CN105675677B - A kind of preparation method of the Cholesterol Biosensor based on spiro material - Google Patents
A kind of preparation method of the Cholesterol Biosensor based on spiro material Download PDFInfo
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- CN105675677B CN105675677B CN201610048350.4A CN201610048350A CN105675677B CN 105675677 B CN105675677 B CN 105675677B CN 201610048350 A CN201610048350 A CN 201610048350A CN 105675677 B CN105675677 B CN 105675677B
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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
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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
Abstract
The invention discloses a kind of preparation method of the Cholesterol Biosensor based on spiro material.The preparation method includes：The first step, clean conductive substrates are taken, the even spread dielectric layer on the conducting surface of its basalis, dry semi-finished product, the material of its dielectric layer is by spiro material and bio-adhesive materials according to volume ratio 1：1- 4 is mixed；Second step：Prepare 0.5 20mg mL‑1Cholesterol oxidase solution, take 27 μ L cholesterol oxidase solution to be uniformly coated on dielectric layer, re-dry formed enzyme layer, that is, complete biology sensor preparation.Preparation method of the present invention is simple, and material usage is few, and cost is low, can be applied to the production of the cholesterol electrochemistry detecting apparatus of low cost, has far reaching significance to portable family and clinical cholesterol detection.
Description
Technical field
The present invention relates to the preparation of biology sensor, more particularly to a kind of cholesterol enzyme bio-sensing based on spiro material
The preparation of device.
Background technology
Cholesterol is a kind of fatty material for being widely present in organism.It helps to form cell membrane and protects lactation
Zooblast is destroyed by foreign substance.The serum total cholesterol level of Healthy People should be less than 200 mgdL-1(5.2mM), it is high
Cholesterol levels can cause fatal disease, such as atherosclerosis, cerebral thrombosis, coronary heart disease and cardiac muscle stalk
Extremely, so, analysis one extremely sensitive Cholesterol Biosensor of exploitation, the detection of special cholesterol concentration, clinic is examined
Disconnected is highly significant.
Recently, in order to meet disease human needs, cholesterol detection technology is quickly grown.There are many methods to be used for detecting
Cholesterol in blood plasma, such as fluorescence method, high performance liquid chromatography and electrochemical process.And electrochemica biological sensor is because of its cost
It is low, quick and easy to cause very big concern.The electrode that increasing researcher is directed to preparing special modification is consolidated for courage
The detection of alcohol.
Most of Cholesterol Biosensor is to be based on hydrogen peroxide (H2O2) electrochemical reduction and prepare.For example, courage
Sterol oxidizing ferment (ChOx) and horseradish peroxidase are modified on a biosensor, and then are catalyzed and are produced hydrogen peroxide and 4-
Cholestene -3- ketone is the redox products of cholesterol.Therefore, in cholesterol detection, it is most attraction to improve enzymatic activity
's.
Carboxymethyl cellulose (CMC) is a kind of good filmogen, and biomolecule can firmly be adsorbed in fiber
On cellulose fiber, so as to fixed.
Spiro material because it has two mutually perpendicular planes, unbodied property and multiple modification website etc. properties and
Great absorbability.Spiro-compound not only shows significant anti-albuminuria, anti-fat, antitumor properties, but also can answer
For asymmetry catalysis, electrochemical sensor, refractory material, luminescent material, photovoltaic cell etc..
In in the past few decades, spiro-compound is organic because its outstanding luminous and electric property is used as blue phosphorescent
The host material of luminescent device (PhOLEDs).It is well known that the key of the electrochemical sensor prepared is the electricity of electrode
Performance.Cholesterol sensor should have the attributes such as cavity transmission ability, non-dispersive and the transport of two-phase carrier.In recent years, standard
Electrochemical techniques, it is the accurate method for determining biomaterial validity such as cyclic voltammetry (CV).The purpose of this patent is to propose
One new electrode modified using spiro-compound is simultaneously used for the detection of cholesterol.
The content of the invention
In view of the shortcomings of the prior art, object of the present invention is to provide a kind of cholesterol biology based on spiro material
The preparation method of sensor, with reference to electrochemical techniques, simple, outstanding electric property and sensor are synthesized using there is spiro material
The characteristic such as simple is prepared, acquisition technique is simple, the mode of production of the Cholesterol Biosensor of low cost.
To solve prior art problem, the technical scheme that the present invention takes is：
A kind of preparation method of the Cholesterol Biosensor based on spiro material, comprises the following steps：
Step 1, clean conductive substrates are taken, the even spread dielectric layer on the conducting surface of its basalis, then relatively wet
It is dry semi-finished product at 18 DEG C -28 DEG C to spend for 30%-40%, temperature, and its dielectric layer is by spiro material and bioadhesive material
Material is according to volume ratio 1：It is coated with and forms after 1-4 mixing；
Step 2,0.5-20mg mL are prepared-1Cholesterol oxidase solution, take 2-7 μ L cholesterol oxidase solution
It is uniformly coated on dielectric layer, then in the case where relative humidity is 30%-40%, temperature is 18 DEG C -28 DEG C, drying forms enzyme layer, that is, completes
The preparation of biology sensor.
Improved as preparation method to be, described spiro-compound is 2- cyano group spiral shell [fluorenes -9,9'- xanthene] (spiro
[fluorene-9,9'-xanthene] -2-carbonitrile, CNSFX), 2,7- dicyanos spiral shell [fluorenes -9,9'- xanthene]
(spiro [fluorene-9,9'-xanthene] -2,7-dicarbonitrile, DCNSFX), 2'- cyano group spiral shell [fluorenes -9,9'-
Xanthene] (spiro [fluorene-9,9'-xanthene] -2'-carbonitrile, 2'-CNSFX), 2', 7'- dicyanos
Spiral shell [fluorenes -9,9'- xanthene] (spiro [fluorene-9,9'-xanthene] -2', 7'-dicarbonitrile, 2', 7'-
DCNSFX), 2,3', 6', 7- four cyano spiral shell [fluorenes -9,9'- xanthenes] (2,3', 6', 7- tetracarbonitrilespiro
[fluorene-9,9'-xanthene], TTCNSFX), 2'- cyano group spiral shell [dibenzo [c, h] xanthene -7,9'- fluorenes] (2'-
Carbonitrilespiro [dibenzo [c, h] xanthene-7,9'-fluorene], CNSFDBX), 2', 7'- dicyano spiral shells
[dibenzo [c, h] xanthene -7,9'- fluorenes] (2', 7'- dicarbonitrilespiro [dibenzo [c, h] xanthene-
7,9'-fluorene], DCNSFDBX), and structural formula is as follows：
Improved as preparation method to be, step 1 dielectric layer is molten by the suspension and bioadhesive of spiro-compound
Liquid is according to volume ratio 1：It is coated with and forms after 2 mixing.
Improved as preparation method to be, the bioadhesive described in step 1 is carboxymethyl cellulose, chitosan or complete
Fluorinated film.
It is improved as preparation method to be, in step 2 preparation of cholesterol oxidase solution be divided into two steps：The first step, claim
0.05-2mg cholesterol oxidase is taken, it is 5-9 phosphate buffer to be dissolved in 10 μ L, pH, obtains the storing solution of cholesterol oxidase；
Second step, the storing solution of 1 μ L cholesterol oxidase is taken, add 9 μ L, pH and be made up of 5-9 phosphate buffer.
It is preferably as cholesterol oxidase solution, the pH of phosphate buffer is 7 described in the first step and second step.
Beneficial effect
Compared with prior art, the present invention has had the advantage that：
First, it is traditionally used for most of biology sensor of cholesterol detection and uses cholesterol oxidase (ChOx)
With the enzyme pair of peroxidase etc., and the present invention using new spiro material, the alternative peroxidase of spiro-compound, urge
Change hydrogen peroxide to decompose, and collect, transmit electronics caused by hydrogen oxide decomposition, accelerate enzyme's reaction speeding.
Secondly, only using one kind, sensor is both reduced relative to the biology sensor of two traditional enzyme systems, the present invention
Cost, the stability of sensor is improved again.
Finally, sensor of the invention, preparation method is simple, the environment of requirement is loose in terms of preparation, to portable family
Exploitation, the popularization of front yard and clinical cholesterol detection equipment have far reaching significance.
Brief description of the drawings
Fig. 1 is the structural representation of the Cholesterol Biosensor based on spiro material, wherein, 1, basalis, 2, medium
Layer, 3, enzyme layer；
Fig. 2 is the detection architecture explanation figure of the Cholesterol Biosensor based on spiro material, wherein, 1, reference electrode,
2nd, to electrode, 3, working electrode, i.e. bare electrode or spiro material electrode, during detection, buffer solution or courage are added into cylindrical container
Sterol solution.
Fig. 3 is in 50mVs-1Under conditions of sweep speed, the spiro material electrode of preparation pH be 5.8,6.0,7.0,7.4,
8.0 KCl containing 0.1M 5mM [Fe (CN)6]-4/-3Cyclic voltammetry curve in solution, when pH value of solution is 7.0, electrode letter
It is number optimal.
Fig. 4 is in 50mVs-1Under conditions of sweep speed, pH value of solution are 7.0, the spiro material electrode DCNSFDBX/ of preparation
ITO neutralizes the cyclic voltammetry curve of (b) in hydrogen peroxide solution in PBS (a)；
Fig. 5 is in 50mVs-1Under conditions of sweep speed, pH value of solution are 7.0, the spiro material electrode ChOx/ of preparation
DCNSFDBX/ITO is in PBS (a), the spiro material electrode DCNSFDBX/ITO (b) and ChOx/DCNSFDBX/ of preparation
Cyclic voltammetry curves of the ITO (c) in cholesterol solution；
Fig. 6 adds cholesterol solution, the spiro material electricity of preparation into the phosphate buffer that pH is 7.0 under -0.50 V
Pole ChOx/DCNSFDBX/ITO chronoamperogram.
Embodiment
With reference to embodiment, the invention will be further described.Following examples are only intended to clearly illustrate this
The performance of invention, and the following examples can not be limited only to.
Embodiment 1
The first step：The aaerosol solution of spiro-compound is prepared, that is, weighs 5mg spiro-compound, the pH for adding 1mL is 7
Phosphate buffer, ultrasonic 20min is stand-by；Prepare carboxymethyl cellulose（CMC）Solution, that is, weigh 1mg carboxymethyl cellulose
（CMC）, the pH for adding 110 μ L is 7 phosphate buffer, and ultrasound mixes, stand-by；By the aaerosol solution of spiro-compound and carboxylic first
Base cellulose（CMC）Solution is with volume ratio 1：2 ratio mixing, ultrasound make its mixed liquor that is uniformly dispersed to obtain.
Take clean fin oxide condutire glass（FTO）, its size is 20mm × 5mm, takes 10 μ L mixed liquor points in FTO substrates
On the conducting surface of layer 1, dried indoors under environment, form the uniform dielectric layer 2 of a thickness.
Second step：1mg cholesterol oxidase (ChOx) is weighed, the pH for being dissolved in 10 μ L is 7 phosphate buffers, is consolidated as courage
The storing solution of alcohol oxidase (ChOx), the storing solution of 1 μ L cholesterol oxidase (ChOx) is taken, the pH for adding 9 μ L delays for 7 phosphoric acid
Fliud flushing, concentration are 10mg mL-1。
Take 5 μ L cholesterol oxidase (ChOx) using liquid point on dielectric layer 2, it is equally dry under environment indoors
Enzyme layer 3, that is, complete the preparation of biology sensor.
Embodiment 2
The first step：The aaerosol solution of spiro-compound is prepared, that is, weighs 5mg spiro-compound, the pH for adding 1mL is 7
Phosphate buffer, ultrasonic 20min is stand-by；The chitosan solution that mass ratio is 1% is prepared, that is, weighs 10mg chitosan, adds
Enter 980 μ L deionized water and 10 μ L acetic acid, ultrasound mixes, stand-by；The aaerosol solution of spiro-compound is gathered with 1% shell
Sugar juice is with 1：2 ratio mixing, ultrasound make its mixed liquor that is uniformly dispersed to obtain.
Take clean indium tin oxide-coated glass（ITO）, its size is 20mm × 5mm, takes 10 μ L mixed liquor points in ITO bases
On the conducting surface of bottom 1, dried indoors under environment, form the uniform dielectric layer 2 of a thickness.
Second step：1mg cholesterol oxidase (ChOx) is weighed, and the pH for being dissolved in 10 μ L is in 7 phosphate buffer, is made
For cholesterol oxidase (ChOx) storing solution, the storing solution of 1 μ L cholesterol oxidase (ChOx) is taken, the pH for adding 9 μ L is 7
Phosphate buffer as cholesterol oxidase (ChOx) use liquid.
Take 5 μ L cholesterol oxidase (ChOx) using liquid point on dielectric layer 2, it is equally dry under environment indoors
Enzyme layer 3, that is, complete the preparation of biology sensor.
Spiro material electrode DCNSFDBX/ITO performance is detected in phosphate buffer (PBS), hydrogen peroxide solution respectively,
Such as Fig. 4, it is found that spiro material electrode DCNSFDBX/ITO has good reduction characteristic, Jin Erbiao in 1mM hydrogen peroxide solutions
Bright spiro material-DCNSFDBX can be with the reduction of catalyzing hydrogen peroxide；
As Fig. 5 prepare spiro material electrode ChOx/DCNSFDBX/ITO in PBS (a), prepare spiro material electricity
Pole DCNSFDBX/ITO (b) is in cholesterol solution, because being generated without hydrogen peroxide, occurs without redox letter
Number, and the spiro material electrode ChOx/DCNSFDBX/ITO (c) prepared in cholesterol solution because there is cholesterol oxidase
(ChOx) cholesterol generation 4- cholestene -3- ketone and hydrogen peroxide are catalyzed, and due to spiro material-DCNSFDBX presence, is promoted
The electron transmission entered between electrode and cholesterol solution, promote hydrogen peroxide to decompose and produce redox signal；
Be spiro material electrode ChOx/DCNSFDBX/ITO prepared by detection to the responsiveness of cholesterol, constantly to
Quantitative cholesterol solution is added in solution, observes the change of electric current, as shown in fig. 6, the response of electrode pair cholesterol is strong.And
This sensor, which is calculated, has the range of linearity wide（0.3-400 mgdL-1）, good linearity the advantages that, its detection is limited to 0.497 μM,
Sensitivity is 12.319 μ A Mm-1cm-2, signal to noise ratio 3.
The detailed description for embodiment only provides as example above, not limits the diversified implementation of the present invention with this
Mode.
Claims (5)
1. a kind of preparation method of the Cholesterol Biosensor based on spiro material, it is characterised in that comprise the following steps：
Step 1, clean conductive substrates are taken, the even spread dielectric layer (2) on the conducting surface of its basalis (1), then relative
Humidity is 30%-40%, temperature is dry semi-finished product at 18 DEG C -28 DEG C, its dielectric layer by spiro-compound suspension
With bioadhesive agent solution according to volume ratio 1：1- 4 mixes coating and formed；
Step 2,0.5-20mgmL is prepared-1Cholesterol oxidase solution, take 2-7 μ L cholesterol oxidase solution uniformly to apply
Cloth is on dielectric layer, then in the case where relative humidity is 30%-40%, temperature is 18 DEG C -28 DEG C, drying forms enzyme layer (3), that is, completes
The preparation of biology sensor；Described spiro-compound be 2- cyano group spiral shell [fluorenes -9,9'- xanthenes], 2,7- dicyanos spiral shell [fluorenes -
9,9'- xanthenes], 2'- cyano group spiral shell [fluorenes -9,9'- xanthenes], 2', 7'- dicyanos spiral shell [fluorenes -9,9'- xanthenes], 2,3',
6', 7- four cyano spiral shell [fluorenes -9,9'- xanthenes], 2'- cyano group spiral shell [dibenzo [c, h] xanthene -7,9'- fluorenes] or 2', 7'- bis-
It is any in cyano group spiral shell [dibenzo [c, h] xanthene -7,9'- fluorenes].
2. a kind of preparation method of the Cholesterol Biosensor based on spiro material, its feature exist according to claim 1
In：Step 1 dielectric layer is by the suspension of spiro-compound with bioadhesive agent solution according to volume ratio 1：2 mixing after be coated with and
Into.
3. a kind of preparation method of the Cholesterol Biosensor based on spiro material, its feature exist according to claim 1
In：Bioadhesive described in step 1 is carboxymethyl cellulose, chitosan or perfluorinated membranes.
4. a kind of preparation method of the Cholesterol Biosensor based on spiro material, its feature exist according to claim 1
In, in step 2 preparation of cholesterol oxidase solution be divided into two steps：The first step, 0.05-2mg cholesterol oxidase is weighed, it is molten
It is 5-9 phosphate buffer in 10 μ L, pH, obtains the storing solution of cholesterol oxidase；Second step, take 1 μ L cholesterol oxidase
Storing solution, add 9 μ L, pH be made up of 5-9 phosphate buffer.
5. a kind of preparation method of Cholesterol Biosensor based on spiro material according to claim 4, its feature
It is：The pH of phosphate buffer is 7 described in the first step and second step.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1374518A (en) * | 2001-03-07 | 2002-10-16 | 松下电器产业株式会社 | Physiological sensor and stroma Quantitative method |
| CN1749254A (en) * | 2005-09-01 | 2006-03-22 | 复旦大学 | Double spiro material containing heteroaton and its synthetic method and use |
| CN101208598A (en) * | 2005-06-24 | 2008-06-25 | 松下电器产业株式会社 | Biosensor |
| CA2512282C (en) * | 2002-12-31 | 2010-04-27 | Council Of Scientific And Industrial Research | Process for preparation of enzyme electrode |
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- 2016-01-26 CN CN201610048350.4A patent/CN105675677B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1374518A (en) * | 2001-03-07 | 2002-10-16 | 松下电器产业株式会社 | Physiological sensor and stroma Quantitative method |
| CA2512282C (en) * | 2002-12-31 | 2010-04-27 | Council Of Scientific And Industrial Research | Process for preparation of enzyme electrode |
| CN101208598A (en) * | 2005-06-24 | 2008-06-25 | 松下电器产业株式会社 | Biosensor |
| CN1749254A (en) * | 2005-09-01 | 2006-03-22 | 复旦大学 | Double spiro material containing heteroaton and its synthetic method and use |
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