的特征多。实际上，实施例的特征要少于上述披露的单个实施例的全部特征。
一些实施例中使用了描述成分、属性数量的数字，应当理解的是，此类用于实施例描述的数字，在一些示例中使用了修饰词“大约”、“近似”或“大体上”来修饰。除非另外说明，“大约”、“近似”或“大体上”表明所述数字允许有±20％的变化。相应地，在一些实施例中，说明书和权利要求中使用的数值参数均为近似值，该近似值根据个别实施例所需特点可以发生改变。在一些实施例中，数值参数应考虑规定的有效数位并采用一般位数保留的方法。尽管本说明书一些实施例中用于确认其范围广度的数值域和参数为近似值，在具体实施例中，此类数值的设定在可行范围内尽可能精确。
针对本说明书引用的每个专利、专利申请、专利申请公开物和其他材料，如文章、 书籍、说明书、出版物、文档等，特此将其全部内容并入本说明书作为参考。与本说明书内容不一致或产生冲突的申请历史文件除外，对本说明书权利要求最广范围有限制的文件(当前或之后附加于本说明书中的)也除外。需要说明的是，如果本说明书附属材料中的描述、定义、和/或术语的使用与本说明书所述内容有不一致或冲突的地方，以本说明书的描述、定义和/或术语的使用为准。
最后，应当理解的是，本说明书中所述实施例仅用以说明本说明书实施例的原则。其他的变形也可能属于本说明书的范围。因此，作为示例而非限制，本说明书实施例的替代配置可视为与本说明书的教导一致。相应地，本说明书的实施例不仅限于本说明书明确介绍和描述的实施例。

Claims (25)

  1. 一种生物传感器，其特征在于，
    所述生物传感器包括工作电极，所述工作电极包括酶膜层，其中，
    所述酶膜层中包括用氧化天然高分子化合物作为交联剂固定的敏感基元，其中，所述氧化天然高分子化合物是由天然高分子化合物经氧化处理得到的。
  2. 如权利要求1所述的生物传感器，其特征在于，所述天然高分子化合物包括纤维素及其衍生物、海藻酸钠、壳聚糖及其衍生物、透明质酸、魔芋甘露聚糖、淀粉及其衍生物、果胶和卡拉胶中的至少一种。
  3. 如权利要求1所述的生物传感器，其特征在于，所述氧化天然高分子化合物为双醛化天然高分子化合物。
  4. 如权利要求3所述的生物传感器，其特征在于，所述氧化天然高分子化合物的氧化度为5％～85％。
  5. 如权利要求4所述的生物传感器，其特征在于，所述氧化天然高分子化合物的氧化度为30％～60％。
  6. 如权利要求1所述的生物传感器，其特征在于，所述敏感基元包括葡萄糖氧化酶、乳酸氧化酶、尿酸酶、或醇氧化酶中的一种。
  7. 如权利要求1所述的生物传感器，其特征在于，所述酶膜层还包括酶稳定剂。
  8. 如权利要求1或7所述的生物传感器，其特征在于，所述酶膜层的厚度范围在0.1μm-20μm之间。
  9. 如权利要求8所述的生物传感器，其特征在于，所述酶膜层的厚度范围在1μm-5μm之间。
  10. 如权利要求1所述的生物传感器，其特征在于，所述生物传感器还包括柔性基底、参比电极、对电极中的至少一种。
  11. 如权利要求10所述的生物传感器，其特征在于，所述柔性基底的厚度范围在10μm～300μm之间。
  12. 如权利要求1所述的生物传感器，其特征在于，所述工作电极还包括金属层、保护层中的至少一种。
  13. 如权利要求12所述的生物传感器，其特征在于，所述工作电极的所述金属层由内到外依次为第一金属层、第二金属层和第三金属层；其中所述第一金属层为Cr、Ti、Ni-Cr中的至少一种；所述第二金属层为金、铜、银中的至少一种；所述第三金属层为铂、铂铱、铂碳中的至少一种。
  14. 如权利要求12所述的生物传感器，其特征在于，所述保护层包括聚氨酯、聚乙二醇、醋酸纤维素、聚乙烯醇、聚乙烯吡咯烷酮、聚乙烯中的至少一种。
  15. 一种生物传感器的制备方法，所述方法包括：
    对天然高分子化合物进行氧化处理，得到氧化天然高分子化合物；以及
    将所述氧化天然高分子化合物作为交联剂，将敏感基元固定在所述生物传感器的工作电极中。
  16. 如权利要求15所述的制备方法，其特征在于，所述对天然高分子化合物进行氧化处理，得到氧化天然高分子化合物具体包括以下步骤：
    将所述天然高分子化合物溶于第一溶剂中，得到天然高分子化合物溶液；
    将氧化剂溶于第二溶剂中，得到氧化性溶液；以及
    将所述天然高分子化合物溶液和所述氧化性溶液混合，氧化反应后得到所述氧化天然高分子化合物。
  17. 如权利要求15或16所述的制备方法，其特征在于，所述氧化天然高分子化合物的氧化度为5％～85％。
  18. 如权利要求17所述的制备方法，其特征在于，所述氧化天然高分子化合物的氧化度为30％～60％。
  19. 如权利要求15所述的制备方法，其特征在于，所述工作电极包括酶膜层，所述酶膜层中包括用所述氧化天然高分子化合物固定的所述敏感基元。
  20. 如权利要求19所述的制备方法，其特征在于，进一步包括制备所述酶膜层的方法；其中，
    所述酶膜层的制备方法包括以下步骤：
    将酶稳定剂、所述敏感基元和所述氧化天然高分子化合物于第三溶剂中混合，得到混合溶液；以及
    将所述混合溶液涂覆在金属层上，交联反应后得到所述酶膜层。
  21. 如权利要求20所述的制备方法，其特征在于，
    在所述混合溶液中，所述敏感基元的浓度为0.5％～20％；
    所述氧化天然高分子化合物的浓度为0.1％～20％；
    所述酶稳定剂的浓度为0.5％～20％；
    所述交联反应的温度为0℃～37℃。
  22. 如权利要求21所述的制备方法，其特征在于，
    在所述混合溶液中，所述敏感基元的浓度为2％～10％；
    所述氧化天然高分子化合物的浓度为5％～15％；
    所述酶稳定剂的浓度为0.5％～5％；
    所述交联反应的温度为5℃～15℃。
  23. 如权利要求1至22中任一项所述的生物传感器在生物传感中的应用。
  24. 如权利要求23所述的生物传感器在生物传感中的应用，其特征在于，所述生物传感器包括免疫传感器、酶传感器、核酸传感器、或细胞传感器。
  25. 如权利要求24所述的生物传感器在生物传感中的应用，其特征在于，所述生物传感器用于检测葡萄糖、尿酸、乳酸或醇类物质中的至少一种。
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