CN105954335A - Method for preparing nano metal and nano metal oxide-based cholesterol biosensor - Google Patents
Method for preparing nano metal and nano metal oxide-based cholesterol biosensor Download PDFInfo
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- CN105954335A CN105954335A CN201610275333.4A CN201610275333A CN105954335A CN 105954335 A CN105954335 A CN 105954335A CN 201610275333 A CN201610275333 A CN 201610275333A CN 105954335 A CN105954335 A CN 105954335A
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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
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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
Abstract
The invention provides a method for preparing a nano metal and nano metal oxide-based cholesterol biosensor. The preparation method comprises the following steps of pre-treating a washed conductive substrate, uniformly coating a metal nano layer on the conductive surface of the substrate, drying and modifying the first layer of an electrode; mixing up a nano metal oxide and the biological adhesive in the volume ratio of 1: 1-3, uniformly coating 5-20 mu L of a mixed liquid on the metal nano layer to obtain a metal oxide nano layer, and drying again to obtain a semi-finished product; preparing 0.5-20 mg/mL of cholesterol oxidase solution, uniformly coating 2-7 mu L of the cholesterol oxidase solution on the metal oxide nano layer, and drying to form an enzyme layer. In this way, a biosensor is prepared. The invention has the advantages of being economical, simple, rapid, sensitive and the like. Meanwhile, the cholesterol biosensor is simple in preparation and can be used for the quantitative detection of cholesterol. The method has profound significance on the development of highly sensitive cholesterol biosensors.
Description
Technical field
The present invention relates to the preparation of biosensor, particularly relate to the preparation of a kind of cholesterol enzyme biosensor based on nano metal and nano-metal-oxide.
Background technology
Cholesterol is a kind of adipose material of character class, has important biological function, and it is not only involved in forming cell membrane, and is synthetic bile acid, vitamin D and the raw material of steroid hormone.Cholesterol can also be converted into bile acid, steroid hormone, 7-DHC, and 7-DHC through metabolism will be changed into vitamin D3, so cholesterol is not harmful material through ultraviolet irradiation.
But, serum cholesterol levels is the highest, the most likely obtains the disease such as atherosclerosis, heart disease.200 mgdL should be less than in cholesterol in serum normal contents-1(5.2mM), more than 240 mgdL-1Lipids contents will be considered high.It practice, the biosensor for cholesterol detection concentration has been delivered, but biosensor based on metal nanoparticle receives much concern, this is because metal nano material has the feature of uniqueness, such as catalysis activity, optics, electronics and magnetic characteristic etc..And in numerous metals, be often used as electrode material mostly is gold, silver, platinum and palladium etc..Utilizing physical property and the chemical property of these metal nano materials uniquenesses, the performance of biosensor has been increased to a new level at present.Be possible not only to make its rapid, miniaturization and multifunction, and it also have highly sensitive, selectivity good, stability is high, fast response time, detection range width and load resistance advantages of higher.
And metal-oxide, if zinc oxide (ZnO) is a very important type oxide semi-conducting material, belong to wide bandgap semiconductor, it can be the more avtive spot of fixing offer of enzyme, can be with efficient absorption enzyme and the activity keeping enzyme, the electron transfer rate being effectively improved between enzyme active center and electrode, it is achieved Direct electron transfer, the highly sensitive electrochemical sensing performance of final acquisition.The ZnO of nanoscale has rich and varied microscopic appearance, also there is the features such as high-specific surface area, good biocompatibility, avirulence, chemistry and photochemical stability, electro-chemical activity and high electron transport ability simultaneously, ZnO nano-structure is made to have important application on the devices such as photoelectricity, surface acoustic wave, Flied emission, piezoelectricity, solaode and sensing, by means such as the regulation and control of nanostructured and doping, it is also possible to further performance be optimized and improve.
Summary of the invention
For the deficiencies in the prior art, object of the present invention is to provide a kind of preparation method based on metal nano material Yu the Cholesterol Biosensor of nano-metal-oxide, combined with electrochemical technology, the characteristic such as utilize that the synthesis of metal nano material and nano-metal-oxide is simple, electric property is excellent and sensor preparation is simple, it is thus achieved that technique is simple, the mode of production of the Cholesterol Biosensor of low cost.
For solving prior art problem, the technical scheme that the present invention takes is:
A kind of based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, comprise the following steps:
Step 1, after taking clean conductive substrates pretreatment, even spread metal nano layer on the conducting surface of its basal layer, then relative temperature be 30%-40%, temperature be at 18 DEG C-28 DEG C be dried, complete electrode ground floor modify；
Step 2, nano-metal-oxide is mixed with biological adhesive 1:1-3 by volume, take 5-20 μ L mixed liquor and be spread evenly across on metal nano layer to obtain metal oxide nano layer, again relative temperature be 30%-40%, temperature be to be dried at 18 DEG C-28 DEG C to obtain semi-finished product；
Step 3, prepares 0.5-20mg
mL-1Cholesterol oxidase solution, the cholesterol oxidase solution taking 2-7 μ L is uniformly coated on metal oxide nano layer, equally relative temperature be 30%-40%, temperature be under 18 DEG C of-28 DEG C of environment be dried formed enzyme layer, i.e. complete the preparation of biosensor.
As preparation method improve, during in step 1, the material of metal nano layer is gold nano grain, silver nano-grain or nano silver wire any one.
Improving as preparation method, in step 2, the material of metal oxide nano layer is nano zine oxide.
As preparation method improve, during in step 2, biological adhesive is carboxymethyl cellulose, chitosan or perfluorinated membranes any one.
Improving as preparation method, in step 2, metal-oxide and biological adhesive by volume 1:1 mixes.
Improving as preparation method, in step 3, the volume of taken cholesterol oxidase solution is 5 μ L.
As what preparation method was improved it is, in step 3, the preparation of cholesterol oxidase solution is divided into two steps: the first step, weighs the cholesterol oxidase (ChOx) of 0.05-2mg, is dissolved in 10 μ L, pH is the phosphate buffer of 5-9, obtains the storing solution of cholesterol oxidase (ChOx)；Second step, takes the storing solution of the cholesterol oxidase (ChOx) of 1 μ L, adds 9 μ L, and pH is that the phosphate buffer of 5-9 is made.
Preparing preferably as cholesterol oxidase solution, the pH of described phosphate buffer is 7.
Beneficial effect
The method of this preparation Cholesterol Biosensor that the present invention provides has a biggest advantage:
First, with self-assembly method by cholesterol oxidase, it is fixed on zinc oxide (ZnO) material and the electrode surface of golden nanometer particle modification, makes a kind of novel high-sensitive electrochemical sensor, achieve the detection to cholesterol under electronegative potential,, this transducer sensitivity is high, favorable reproducibility, stable performance, and good to the selectivity of cholesterol, fast, convenient.
Secondly, make use of the premium properties of zinc oxide (ZnO), the feature such as electro-chemical activity and high electron transport ability cleverly, add chemical stability and the catalysis activity of golden nanometer particle, the highly sensitive electrochemical sensing performance of final acquisition simultaneously.
Finally, invention design replaces precious metal nanoparticle fixing making enzyme electrode by cheap zinc oxide (ZnO) nano material, not only maintain nanoparticle the strong of enzyme is adsorbed and the activity of enzyme, and greatly reduce the cost of electrode fabrication, clinical diagnosis is critically important, economy of the present invention, simple, quick, sensitive, and preparation is simple, can be with the batch production of low cost, the application to there being electrochemica biological sensor has far reaching significance.
Accompanying drawing explanation
Fig. 1, with naked ITO electrode (curve a)；ZnO/ITO electrode (the curve b) that ChOx modifies；Au/CMC/ITO electrode that ChOx modifies (ZnO/Au/CMC/ITO electrode that curve c) and the ChOx modify (signal of (pH7.0) in the cholesterol solution of curve d) detection 200mg/dL.
Fig. 2, carries out specific performance detection respectively with glucose, oxalic acid, citric acid, Cys, ascorbic acid and cholesterol to ChOx/ZnO/Au/CMC/ITO electrode.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.Following example are only intended to clearly illustrate the performance of the present invention, and can not be limited only to the following examples.
Embodiment 1
Step 1, take clean fin oxide condutire glass (ITO), its a size of 20mm × 5mm, pretreatment, the fin oxide condutire glass (ITO) that will clean be immersed in 70 DEG C the water of volume ratio 5:1:1, the hydrogen peroxide of 30%, 30% ammonia in 90min, then rinse, 50 DEG C of oven dried with flowing water.Afterwards fin oxide condutire glass (ITO) electrode of pretreatment is immersed in 12h in 3-aminopropyl triethoxysilane (APTES) ethanol solution of 2%, forms amine silicon single-layer film.Wash three times with dehydrated alcohol, dry, complete the process to fin oxide condutire glass (ITO).By ultrasonic 5-10 minute of gold nano grain (AuNPs), then take gold nano grain (AuNPs) the solution point of 2 μ L on the conducting surface of the fin oxide condutire glass (ITO) of pretreatment, be dried under indoor environment, form the thin uniform gold nano grain film of thick layer.
Step 2, prepares carboxymethyl cellulose (CMC) solution, i.e. weighs the carboxymethyl cellulose (CMC) of 1mg, add the phosphate buffer that pH is 7 of 110 μ L, and ultrasonic mixing is stand-by；By 20 mg mL-1Nano oxidized zinc solution mix with the ratio of volume ratio 1:1 with carboxymethyl cellulose (CMC) solution, ultrasonic make it be uniformly dispersed.Take the mixed solution point of 10 L burnett's solutions and carboxymethyl cellulose (CMC) solution on gold nano grain film, be dried under indoor environment and to obtain semi-finished product.
Step 3, weigh the cholesterol oxidase (ChOx) of 1mg, the pH being dissolved in 10 μ L is 7 phosphate buffers, storing solution as cholesterol oxidase (ChOx), take the storing solution of the cholesterol oxidase (ChOx) of 1 μ L, the pH adding 9 μ L is 7 phosphate buffers, and concentration is 10mg mL-1。,
Take the use liquid point of cholesterol oxidase (ChOx) of 5 μ L on metal oxide nano layer, be dried under indoor environment equally and to obtain enzyme layer, i.e. complete the preparation of biosensor.
As it is shown in figure 1, in the cholesterol solution of 200mg/dL (pH7.0), respectively with naked ITO electrode (curve a)；ZnO/ITO electrode (the curve b) that ChOx modifies；((curve d) detects the ZnO/Au/CMC/ITO electrode that curve c) and ChOx modifies the Au/CMC/ITO electrode that ChOx modifies, and finds that the electrode that metal oxide nano layer is modified Sensitive Detection can go out cholesterol concentration.
Embodiment 2
Step 1, take clean fin oxide condutire glass (ITO), its a size of 20mm × 5mm, pretreatment, the fin oxide condutire glass (ITO) that will clean be immersed in 70 DEG C the water of volume ratio 5:1:1, the hydrogen peroxide of 30%, 30% ammonia in 90min, then rinse, 50 DEG C of oven dried with flowing water.Afterwards fin oxide condutire glass (ITO) electrode of pretreatment is immersed in 12h in 3-aminopropyl triethoxysilane (APTES) ethanol solution of 2%, forms amine silicon single-layer film.Wash three times with dehydrated alcohol, dry, complete the process to fin oxide condutire glass (ITO).: take the fin oxide condutire glass (ITO) of process, by ultrasonic 5-10 minute of gold nano grain (AuNPs), then gold nano grain (AuNPs) the solution point of 2 μ L is taken on the conducting surface of the fin oxide condutire glass (ITO) of pretreatment, it is dried under indoor environment, forms the thin more uniform gold nano grain film of thick layer.
Step 2, preparation mass ratio is the chitosan solution of 1%, i.e. weighs the chitosan of 10mg, adds deionized water and the acetic acid of 10 μ L of 980 μ L, and ultrasonic mixing is stand-by；By 20 mg mL-1Burnett's solution and 1% chitosan solution mix with the ratio of 1:1, ultrasonic make it be uniformly dispersed.Take 10 L burnett's solutions and 1% the mixed solution point of chitosan solution on gold nano grain film, be dried under indoor environment, form the hybrid films of the more uniform nano zine oxide of second layer thickness and chitosan.
Step 3, weigh the cholesterol oxidase (ChOx) of 1mg, the pH being dissolved in 10 μ L is 7 phosphate buffers, storing solution as cholesterol oxidase (ChOx), take the storing solution of the cholesterol oxidase (ChOx) of 1 μ L, the pH adding 9 μ L is 7 phosphate buffers, and concentration is 10mg mL-1。,
Take the use liquid point of cholesterol oxidase (ChOx) of 5 μ L on metal oxide nano layer, be dried under indoor environment equally and to obtain enzyme layer, i.e. complete the preparation of biosensor.
As in figure 2 it is shown, the ZnO/Au/CMC/ITO electrode having modified ChOx has specificity to the detection of cholesterol, for other interference factors, such as glucose, oxalic acid, citric acid, Cys, ascorbic acid, all can exclusive PCR, keep the specificity of detection.
Detailed description for embodiment is only used as example above provides, and not limits the diversified embodiment of the present invention with this.
Claims (8)
1. one kind based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, it is characterised in that comprise the following steps:
Step 1, after taking clean conductive substrates pretreatment, even spread metal nano layer on the conducting surface of its basal layer, then relative temperature be 30%-40%, temperature be at 18 DEG C-28 DEG C be dried, complete electrode ground floor modify；
Step 2, nano-metal-oxide is mixed with biological adhesive 1:1-3 by volume, take 5-20 μ L mixed liquor and be spread evenly across on metal nano layer to obtain metal oxide nano layer, again relative temperature be 30%-40%, temperature be to be dried at 18 DEG C-28 DEG C to obtain semi-finished product；
Step 3, prepares 0.5-20mg mL-1Cholesterol oxidase solution, the cholesterol oxidase solution taking 2-7 μ L is uniformly coated on metal oxide nano layer, equally relative temperature be 30%-40%, temperature be under 18 DEG C of-28 DEG C of environment be dried formed enzyme layer, i.e. complete the preparation of biosensor.
The most according to claim 1 based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, it is characterised in that: during in step 1, the material of metal nano layer is gold nano grain, silver nano-grain or nano silver wire any one.
It is the most according to claim 1 based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, it is characterised in that: in step 2, the material of metal oxide nano layer is nano zine oxide.
The most according to claim 4 based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, it is characterised in that: during in step 2, biological adhesive is carboxymethyl cellulose, chitosan or perfluorinated membranes any one.
The most according to claim 2 based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, it is characterised in that: in step 2, metal-oxide and biological adhesive by volume 1:1 mixes.
It is the most according to claim 1 based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, it is characterised in that: in step 3, the volume of taken cholesterol oxidase solution is 5 μ L.
The most according to claim 1 based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, it is characterized in that, in step 3, the preparation of cholesterol oxidase solution is divided into two steps: the first step, weigh the cholesterol oxidase of 0.05-2mg, it is dissolved in 10 μ L, pH is the phosphate buffer of 5-9, obtains the storing solution of cholesterol oxidase；Second step, takes the storing solution of the cholesterol oxidase of 1 μ L, adds 9 μ L, and pH is that the phosphate buffer of 5-9 is made.
It is the most according to claim 7 based on nano metal and the preparation method of the Cholesterol Biosensor of nano-metal-oxide, it is characterised in that: the pH of described phosphate buffer is 7.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115266868A (en) * | 2022-08-22 | 2022-11-01 | 苏州大学 | Specific electrochemical sensor based on metal oxide and construction method and application thereof |
| CN115607738A (en) * | 2022-10-31 | 2023-01-17 | 中国人民解放军陆军军医大学 | Cardiovascular implant based on bionic endothelial cell antithrombotic function and preparation method thereof |
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- 2016-07-06 CN CN201610275333.4A patent/CN105954335A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115266868A (en) * | 2022-08-22 | 2022-11-01 | 苏州大学 | Specific electrochemical sensor based on metal oxide and construction method and application thereof |
| CN115607738A (en) * | 2022-10-31 | 2023-01-17 | 中国人民解放军陆军军医大学 | Cardiovascular implant based on bionic endothelial cell antithrombotic function and preparation method thereof |
| CN115607738B (en) * | 2022-10-31 | 2023-07-14 | 中国人民解放军陆军军医大学 | Cardiovascular implant based on bionic endothelial cell antithrombotic function and preparation method thereof |
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Application publication date: 20160921 |