CN116359311A - Dynamic lactic acid sensor film and preparation method thereof - Google Patents

Dynamic lactic acid sensor film and preparation method thereof Download PDF

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Publication number
CN116359311A
CN116359311A CN202310117045.6A CN202310117045A CN116359311A CN 116359311 A CN116359311 A CN 116359311A CN 202310117045 A CN202310117045 A CN 202310117045A CN 116359311 A CN116359311 A CN 116359311A
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enzyme
lactic acid
layer
solution
peba
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郭劲宏
褚正康
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Chongqing Lianxin Zhikang Biotechnology Co ltd
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Chongqing Lianxin Zhikang Biotechnology Co ltd
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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
    • G01N27/416—Systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • 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
    • 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
    • C12Q1/004—Enzyme electrodes mediator-assisted
    • 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
    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
    • A61B2562/12—Manufacturing methods specially adapted for producing sensors for in-vivo measurements
    • 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)

Abstract

The invention relates to the technical field of sensors, in particular to a dynamic lactic acid sensor film, which comprises the following components: the lactic acid and oxygen in the liquid to be detected sequentially pass through the diffusion layer and the anti-interference layer to be contacted with the enzyme layer, the enzyme layer reacts with the lactic acid to generate hydrogen peroxide, the hydrogen peroxide is diffused to the metal catalytic layer to be reduced, and the metal conductive layer is contacted with the metal layer. Also relates to a preparation method of the dynamic lactic acid sensor film, which comprises the following steps: s1: proportioning PEBA and DMF to obtain PEBA solution; the mass ratio of PEBA to DMF is 1:1-1:20; s2: stirring and heating the prepared PEBA solution; the composition and proportion of the blend are flexibly designed, and the biocompatibility, the oxygen permeability, the lactic acid permeability and the mechanical property of the dynamic lactic acid sensor can be accurately regulated and controlled through a simple process, so that the detection range, the repeatability, the service life, the mass production and the like are improved.

Description

Dynamic lactic acid sensor film and preparation method thereof
Technical Field
The invention relates to the technical field of sensors, in particular to a dynamic lactic acid sensor film and a preparation method thereof.
Background
Lactic acid is the final product of glucose in the anaerobic metabolic process in human body, and the oxygen demand of reaction tissues and the oxygen supply capacity of blood are balanced, so that acute critical diseases and chronic disease managers are extremely easy to increase lactic acid to cause lactic acidosis, thereby endangering life. Such as: serious hypoxia of tissues caused by heart and lung dysfunction, vascular obstruction, shock, anemia, heart failure, asphyxia, carbon monoxide poisoning and the like, and abnormal increase of lactic acid; the biguanide drugs are taken by diabetes, so that lactic acid is accumulated in the body; systemic diseases such as malignant tumor and liver and kidney dysfunction cause liver and kidney dysfunction of the organism, and excessive lactic acid in the body cannot be metabolized and discharged out of the body. Currently, extracorporeal blood detection is a method for monitoring lactic acid, and it is difficult to obtain real-time accurate lactic acid change information even when blood is frequently drawn. The dynamic lactic acid detection implanted in the body can continuously monitor the lactic acid content of a patient, provide timely and accurate guidance, and better ensure smooth operation, reasonable administration of chronic disease management and diagnosis.
Dynamic lactic acid sensors based on the first generation principle are still in use in many product studies. They utilize lactate oxidase to oxidize lactic acid to hydrogen peroxide in the presence of oxygen, the hydrogen peroxide being further reduced by a catalyst such as nano platinum, and the resulting redox current being related to the lactic acid content. Wherein the diffusion layer is one of the important film layers of the dynamic lactic acid sensor, usually used as an outer surface film, and is contacted with the measuring solution. The film layer controls the amount of lactic acid and oxygen entering the film on one hand, and the image sensor detects linearity, sensitivity and balancing time; on the other hand, the compatibility with tissue fluid, the stability of the image sensor and the side effect on human body are determined. Considering that the lactic acid content in tissue fluid is tens times higher than that of oxygen, and that the water solubility (i.e. hydrophilic) of lactic acid, the non-water solubility (i.e. hydrophobic) of oxygen and the hydrophilic characteristic of tissue fluid, the current research is mainly focused on introducing hydrophilic segments and hydrophobic segments into a polymer diffusion membrane simultaneously by means of polymerization reaction means such as copolymerization, grafting and the like, it is expected to limit the permeation quantity of lactic acid but improve the permeation quantity of oxygen on the basis of maintaining biocompatibility, adjust the unbalance of the lactic acid and oxygen content in tissue fluid, and optimize the detection linearity, sensitivity and service life. However, the polymerization reaction such as copolymerization and grafting is complex, the reaction condition is severe, the repeatability is poor, and stable mass production is not easy. In addition, the organic monomer and the initiator remain in the reaction, and the toxicity risk caused by implantation into the human body is not neglected. Overall, the overall performance controlling effect is not satisfactory.
In the prior art, the polymer with both hydrophilic segments and hydrophobic segments is obtained by polymerizing or grafting the compound through a diffusion layer, the biocompatibility and the lactic acid permeability are regulated by using the hydrophilic segments, and the oxygen permeability is regulated by using the hydrophobic segments.
Regarding copolymerization: the components participating in the copolymerization reaction comprise two or more monomers and one or more initiators to form a block polymer.
1. The hydrophilic-hydrophobic copolymerization regulation and control mode is as follows: screening and combining monomer types, adjusting the feeding proportion of hydrophilic and hydrophobic monomers, saving the monomer concentration, controlling the reaction degree through temperature, stirring, sample adding modes and the like.
2. The monomers forming the hydrophilic segment typically bear hydrophilic groups and are compatible with water, and the resulting polymer comprises: epoxy functional groups such as polyethylene oxide, polypropylene oxide, and the like; hydroxyl functional groups such as polyethylene glycol, vinyl alcohol, and the like; with carboxyl and amino functions, such as vinyl alanine, acetyl alanine, etc.
3. The monomer forming the hydrophobic segment is provided with a hydrophobic group, the compatibility with water is poor, and the formed polymer comprises: polypropylene, polyethylene, polystyrene, and the like.
4. The block polymer comprises two-stage or three-stage copolymerization.
5. The initiator comprises azo compounds, peroxy compounds and composite compounds. Azo compounds commonly used, such as azobisisobutyronitrile, azobisisoheptonitrile, and dimethyl azobisisobutyrate initiator, etc.; conventional peroxides such as hydrogen peroxide, ammonium persulfate, potassium persulfate, benzoyl peroxide, t-butyl benzoyl peroxide, methyl ethyl ketone peroxide, etc.; common composite initiation systems, such as an azodiisobutyronitrile and azodiisoheptonitrile composite initiation system, a dibenzoyl peroxide and tert-butyl peroxybenzoate composite initiation system and the like.
Regarding grafting: hydrophobic backbone chains and hydrophilic grafting groups are formed by grafting. The components participating in the grafting reaction include two or more monomers forming the backbone chain, and a grafting compound.
1. The hydrophilic-hydrophobic grafting regulation mode is as follows: the proportion of the grafting compound to the framework is adjusted, the molecular weight and the concentration of the grafting compound are controlled by the changes of temperature, stirring, sample adding modes and the like, and the reaction degree is controlled.
2. The grafting compound is provided with hydrophilic groups, mainly polyethylene glycol with hydroxyl functional groups and derivatives thereof, has various molecular weights for selection, and performs grafting reaction in modes of polycondensation reaction, ultraviolet crosslinking and the like.
3. The monomer forming the skeleton chain mainly refers to diisocyanate, dihydric or polyhydric alcohol and a chain extender, and the diisocyanate, the dihydric or polyhydric alcohol and the chain extender react to form polyurethane prepolymer, and then the polyurethane prepolymer reacts with the grafting compound.
The above-described technique has the following problems:
1. the process is complicated, and generally involves harsh reaction conditions such as high temperature, nitrogen protection, high-speed stirring, slow and uniform sample addition and the like. The process control is poor, the effect of the prepared copolymer is not easy to be repeated, and stable batch production is difficult to realize;
2. the conversion rate of the copolymerization reaction is limited, and residual organic monomers and initiators are difficult to be removed cleanly even through repeated filtration, precipitation and other means. The risk of biotoxicity after implantation in the human body is not negligible.
3. Byproducts are easily generated, and there is an uncertain interference risk.
4. The copolymer is quickly dried to form a film at a higher temperature to obtain a low-crystallinity polymer with a large free volume, which is more beneficial to the passage of oxygen and lactic acid molecules. However, low crystallinity polymers are often accompanied by low mechanical properties, such as insufficient film strength. The current research neglects to design the comprehensive mechanical properties, and the damage of external force of the sensor in the process of implantation into a human body and in the process of use is easy to cause.
Aiming at the current situation, a diffusion membrane which has simple preparation process, proper biocompatibility, high oxygen permeability and low lactic acid permeability is required to be invented, and the detection range, accuracy, repeatability, service life and mass production of the dynamic lactic acid sensor can be remarkably improved.
Disclosure of Invention
The invention provides a dynamic lactic acid sensor membrane and a preparation method thereof, which can solve the problems.
In order to solve the technical problems, the application provides the following technical scheme:
a dynamic lactate sensor film comprising: the device comprises a metal conducting layer, a metal catalytic layer, an enzyme layer, an anti-interference layer and a diffusion layer, wherein lactic acid and oxygen in liquid to be detected sequentially pass through the diffusion layer and the anti-interference layer to be contacted with the enzyme layer, the enzyme layer reacts with lactic acid to generate hydrogen peroxide, the hydrogen peroxide is diffused to the metal catalytic layer to be reduced, and the metal conducting layer is contacted with the metal layer and is used for transmitting an oxidation-reduction current signal to an instrument contact end to convert an electric signal into a digital signal; the diffusion layer is composed of PEBA and an oxygen-rich polymer.
Basic principle and beneficial effect: the lactic acid sensor comprises a substrate, a metal catalytic layer, an enzyme layer, an anti-interference layer and a diffusion layer from inside to outside, wherein lactic acid and oxygen in liquid to be detected sequentially contact the enzyme layer through the diffusion layer and the anti-interference layer, and under the participation of the oxygen, the lactic acid is subjected to enzyme catalytic oxidation to generate hydrogen peroxide. Further, hydrogen peroxide diffuses into the metal catalytic layer to be reduced, and the conductive metal in contact with the hydrogen peroxide transmits a redox current signal to the instrument contact end, converts an electric signal into a digital signal, and outputs a detection concentration value. Wherein the substrate is both a conductive medium for electrical signals and a carrier for the sensor. Based on PEBA as a substrate and on the basis of keeping self biocompatibility and lactic acid permeability, polymer with good compatibility with PEBA and rich oxygen is added in a blending way, and meanwhile, the biocompatibility, lactic acid and oxygen permeation balance and mechanical property of the diffusion membrane are regulated and controlled, so that the detection range, accuracy, service life and batch production operability of the dynamic lactic acid sensor are remarkably improved.
The selected diffusion film substrate material PEBA is formed by polycondensation reaction of polyamide containing dihydroxyl and polyether glycol, has good film forming performance, and has the rigidity of polyamide and the softness of polyether. The rigid polyamide is a guarantee of mechanical properties of the blend diffusion membrane, has high strength and toughness similar to those of the polyamide, and can well resist external destructive forces randomly occurring in the implantation process and the use process of the dynamic lactic acid sensor. The soft polyether glycol has good biocompatibility and lactic acid permeability due to hydrophilicity on one hand, and has larger free volume due to flexibility on the other hand, and provides higher oxygen permeability. The comprehensive performance of the diffusion membrane can be controlled to a great extent by the simple operation of adjusting the composition of the rigid polyamide and the flexible polyether glycol;
considering that PEBA soft polyether glycol is helpful for permeation of oxygen and lactic acid, a state of high oxygen permeation and low lactic acid permeation is difficult to achieve by single use, and therefore, a strategy of blending PEBA and an oxygen-enriched polymer is introduced. After the oxygen-enriched polymer is added, compared with the permeation of lactic acid, the oxygen enters the membrane at a speed of ten times or even tens of times, so that the content of the oxygen and the lactic acid in tissue fluid is well adjusted to be in an equilibrium state, the effective catalysis of lactic acid oxidase is ensured, and the detection sensitivity, the linear range and the validity period of the dynamic lactic acid sensor are improved. In addition, the selected oxygen-enriched polymer has good compatibility with the PEBA substrate, avoids the phase separation phenomenon after the membrane is dried, and maintains the mechanical properties of the membrane.
Aims at solving the problems of complex procedures, and multiple side reactions of modification means such as copolymerization, grafting and the like. The blending regulation strategy is adopted, so that the method is simple and easy to operate, has no residual interference and is beneficial to batch production;
from the standpoint of ensuring the biocompatibility and mechanical properties of the diffusion membrane, PEBA is selected as a substrate, and is the main component of the diffusion membrane. The polyamide/polyether glycol copolymer is formed by polycondensation reaction of polyamide containing dihydroxyl and polyether glycol, has good film forming performance, and has the rigidity of polyamide and the softness of polyether. Rigid polyamides are a guarantee of the mechanical properties of the blended diffusion film. The soft polyether glycol has good biocompatibility and lactic acid permeability due to hydrophilicity on the one hand. In addition, the flexible PEBA of the polyether alcohol has a larger free volume and also has a higher oxygen permeability. The biocompatibility and the mechanical property of the membrane can be regulated and controlled by the simple operation of regulating the composition of the rigid polyamide and the flexible polyether glycol, and the oxygen permeability can be regulated to a certain extent;
the adopted blending strategy can not introduce small molecular organic matters, and the risk of toxicity and interference caused after implantation into a human body is low.
PEBA composed of different polyamides and polyether diols has been commercially available in a mature form, providing support to a certain extent for simplified and stable diffusion membrane manufacturing processes.
According to the scheme, the diffusion membrane of the dynamic lactic acid sensor has biocompatibility, high oxygen permeability, low lactic acid permeability and good mechanical property, the diffusion membrane is used as a substrate after the hydrophilicity, the biocompatibility and the mechanical property are regulated and controlled in PEBA, and then oxygen-enriched polymer with good PEBA compatibility is mixed to improve the oxygen permeability, so that the precise regulation and control of the comprehensive performance of the diffusion membrane are facilitated. The composition and proportion of the blend are flexibly designed, and the biocompatibility, the oxygen permeability, the lactic acid permeability and the mechanical property of the dynamic lactic acid sensor can be accurately regulated and controlled through a simple process, so that the detection range, the repeatability, the service life, the mass production and the like are improved.
Further, the enzyme layer also comprises enzyme, enzyme protective agent, enzyme cross-linking agent, hydrogel film-forming modifier and hydrogel mixed cross-linking agent; the enzyme is selected from lactic acid oxidase, and the mass ratio of the enzyme in the enzyme liquid is 1% -15%; the protective agent is selected from bovine serum albumin, and the ratio of the protective agent to enzyme is 0.1:1-10:1 range; the enzyme cross-linking agent is glutaraldehyde, the hydrogel is polyvinyl alcohol, and the mass ratio of the hydrogel to the enzyme solution is 0.1-10%; the hydrogel mixed crosslinking agent is selected from two or more of boric acid, glutaraldehyde, glyoxal, citric acid, oxalic acid, tannic acid, fumaric acid, maleic anhydride, 2, 3-dimethyl maleic anhydride and 2, 3-diphenyl maleic anhydride.
The beneficial effects are that: the enzyme layer is used for providing enzyme to catalyze and oxidize lactic acid to generate hydrogen peroxide, and the normal operation of lactic acid reaction can be ensured in use and the reaction efficiency can be improved through the proportioning and the use of the enzyme, the enzyme protecting agent, the enzyme cross-linking agent, the hydrogel film-forming modifier and the hydrogel mixed cross-linking agent.
Further, the ratio of the enzyme to the enzyme solution is 5%, the ratio of the protective agent to the enzyme is 2:1, and the mass ratio of the hydrogel to the enzyme solution is 2.5%.
Further, the interference layer is made of cellulose acetate, and the mass concentration of the interference layer is in the range of 0.1% -5%.
Further, the mass ratio of PEBA to oxygen-enriched polymer is controlled at 10:1-1:1 range.
A method for preparing a dynamic lactic acid sensor film, comprising the following steps:
s1: proportioning PEBA and DMF to obtain PEBA solution; the mass ratio of PEBA to DMF is 1:1-1:20;
s2: stirring and heating the prepared PEBA solution; stirring speed is 500rpm-900rpm, and heating temperature is: heating for 2-4h at 80-100 ℃; obtaining PEBA solution with concentration of 5% -20% after dissolution;
s3: preparing an oxygen-enriched polymer solution by using the oxygen-enriched polymer and DMF; the mass ratio of the oxygen-enriched polymer to DMF is 1:9-1:99;
s4: stirring and heating the prepared oxygen-enriched polymer solution; stirring speed is 500rpm-900rpm, and heating temperature is: heating for 2-4h at 80-100 ℃; obtaining PS solution with concentration of 1% -10% after dissolution;
s5: the PEBA solution and the oxygen-enriched polymer solution are mixed according to the mass ratio of 1:1, uniformly mixing to obtain a diffusion film solution;
s6: the needle electrode coated with the enzyme layer and the anti-interference layer is lifted for 1 time in the diffusion film solution by adopting a dipping and lifting process;
s7: and placing the coated electrode in a constant temperature and humidity box with the temperature of 37 ℃ and the humidity of 60% for 72 hours to obtain the diffusion film.
Further, PEBA described in step S1 selects one of PEBAX 1657, PEBAX 2533, PEBAX 3533, PEBAX4033, PEBAX 5333, PEBAX 6333, PEBAX7033, PEBAX 7233, and PEBAX 7233.
Further, the oxygen-rich polymer in step S3 is selected from one of polysulfone, cellulose acetate, polydimethylsiloxane or polytrimethylsilane-1-propyne.
Further, the PEBA selects PEBAX 1657 comprising 40% rigid chains and 60% soft chains, and the oxygen-rich polymer selects polysulfone.
Drawings
Fig. 1 is a cross-sectional view of a dynamic lactate sensor film and an embodiment of a method of making the same.
Detailed Description
The following is a further detailed description of the embodiments:
an embodiment as shown in figure 1 of the drawings,
a dynamic lactic acid sensor film is applied to a lactic acid sensor and comprises a metal conducting layer, a metal catalytic layer, an enzyme layer, an anti-interference layer and a diffusion layer from inside to outside. Specifically, lactic acid and oxygen in the liquid to be detected sequentially pass through the diffusion layer and the anti-interference layer to be contacted with the enzyme layer, and under the participation of the oxygen, the lactic acid is catalyzed and oxidized by the enzyme to generate hydrogen peroxide. Further, hydrogen peroxide is diffused to the metal catalyst 2 to be reduced, and the metal conducting layer contacted with the hydrogen peroxide transmits a redox current signal to the contact end of the instrument, converts an electric signal into a digital signal and outputs a detection concentration value. The metal conducting layer is not only the conducting medium of the electric signal, but also the bearing support of the sensor, and is in a thin needle shape. The component of the material is inert metal with strong conductivity, preferably platinum iridium wire.
The enzyme layer provides an enzyme to catalyze the oxidation of lactic acid to hydrogen peroxide. Besides enzyme, the enzyme protective agent, enzyme cross-linking agent, hydrogel film-forming modifier, hydrogel mixed cross-linking agent and the like are included. Wherein the enzyme is the most core component and is selected from lactic acid oxidase, and the mass ratio of the enzyme in the enzyme liquid is 1% -15%, preferably 5%. The protective agent is selected from bovine serum albumin, and the ratio of the protective agent to enzyme is 0.1:1-10: within the range of 1, preferably 2:1. the enzyme cross-linker is a molecule with aldehyde groups, preferably glutaraldehyde. The hydrogel film-forming modifier is a hydrogel with good compatibility with enzyme, preferably polyvinyl alcohol, and the mass ratio of the hydrogel film-forming modifier in the enzyme solution is 0.1% -10%, preferably 2.5%. The hydrogel mixed crosslinking agent is selected from two or more of boric acid, glutaraldehyde, glyoxal, citric acid, oxalic acid, tannic acid, fumaric acid, maleic anhydride, 2, 3-dimethyl maleic anhydride and 2, 3-diphenyl maleic anhydride.
The anti-interference layer can prevent molecules such as vitamin C, cholesterol, uric acid, urea and the like from entering the membrane by physical or chemical action, so that interference is reduced. Selected from cellulose acetate. The mass concentration is in the range of 0.1% -5%, preferably 1%.
The diffusion layer is composed of PEBA and an oxygen-enriched polymer, and plays a vital role in the performance of the dynamic lactic acid sensor. The sensor is used as an outer surface membrane to be contacted with a measuring solution, so that the amount and the speed of lactic acid and oxygen entering the membrane are controlled, and the compatibility of the sensor and tissue fluid is determined. PEBA is selected from PEBAX 1657, PEBAX 2533, PEBAX 3533, PEBAX4033, PEBAX 5333, PEBAX 6333, PEBAX7033, PEBAX 7233, etc. of different types of the company aclamate. PEBAX 1657 containing 40% rigid chains and 60% soft chains is preferred. The oxygen-enriched polymer is selected from Polysulfone (PS), cellulose acetate, polydimethylsiloxane (PDMS), and polytrimethylsilane-1-propyne. PDMS and PS are preferred. Wherein the mass ratio of PEBA to oxygen-enriched polymer is controlled at 10: 1-1:1. The solvent is selected from N-N-Dimethylformamide (DMF), dimethyl sulfoxide, propanol, isopropanol, and one or more of the mixture.
Example two
Embodiment two is a method for manufacturing a dynamic lactate sensor film in embodiment one, comprising the following steps:
a mixed solvent of 0.5g PEBAX 1657 and 4.5g DMF was weighed out, and heated at 90℃for 3 hours under stirring at 600rpm to obtain a 10% PEBAX 1657 solution which was completely dissolved.
0.05g of PS and 4.95g of DMF were weighed and heated at 90℃for 2h with stirring at 600rpm to give a 1% PS solution which was completely dissolved.
10% PEBAX 1657 solution and 1% PS solution are mixed according to the mass ratio of 1:1, uniformly mixing to obtain a diffusion film solution.
And (3) adopting a dipping and pulling process, and pulling the needle electrode coated with the enzyme layer and the anti-interference layer in the diffusion film solution for 1 time.
And placing the coated electrode in a constant temperature and humidity box with the temperature of 37 ℃ and the humidity of 60% for 72 hours to obtain the diffusion film.
Example III
The third embodiment is different from the second embodiment in that: only the solution DMF in the step (1) is adjusted to be a mixed solvent of DMF and isopropanol, and the ratio of DMF to isopropanol is 9:1.
example IV
The fourth embodiment is different from the second embodiment in that: only the concentration of the 1% ps solution in step (2) was adjusted to 5%.
Example five
The fifth embodiment is different from the second embodiment in that: only the concentration of the 1% ps solution in step (2) was adjusted to 10%.
Example six
The difference between the sixth embodiment and the second embodiment is that only the number of 1% ps pulls in the step (4) is adjusted to 3, and every two adjacent times are separated by 3min.
Example seven
Embodiment seven differs from embodiment two in that: and (3) only adjusting the drying time in the step (5) to 120 hours.
Example eight
Embodiment eight differs from embodiment two in that: only the solvent in steps (1) and (2) was adjusted to isopropanol, and PS in (2) was adjusted to PDMS.
Example nine
Embodiment nine is a specific implementation manner of embodiment one:
(1) Sequentially polishing, pickling, alkali washing, washing with purified water, ultrasonically washing, and drying in an oven at 80 ℃ for 30min;
(2) 20mL of a 0.05% chloroplatinic acid solution and 0.4mL of a 0.03mol/L sodium borate solution were prepared. Soaking the polished platinum iridium wires in the step (1) in a chloroplatinic acid solution, then slowly adding sodium borate solution dropwise into the chloroplatinic acid solution at a constant temperature and a constant speed of 600rpm at 60 ℃, continuously heating and stirring for 60min, taking out the platinum iridium wires, transferring into a 200 ℃ oven for drying for 10min, flushing with ultrapure water, roasting for 2min again, and drying for later use.
(3) 2.76g of sodium dihydrogen phosphate is weighed and dissolved in ultrapure water, and the solution is marked as A solution after being stirred uniformly and the volume is fixed to 100 mL; 7.16g of disodium hydrogen phosphate was weighed and dissolved in ultrapure water, and after stirring uniformly, the volume was fixed to 100mL, and the solution was labeled as solution B. Mixing 9.5mL of A solution and 15.5mL of B solution uniformly, and diluting to 100mL of constant volume by using ultrapure water to obtain 0.05mol/L phosphate buffer solution with pH of 7.0, wherein the phosphate buffer solution is marked as PB solution.
(4) The lactate oxidase and bovine serum albumin were taken out from the refrigerator refrigerating chamber and rewet at room temperature for 30min, then 0.100g lactate oxidase, 0.200g bovine serum albumin, 0.01g glutaraldehyde, 4.7900g PB were weighed, stirred under magnetic stirring at 500rpm for 1h, and labeled as solution 1.
(5) Weighing 0.4g of polyvinyl alcohol and 19.6g of water, heating for 2 hours at 100 ℃ under magnetic stirring at 600rpm by adopting a condensing reflux mode, and naturally cooling, wherein the polyvinyl alcohol is dissolved to be in a clear and transparent state.
(6) And stirring the solution 1 and the dissolved polyvinyl alcohol solution at normal temperature for 1h at a stirring speed of 200rpm until the solution and the dissolved polyvinyl alcohol solution are uniformly mixed.
(7) And (3) adopting a dipping and pulling method, pulling the platinum iridium wire in the step (2) in the solution in the step (6) for 2 times at intervals of 3min, and then drying in a 37 ℃ incubator for 2h for later use.
(8) 0.025g of cellulose acetate and 4.975g of cyclohexanone were weighed, and heated at 90℃for 30 minutes under stirring at 600rpm, to obtain a 0.5% cellulose acetate solution.
(9) And (3) adopting a dipping and pulling method, pulling the platinum iridium wire in the step (7) for 1 time when in solution in the step (8), and then drying the platinum iridium wire in a 37 ℃ incubator for 24 hours for later use.
(10) A mixed solvent of 0.5g PEBAX 1657 and 4.5g DMF was weighed out, and heated at 90℃for 3 hours under stirring at 600rpm to obtain a 10% PEBAX 1657 solution which was completely dissolved.
(11) 0.05g PS and 4.95g DMF were weighed and heated at 90℃for 2h with stirring at 600rpm to give a 1% PS solution which was completely dissolved.
(12) 10% PEBAX 1657 solution and 1% PS solution are mixed according to the mass ratio of 1:1, uniformly mixing to obtain a diffusion film solution.
(13) And (3) adopting a dipping and pulling method, and pulling the platinum iridium wire in the step (9) in the solution in the step (10) for 1 time.
(14) And (3) placing the coated electrode in a constant temperature and humidity box with the temperature of 37 ℃ and the humidity of 60% for 72 hours to obtain the dynamic lactic acid sensor.
The beneficial effects are that: 1. the dynamic lactic acid sensor has simple manufacturing process, reliable repeatability and easy mass production;
2. the dynamic lactic acid sensor has the advantages of accurate comprehensive performance regulation and control, wide detection range, high sensitivity and accuracy and long validity period;
3. the dynamic lactic acid sensor has good strength and toughness, and can resist random external damage in the storage, implantation and use processes.
4. The dynamic lactic acid sensor has excellent biocompatibility, strong anti-interference capability of cells and the like, and small side effect on human bodies.
1. The test scheme and the data processing mode are as follows:
1) Preparing PB buffer solutions with lactic acid content of 0.5mmo/L, 1.5mmo/L, 4.5mmo/L, 9mmo/L and 13.5mmo/L respectively, and marking as L1, L2, L3, L4 and L5;
2) Taking the day of the completion of the sensor drying as the starting time point, testing L1, L2, L3, L4 and L5 on days 1, 3, 5, 7 and 9 respectively, controlling the indoor temperature to be constant at 25 ℃ (deviation +/-1.5 ℃), the humidity to be constant at 60%RH (deviation +/-3%RH), and repeating the test for 5 times on each buffer solution;
3) Within 9 days of continuous testing, during the non-testing period, the sensor is soaked in the L2 solution to simulate the state of continuous soaking in tissue fluid after the sensor is implanted;
2. summary of experimental conditions for the sensors of examples 2,3, 4, 5, 6, 7, 8, see table 1:
Figure BDA0004078929840000091
test currents (nA) for the sensors of table 13, examples 2,3, 4, 5, 6, 7, 8 at different times are shown in table 2:
Figure BDA0004078929840000101
TABLE 2
4. According to the data of table 2, with the concentration on the abscissa and the measured current (average of 5 repeated test currents) on the ordinate, a unitary linear fit was performed to obtain e=a×c+b, a representing the sensitivity of the sensor. The greater the sensitivity and the less the attenuation, the more accurate and long-term stability the sensor can maintain. The sensitivity of the sensors of examples 2,3, 4, 5, 6, 7, 8 at different times and their attenuation are shown in table 3:
Figure BDA0004078929840000102
signal graphs for table 35, example 9 sensor tests L1, L2, L3, L4, L5 are shown in table 4:
Figure BDA0004078929840000103
TABLE 4 Table 4
Examples ten
The tenth embodiment is different from the first embodiment in that a dipping-pulling process is adopted to pull the needle-shaped electrode coated with the enzyme layer and the anti-interference layer in the diffusion membrane solution 1 time and simultaneously ultrasonically oscillate the enzyme solution.
In the process of preparing the enzyme film, the uniformity of the enzyme film directly influences the measurement accuracy of the later-stage sensor; in order to prevent uneven thickness of the enzyme film, an electric field is introduced into the enzyme solution storage area before the coating process is carried out, and electrodes of active sites in the enzyme solution are differentiated under the influence of the electric field and are uniformly arranged under the influence of the electrodes, so that repeated accumulation of a plurality of active sites is reduced, and uneven thickness of the enzyme film in the later stage is avoided. In the hydrogel skeleton formed by mixing the cross-linking agent and the active site, the active site can be arranged according to the electrode direction by guiding the hydrogel skeleton, but the active site is deflected at the position (namely, the active site with inconsistent electrode can be deflected) due to the fixation of the cross-linking agent, the skeleton network can be driven to deflect simultaneously, the unification of the electrode orientation of the active site can occur temporarily, when the electric field force disappears at the later stage, the hydrogel skeleton can generate a restoring force, namely, the deflection at the earlier stage of restoration, the thickness of the enzyme film can be locally increased again by the twisted skeleton network, and the uneven condition is caused. The ultrasonic wave is introduced, the connection between the active sites differentiated by the electrodes and the mixed cross-linking agent is broken through microwave oscillation, under the condition that a skeleton network of the broken active sites is unchanged, the electrodes turn towards the non-uniform active sites, after the ultrasonic wave is removed, the active sites keep unchanged in direction under the continuous action of an electric field, are connected with the surrounding skeleton network again to form a skeleton network with the same active site direction, the problem that recovery deflection occurs in the skeleton network is reduced, and the enzyme film is more uniform.
The foregoing is merely an embodiment of the present invention, the present invention is not limited to the field of this embodiment, and the specific structures and features well known in the schemes are not described in any way herein, so that those skilled in the art will know all the prior art in the field before the application date or priority date, and will have the capability of applying the conventional experimental means before the date, and those skilled in the art may, in light of the teaching of this application, complete and implement this scheme in combination with their own capabilities, and some typical known structures or known methods should not be an obstacle for those skilled in the art to practice this application. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the utility of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims (9)

1. A dynamic lactate sensor film, comprising: the device comprises a metal conducting layer, a metal catalytic layer, an enzyme layer, an anti-interference layer and a diffusion layer, wherein lactic acid and oxygen in liquid to be detected sequentially pass through the diffusion layer and the anti-interference layer to be contacted with the enzyme layer, the enzyme layer reacts with lactic acid to generate hydrogen peroxide, the hydrogen peroxide is diffused to the metal catalytic layer to be reduced, and the metal conducting layer is contacted with the metal layer and is used for transmitting an oxidation-reduction current signal to an instrument contact end to convert an electric signal into a digital signal; the diffusion layer is composed of PEBA and an oxygen-rich polymer.
2. A dynamic lactate sensor film according to claim 1, wherein: the enzyme layer also comprises enzyme, an enzyme protective agent, an enzyme cross-linking agent, a hydrogel film-forming modifier and a hydrogel mixed cross-linking agent; the enzyme is selected from lactic acid oxidase, and the mass ratio of the enzyme in the enzyme liquid is 1% -15%; the protective agent is selected from bovine serum albumin, and the ratio of the protective agent to enzyme is 0.1:1-10:1 range; the enzyme cross-linking agent is glutaraldehyde, the hydrogel is polyvinyl alcohol, and the mass ratio of the hydrogel to the enzyme solution is 0.1-10%; the hydrogel mixed crosslinking agent is selected from two or more of boric acid, glutaraldehyde, glyoxal, citric acid, oxalic acid, tannic acid, fumaric acid, maleic anhydride, 2, 3-dimethyl maleic anhydride and 2, 3-diphenyl maleic anhydride.
3. A dynamic lactate sensor film according to claim 2, wherein: the ratio of the enzyme to the enzyme solution is 5%, the ratio of the protective agent to the enzyme is 2:1, and the mass ratio of the hydrogel to the enzyme solution is 2.5%.
4. A dynamic lactate sensor film according to claim 1, wherein: the interference layer is made of cellulose acetate, and the mass concentration of the interference layer is within the range of 0.1% -5%.
5. A dynamic lactate sensor film according to claim 1, wherein: the mass ratio of PEBA to oxygen-enriched polymer is controlled at 10:1-1:1 range.
6. A preparation method of a dynamic lactic acid sensor membrane is characterized by comprising the following steps: the method comprises the following steps:
s1: proportioning PEBA and DMF to obtain PEBA solution; the mass ratio of PEBA to DMF is 1:1-1:20;
s2: stirring and heating the prepared PEBA solution; stirring speed is 500rpm-900rpm, and heating temperature is: heating for 2-4h at 80-100 ℃; obtaining PEBA solution with concentration of 5% -20% after dissolution;
s3: preparing an oxygen-enriched polymer solution by using the oxygen-enriched polymer and DMF; the mass ratio of the oxygen-enriched polymer to DMF is 1:9-1:99;
s4: stirring and heating the prepared oxygen-enriched polymer solution; stirring speed is 500rpm-900rpm, and heating temperature is: heating for 2-4h at 80-100 ℃; obtaining PS solution with concentration of 1% -10% after dissolution;
s5: the PEBA solution and the oxygen-enriched polymer solution are mixed according to the mass ratio of 1:1, uniformly mixing to obtain a diffusion film solution;
s6: the needle electrode coated with the enzyme layer and the anti-interference layer is lifted for 1 time in the diffusion film solution by adopting a dipping and lifting process;
s7: and placing the coated electrode in a constant temperature and humidity box with the temperature of 37 ℃ and the humidity of 60% for 72 hours to obtain the diffusion film.
7. The method for preparing the dynamic lactic acid sensor membrane according to claim 6, wherein the method comprises the following steps: PEBA described in step S1 selects one of PEBAX 1657, PEBAX 2533, PEBAX 3533, PEBAX4033, PEBAX 5333, PEBAX 6333, PEBAX7033, PEBAX 7233, and PEBAX 7233.
8. The method for preparing the dynamic lactic acid sensor membrane according to claim 7, wherein: the oxygen-enriched polymer in the step S3 is selected from one of polysulfone, cellulose acetate, polydimethylsiloxane or polytrimethylsilane-1-propyne.
9. The method for preparing the dynamic lactic acid sensor membrane according to claim 8, wherein: the PEBA selects PEBAX 1657 comprising 40% rigid chains and 60% soft chains, and the oxygen-rich polymer selects polysulfone.
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