CN112240901B - Simple preparation method of glycerol biosensor chip - Google Patents
Simple preparation method of glycerol biosensor chip Download PDFInfo
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- CN112240901B CN112240901B CN202011134405.6A CN202011134405A CN112240901B CN 112240901 B CN112240901 B CN 112240901B CN 202011134405 A CN202011134405 A CN 202011134405A CN 112240901 B CN112240901 B CN 112240901B
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 title claims abstract description 135
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 49
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 31
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 claims abstract description 22
- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 claims abstract description 20
- 239000002002 slurry Substances 0.000 claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 17
- 238000007650 screen-printing Methods 0.000 claims abstract description 17
- 239000002105 nanoparticle Substances 0.000 claims abstract description 15
- GEYXPJBPASPPLI-UHFFFAOYSA-N manganese(III) oxide Inorganic materials O=[Mn]O[Mn]=O GEYXPJBPASPPLI-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000002243 precursor Substances 0.000 claims abstract description 8
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 7
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 7
- 238000007639 printing Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 48
- 229910052799 carbon Inorganic materials 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 14
- 108010054790 glycerol-3-phosphate oxidase Proteins 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 102000057621 Glycerol kinases Human genes 0.000 claims description 11
- 108700016170 Glycerol kinases Proteins 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 11
- 239000000843 powder Substances 0.000 claims description 10
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 6
- 238000001354 calcination Methods 0.000 claims description 6
- 229910001437 manganese ion Inorganic materials 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 6
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 5
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 5
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 5
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000005642 Oleic acid Substances 0.000 claims description 5
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 5
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 5
- BZSXEZOLBIJVQK-UHFFFAOYSA-N 2-methylsulfonylbenzoic acid Chemical compound CS(=O)(=O)C1=CC=CC=C1C(O)=O BZSXEZOLBIJVQK-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000006185 dispersion Substances 0.000 claims description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- 239000003929 acidic solution Substances 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 2
- 239000000919 ceramic Substances 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 239000010949 copper Substances 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
- 239000010931 gold Substances 0.000 claims description 2
- VASIZKWUTCETSD-UHFFFAOYSA-N manganese(II) oxide Inorganic materials [Mn]=O VASIZKWUTCETSD-UHFFFAOYSA-N 0.000 claims description 2
- 239000011858 nanopowder Substances 0.000 claims description 2
- 229910052697 platinum Inorganic materials 0.000 claims description 2
- 239000005020 polyethylene terephthalate Substances 0.000 claims description 2
- 229910052709 silver Inorganic materials 0.000 claims description 2
- 239000004332 silver Substances 0.000 claims description 2
- 238000000967 suction filtration Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims 1
- 239000002245 particle Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 8
- 230000002194 synthesizing effect Effects 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- 238000000855 fermentation Methods 0.000 abstract description 4
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- 239000002028 Biomass Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 239000002057 nanoflower Substances 0.000 abstract 1
- 230000004044 response Effects 0.000 description 9
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000012286 potassium permanganate Substances 0.000 description 4
- 238000010923 batch production Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000002003 electrode paste Substances 0.000 description 3
- 239000011267 electrode slurry Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000011268 mixed slurry Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
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- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 2
- 239000007772 electrode material Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- LQKOJSSIKZIEJC-UHFFFAOYSA-N manganese(2+) oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Mn+2].[Mn+2].[Mn+2].[Mn+2] LQKOJSSIKZIEJC-UHFFFAOYSA-N 0.000 description 2
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 description 2
- 235000011151 potassium sulphates Nutrition 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 229910002804 graphite Inorganic materials 0.000 description 1
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- 238000009776 industrial production Methods 0.000 description 1
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- 230000002452 interceptive effect Effects 0.000 description 1
- 230000007257 malfunction Effects 0.000 description 1
- PPNAOCWZXJOHFK-UHFFFAOYSA-N manganese(2+);oxygen(2-) Chemical compound [O-2].[Mn+2] PPNAOCWZXJOHFK-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- Chemical Kinetics & Catalysis (AREA)
- Nanotechnology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention relates to a simple preparation method of a glycerol biosensor chip. Firstly, trimanganese tetroxide/graphene oxide nano sensing slurry with a nano flower structure is synthesized, and then the nano sensing slurry is printed by adopting a screen printing technology to prepare a biological sensing chip for converting a glycerol concentration signal into an electric signal to be output. The biosensor chip can realize accurate detection of biomass glycerol in clinical medicine, food safety, fermentation industry and bioengineering. The synthesis steps of the biosensor chip related by the invention are as follows: preparing and synthesizing a precursor manganese dioxide reaction solution A, B, centrifuging, drying and the like; preparing nano flower-like trimanganese tetroxide; synthesizing graphene oxide nanoparticles; and (3) printing a sensing chip based on manganous manganic oxide/graphene oxide.
Description
Technical Field
The invention relates to a simple and convenient preparation method of a glycerol biosensor chip, and belongs to the technical field of glycerol detection devices.
Background
Since the fields of food safety, medical treatment and health, biological fermentation and the like are closely related to the civilian life, in recent years, the detection of related substances is more and more paid more attention by governments and common people. The realization of accurate and rapid sensing detection of key biological substances has important significance for the development of the fields. Compared with various sensors, the electrochemical sensor has the advantages of convenience in operation, low cost, stable performance, high accuracy and the like, and is widely applied to various industries. Few mature products are available for the detection of biomass. This is mainly because the detection system for the presence of biomass is often complex in composition and has a large number of interfering substances, which easily causes malfunction or misalignment of the sensor. The core of the biosensor is a sensing electrode, so that the development of an electrode with high stability and good performance is particularly important for practical application.
At present, the development trend of the electrochemical biosensing electrode is miniaturization, integration and batch production. The screen printing technology prints ink into the electrode chip through the pressure of a scraper and a model of a screen plate, and the high accuracy and adaptability of the screen printing technology enable the screen printing technology to be widely applied. Wherein the screen printing electrode chip meets the trends of low cost, batch production and miniaturization of the electrode chip.
Glycerol in human blood is converted into substances such as triglyceride and the like, and the substances cause burden to human bodies due to overhigh content, so the content of the glycerol is also a very important physiological index in the human bodies. Meanwhile, glycerol is also generated in the industry of alcohol fermentation, so that the accurate detection of the glycerol has a wide market. The current main method is to detect hydrogen peroxide, and the content of glycerol is indirectly reflected by the current change of the hydrogen peroxide. For glycerol detection, no literature reports a preparation method of a related biosensor chip, and the field is still in a blank stage of technical research and development.
Disclosure of Invention
The invention aims to prepare a simple glycerol biosensor chip for detecting the concentration of glycerol in the fermentation process.
The technical scheme of the invention is as follows: a simple glycerol biosensor chip is prepared by the following steps:
1) The preparation and synthesis of the precursor manganese dioxide synthetic solution specifically comprise the following steps: the synthetic liquid A is an aqueous solution containing manganese ions, and the synthetic liquid B is an acidic solution. Wherein the manganese ion-containing solution may be KMnO 4 ，MnSO 4 Wherein the concentration of the two solutions is 1-100 mM, the acid solution can be one of oleic acid, formic acid and sodium chlorate, the concentration of the acid solution is 1-100 mM, and the pH value of the acid solution is 1-7. And (3) filling the solution B with a certain volume into an injection pump, dropwise adding the solution B into the solution A at a certain speed, taking out the synthetic solution after a period of time, centrifuging and drying the synthetic solution to obtain precursor manganese dioxide powder.
2) The synthesis of flower-like manganous oxide nanoparticles specifically comprises the following steps: and putting the precursor manganese dioxide powder into a muffle furnace, and calcining at high temperature to obtain flower-like manganese tetroxide nano-powder.
3) The synthesis of the graphene oxide nanoparticles specifically comprises the following steps: dissolving graphene oxide in water to prepare a certain concentration, crushing for a period of time by using a cell crusher to obtain a graphene oxide dispersion liquid, centrifugally washing the dispersion liquid to remove impurities, performing suction filtration, and drying to obtain graphene oxide nanoparticles.
4) The synthesis of the printing paste specifically comprises the following steps: and mixing the synthesized manganese tetraoxide powder with carbon ink and graphene oxide nano particles according to a certain mass ratio to obtain the nano slurry for printing.
5) The printing of three electrodes specifically includes: the method comprises the steps of screen printing carbon ink on a support to form a counter electrode and a connector, screen printing silver chloride on the support to form a reference electrode, screen printing mixed slurry of the carbon ink, manganese tetraoxide and graphene oxide on the support to form a working electrode, and curing the support for a plurality of hours at a certain temperature.
6) The preparation of the glycerol biosensor chip specifically comprises the following steps: preparing a mixed solution of glycerol kinase and glycerol-3-phosphate oxidase with a certain concentration, adding glutaraldehyde with a certain proportion into the mixed solution, and uniformly mixing. And (4) taking out a certain amount of enzyme solution, uniformly dripping the enzyme solution on the working electrode of the chip prepared in the step (5), putting the chip coated with the enzyme solution in a refrigerator, and drying for several hours at low temperature to obtain the enzyme biosensor chip for detecting the glycerol.
As a further improvement of the invention, in the step 1), the synthetic liquid a is an aqueous solution containing manganese ions, and the synthetic liquid B is an acidic solution. Wherein the manganese ion-containing solution may be KMnO 4 ，MnSO 4 The concentration of the one of the acid solutions is 1-100 mM, the acid solution can be one of oleic acid, formic acid and sodium chlorate, the concentration of the acid solution is 1-100 mM, and the pH value of the acid solution is 1-7.
As a further improvement of the invention, the injection speed of the acid solution in the step 1) is 50-200 mu L min -1 The centrifugal rate is 5000 r. Min -1 -10000 r·min -1 The centrifugation time is 3 min to 30 min, the drying temperature is 50 ℃ to 100 ℃, and the time is 5 ℃ to 10 h.
As a further improvement of the invention, the manganese dioxide calcining temperature in the step 2) is 200-500 ℃, and the calcining time is 2-10 h.
As a further improvement of the invention, the concentration of the graphene oxide in the step 3) is 0.02-0.2 g. L -1 Pulverizing for 10-60 min, centrifuging for 5-30 min at a speed of 5000 r. Min -1 -10000 r·min -1 The drying temperature is 40-80 ℃, and the drying time is 2-5 h.
As a further improvement of the present invention, the mass ratio of the flower-like trimanganese tetroxide nanoparticles to the graphene oxide and the carbon ink used as the working electrode material in the step 4) is 1:1:1-1:100:100.
as a further improvement of the invention, the support in the step 5) is made of PVC, alumina, PET, ceramic, gold, silver, copper, platinum and other materials, the curing time is 1-5 h, and the curing temperature is 40-100 ℃.
As a further improvement of the invention, the concentration of the glycerol kinase and the glycerol-3-phosphate oxidase in the step 6) is 200-2000U. ML -1 Wherein the mass percentage concentration of the glutaraldehyde in the two enzyme solutions is 0.1-10%, and the concentration range of the enzyme solution coated on the working electrode is 5 muL-cm -2 -200 µL·cm -2 And drying the enzyme biosensor chip at 0-20 ℃.
The manganous-manganic oxide serving as an electron mediator is low in detection potential, has excellent anti-interference capability, and is large in specific surface area and high in conductivity. Therefore, the patent takes manganous-manganic oxide and graphene oxide as electrode materials, and synthesizes nano-slurry with high catalytic performance and conductive performance by controlling the nano-structure of the manganous-manganic oxide and the graphene oxide, and the nano-slurry is used for preparing a screen printing electrode chip.
The sensor chip is simple in preparation process and low in cost, can be used for large-scale batch production, has wide market application prospect, and can realize accurate detection of the concentration of glycerol.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the method is characterized in that a sensing chip is obtained by co-fixing glycerol kinase and glycerol-3-phosphate oxidase based on a screen printing technology, manganous-manganic oxide nanoparticles with certain mass are mixed with carbon ink and graphene oxide to form nano composite slurry, the conductivity and the resistivity of the synthesized slurry are controlled by controlling the manganous-manganic oxide nanoparticles, and the specific surface area of the slurry is increased by adding the graphene oxide to improve the conductivity. The glycerol kinase and the glycerol-3-phosphate oxidase are co-fixed on the screen printing electrode chip by utilizing the screen printing electrode chip to obtain the high-performance glycerol biosensor chip.
2. The raw materials used by the invention are cheap and easy to obtain, the preparation method is simple and convenient, and the biosensor chip has a prospect of large-scale industrial production, and simultaneously has good detection performance on glycerol.
Drawings
FIG. 1 is a diagram of a glycerol biosensor chip.
Detailed Description
The technical solution of the present invention will be described in detail with reference to fig. 1.
A simple glycerol biosensor chip is prepared by mixing manganous-manganic oxide, conductive carbon slurry and graphene oxide to obtain mixed slurry, obtaining an electrode through a screen printing technology, and finally loading glycerol kinase and glycerol-3-phosphate oxidase on the surface of the electrode to obtain the high-performance glycerol biosensor chip.
Example 1
A simple glycerol biosensor chip is prepared by the following steps:
1) Dissolving 1.5 g potassium permanganate (national drug group chemical reagent Co., ltd.) in 500 mL water, stirring vigorously for 30 min, and injecting 10 mL oleic acid (Shanghai Michelin Biochemical technology Co., ltd.) solution with concentration of 100 mM into potassium permanganate solution with injection speed of 80 μ L min using injection pump (Oran technology Legato) -1 . The reaction temperature is controlled to be 40 ℃, the synthetic liquid is taken out after 5 h and is centrifuged (GL 21M of Kaida scientific instruments Co., ltd., hunan), the centrifugation speed is 5000 r/min, and the centrifugation time is 15 min. The solid obtained by centrifugation was dried at 80 ℃ for 8 h.
2) Synthesizing flower-like manganous manganic oxide nanoparticles by a thermal oxidation method: the manganese dioxide precursor was calcined in a muffle furnace at 200 ℃ for 5 h.
3) Weighing 1.0 g graphene oxide (Nanjing Jicang nanometer technology Co., ltd. 0.8-3 um) and adding into water to prepare 0.02 g.L -1 The graphite oxide mixed solution of (1) is put into a cell crusher (Shanghai Jingxin industry development Co., ltd., XM-650T) to be pulverizedPulverizing into 200 nm, centrifuging at 8000 r min, and washing -1 And centrifuging for 10 min, removing solid impurities, filtering the centrifugate with microporous membrane (Biyunyan), and oven drying the membrane in an oven at 50 deg.C for 2 h.
4) Weighing 1.0 g manganomanganic oxide powder, and mixing the manganomanganic oxide powder according to the weight ratio of the manganomanganic oxide: and (3) graphene oxide: the carbon slurry is prepared by mixing the following components in a ratio of 1:1:8, mixing to prepare working electrode slurry, and placing the slurry in an oven at 60 ℃ to be dried until the viscosity is 120 dpa.S.
5) 8 g working electrode paste, 5 g silver chloride paste (Himmenn nano-technology Jiangsu Co., ltd.) and 15 g carbon paste (Qian generation electronic materials Co., ltd.) were printed on different positions of a PET substrate (Jiangsu Xianfeng nano-materials Co., ltd.) by a screen printing technique, respectively. Wherein, the carbon ink is printed on the support body through silk screen to form a counter electrode and a connector, the silver chloride is printed on the support body through silk screen to form a reference electrode, and the mixed slurry of the carbon ink, the trimmings tetroxide and the graphene oxide is printed on the support body through silk screen to form a working electrode. As shown in fig. 1, the center dot is the working electrode, the right arc region is the counter electrode, and the left arc region is the reference electrode (the shape of each electrode is directly printed by the shape of the silk screen). And preparing the chip of the three-electrode system.
6) Using PBS solution as a solvent, 0.2U. Mu.L was prepared -1 Glycerol-3-phosphate oxidase solution (Shanghai Michelin Biochemical technology Co., ltd.) and 0.5U. Mu.L -1 Adding glutaraldehyde to make the mass fraction of glutaraldehyde in the two enzyme solutions 0.5%, uniformly loading 5 μ L of glycerol-3-phosphate oxidase solution on the working electrode area, then uniformly dripping 10 μ L of glycerol kinase solution on the working electrode area by using a micro sample injection needle, and drying 12 h at 4 ℃ to obtain the glycerol biosensor chip.
7) A glycerol biosensor chip is shown in figure 1.
Through the timing ampere current test, the following results are obtained: the glycerol prepared by the method is biologicalThe detection sensitivity of the sensor chip to glycerol is 9.31 muA.mM -1 •cm -2 And the detection limit is 15 mu M. After the test, the chip is placed in a buffer solution with the pH value of 7 at the temperature of 4 ℃ for one week, and the response signal of the chip is basically unchanged; after one month, the response signal was 95% of the initial signal; after three months, the response signal was 88% of the initial signal, indicating that the stability of the chip was good.
Examples 2-3 the instrumentation and reagent sources were the same.
Example 2
A simple glycerol biosensor chip comprises the following components:
1) Dissolving 2g potassium permanganate in 500 mL water, stirring vigorously for 30 min, and injecting 10 mL oleic acid solution with concentration of 80 mM into potassium permanganate solution at injection speed of 100 muL.min -1 Controlling the reaction temperature to be 40 ℃, then taking out the synthetic liquid and centrifuging, wherein the centrifugation speed is 5000 r/min, and the centrifugation time is 15 min. The solid obtained by centrifugation was dried at 80 ℃ for 5 h.
2) Synthesizing flower-like manganous-manganic oxide nano-particles by a thermal oxidation method, and calcining a manganese dioxide precursor at 300 ℃ for 2 h.
3) Taking out 2.0 g graphene oxide, dissolving the graphene oxide in water to prepare 0.1 g. L -1 The graphene oxide solution is placed in a cell crusher to be crushed for 30 min, the solution is centrifugally washed to remove solid impurities, a microporous filter membrane is used for filtration, and the filter membrane is placed in an oven to be dried at 80 ℃ for 4 h.
4) Taking 1.0 g manganomanganic oxide powder, mixing the manganomanganic oxide: and (3) graphene oxide: the carbon slurry is prepared by mixing the following components in a ratio of 1:2:7 to prepare working electrode slurry, and placing the slurry in an oven at 60 ℃ to be dried until the viscosity is 120 dpa & S.
5) 8 g working electrode paste, 5 g silver chloride paste and 15 g carbon paste were printed on PET substrates, respectively, using screen printing techniques.
6) The amount of the resulting mixture was 0.2U. Mu.L -1 Glycerol-3-phosphate oxidase and 0.5U. Mu.L -1 The enzyme solution contains 0.25 percent of glutaraldehyde, and 4 mu L of glycerol-3-phosphate oxidase is loaded on a working electrodeAfter that, 12 mu L of glycerol kinase is dripped on the working electrode, and the glycerol biosensor chip is obtained by drying 12 h at 4 ℃.
7) The glycerol biosensor chip is shown in the figure.
Through the timing ampere current test, the following results are obtained: the glycerol biosensor chip prepared by the method has high detection sensitivity of 10.17 muA.mM on glycerol -1 •cm -2 And the detection limit is 12 mu M. After the test, the chip is placed in a buffer solution with the pH value of 7 at the temperature of 4 ℃ for one week, and the response signal of the chip is basically unchanged; after one month, the response signal was 94% of the initial signal; after three months, the response signal was 86% of the initial signal, indicating that the stability of the chip was good.
Example 3
A simple glycerol biosensor chip comprises the following components:
1) Dissolving 1 g potassium sulfate (Shanghai Michelin Biochemical technology Co., ltd.) in 500 mL water, vigorously stirring for 30 min, and injecting 15 mL solution of sodium chlorate (Shanghai Aladdin Biochemical technology Co., ltd.) with concentration of 90 mM into the potassium sulfate solution at an injection rate of 200. Mu.L.min -1 The reaction temperature is controlled to be 40 ℃, then the synthetic liquid is taken out and centrifuged, the centrifugation speed is 5000 r/min, and the centrifugation time is 20 min. The solid obtained by centrifugation was dried at 50 ℃ for 10 h.
2) Synthesizing flower-like manganous-manganic oxide nano-particles by a thermal oxidation method, and calcining a manganese dioxide precursor at 200 ℃ to obtain 8 h.
3) 3.0 g graphene oxide is taken out and dissolved in water to prepare 0.2 g. L -1 The graphene oxide solution is placed in a cell crusher to be crushed for 30 min, the solution is centrifugally washed to remove solid impurities, a microporous filter membrane is used for filtration, and the filter membrane is placed in an oven to be dried at 70 ℃ for 5 h.
4) Taking 1.0 g manganomanganic oxide powder, mixing the manganomanganic oxide: and (3) graphene oxide: the carbon slurry is prepared by mixing the following components in a ratio of 1:3:9 to prepare working electrode slurry, and placing the slurry in an oven at 60 ℃ to be dried until the viscosity is 120 dpa & S.
5) 8 g working electrode paste, 5 g silver chloride paste and 15 g carbon paste were printed on PET substrates, respectively, using screen printing techniques.
6) The amount of the resulting mixture was 0.2U. Mu.L -1 Glycerol-3-phosphate oxidase and 0.5U. Mu.L -1 The glycerol kinase, the enzyme solution contains 0.125% of glutaraldehyde, 10 mul of glycerol-3-phosphate oxidase is loaded on a working electrode, then 10 mul of glycerol kinase is dripped on the working electrode, and at 4 ℃, 12 h is dried to obtain the glycerol biosensor chip.
7) The glycerol biosensor chip is shown in the figure.
Through the timing ampere current test, the following results are obtained: the detection sensitivity of the glycerol biosensor chip prepared by the method to glycerol is higher and is 12.04 muA.mM -1 •cm -2 And the detection limit is 10 mu M. After the test, the chip is placed in a buffer solution with the pH value of 7 at the temperature of 4 ℃ for one week, and the response signal of the chip is basically unchanged; after one month, the response signal was 91% of the initial signal; after three months, the response signal was 80% of the initial signal, indicating that the stability of the chip was good.
Claims (4)
1. A simple glycerol biosensor chip preparation method is characterized by comprising the following steps:
step 1: precursor MnO 2 Preparation of synthetic fluid
Dropwise adding the acidic solution into an aqueous solution containing manganese ions, reacting to obtain a synthetic solution, centrifuging and drying the synthetic solution to obtain manganese dioxide powder;
and 2, step: synthesis of flower-like mangano-manganic oxide nano-particles
Putting manganese dioxide powder into a muffle furnace, and calcining to obtain flower-shaped trimanganese tetroxide nano powder;
and step 3: adding the graphene oxide powder into water, uniformly mixing, crushing by using a cell crusher to obtain a graphene oxide dispersion liquid, centrifugally washing, performing suction filtration, and drying to obtain graphene oxide nanoparticles;
and 4, step 4: synthesis of printing pastes
Mixing flower-like manganous-manganic oxide powder with carbon ink and graphene oxide nano particles to obtain nano slurry;
and 5: printing of three electrodes
Respectively carrying out screen printing on carbon ink, silver chloride and nano slurry at different positions of the support body, and then controlling the temperature to carry out curing;
and 6: preparation of Glycerol biosensor chip
Preparing glycerol kinase solution and glycerol-3-phosphate oxidase solution, adding glutaraldehyde respectively, and mixing uniformly; sequentially dripping the nano slurry on the upper layer, and drying at low temperature to obtain the glycerol detection enzyme biosensor chip;
the solute of the aqueous solution containing manganese ions in the step 1 is KMnO 4 Or MnSO 4 The concentration is 1-100 mM, the acid solution is any one of oleic acid, formic acid and sodium chlorate, the concentration is 1-100 mM, and the pH value of the acid solution is 1-7; the dropping speed of the acid solution is 10-200 mu L/min -1 The centrifugal rate of the synthetic liquid is 5000-10000 r. Min -1 Centrifuging for 3-30 min at 60 deg.C for 5-10 h;
in the step 2, the manganese dioxide is calcined at the temperature of 200-500 ℃ for 2-10 h;
the concentration of the graphene oxide in the dispersion liquid in the step 3 is 0.02-0.2 g. L -1 Particle size 200 nm; the centrifugation time is 5-30 min, and the centrifugation speed is 5000-10000 r. Min -1 The drying temperature is 40-80 ℃, and the drying time is 2-5 h.
2. The simple glycerol biosensor chip preparation method according to claim 1, wherein in step 4, the mass ratio of flower-like trimanganese tetroxide nanoparticles to graphene oxide to carbon ink is 1.
3. The method for preparing the simple glycerol biosensor chip according to claim 1, wherein the support in step 5 is made of any one of PVC, alumina, PET, ceramic, gold, silver, copper and platinum, the curing time is 1-5 h, and the curing temperature is 40-100 ℃.
4. The method for preparing the simple glycerol biosensor chip according to claim 1, wherein the concentrations of the glycerol kinase and the glycerol-3-phosphate oxidase in step 6 are both 200-2000U-mL -1 The mass percentage concentration of the glutaraldehyde is 0.1-10%, and the amount of the enzyme solution coated on the upper layer of the nano-slurry is 5-200 muL-cm -2 The low-temperature drying temperature is 0-20 ℃, and the drying time is 12 h.
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