CN108593719A - A kind of immunosensor based on flexible electrode - Google Patents
A kind of immunosensor based on flexible electrode Download PDFInfo
- Publication number
- CN108593719A CN108593719A CN201810668237.5A CN201810668237A CN108593719A CN 108593719 A CN108593719 A CN 108593719A CN 201810668237 A CN201810668237 A CN 201810668237A CN 108593719 A CN108593719 A CN 108593719A
- Authority
- CN
- China
- Prior art keywords
- flexible electrode
- immunosensor
- electrode
- polylactic acid
- flexible
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
- G01N27/12—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance of a solid body in dependence upon absorption of a fluid; of a solid body in dependence upon reaction with a fluid, for detecting components in the fluid
- G01N27/125—Composition of the body, e.g. the composition of its sensitive layer
- G01N27/126—Composition of the body, e.g. the composition of its sensitive layer comprising organic polymers
-
- 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
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
The invention discloses a kind of immunosensor based on flexible electrode and its applications, belong to biosensor technology field.The immunosensor, including flexible electrode, the flexible electrode include flexible electrode substrate and are fixed on the probe biomolecule of flexible electrode substrate surface, and the composition material of the flexible electrode substrate includes polylactic acid and graphene.With after target substance to be measured specific binding impedance variations occur for probe biomolecule on immunosensor provided by the invention, and target substance solubility to be measured in solution is calculated by detecting impedance variations.The present invention uses electrode basement prepared by polylactic acid and graphene composite material to can be used to immobilized bio-molecule probes for flexible material containing abundant carboxylic group on polylactic acid molecule and keep its bioactivity；The graphene-doped electric conductivity and flexibility for improving electrode material, greatly promotes the performance of sensor in polylactic acid matrix.
Description
Technical field
The present invention relates to biosensor technology fields, and in particular to a kind of immunosensor based on flexible electrode.
Background technology
Immunosensor has many advantages, such as that selectivity is good, small portable, at low cost, is widely used to food security
The fields such as detection, clinical analysis, environmental monitoring, domestic medicine, such as pathogenic bacteria, antibiotic, Urine proteins, tumor marker
Detection.For realizing for trace substances in filed detection, further increasing transducer sensitivity, repeatability, stability has
Important meaning.
When immunosensor is used for successive objective analyte detection, the reproducibility of probe biomolecule is to restrict the type sensor
Big problem when practical application.Therefore development can be mass-produced and the immunosensor of low cost, being used as can disposal type
Sensor, it will when immunosensor being overcome repeatedly to measure need to probe biomolecule carry out regeneration treatment and caused by operation it is numerous
The drawbacks such as trivial, time-consuming effort.
In face of more and more special detection environment, novel sensor technology of the exploitation based on new material, new process has become
For development trend.Flexible sensor based on flexible base material, due to flexibility, ductility, can free bend even
The features such as folding, be easy to carry, wearable, and structure type is versatile and flexible, can arbitrarily be placed according to the requirement of measuring condition,
Suitable for many special applications scenes and environment.
Common flexible material includes polyvinyl alcohol (PVA), polyester (PET), polyimides (PI), poly- naphthalene dimethyl ester second two
Alcohol ester (PEN), the scraps of paper, textile material etc..As electron institute Xia Shanhong seminars of the Chinese Academy of Sciences have developed the flexibility of paper base material
Immunosensor realizes glycosylated hemoglobin by the self-assembled monolayer on paper substrate and the method that introduces nanogold particle
Detect immunosensor；Institutes Of Technology Of Zhejiang Xu Pan acts are prepared for being based on graphene oxide in flexible substrates (ITO/PET)
(GO)/reduction-state graphene oxide (rGO) sensor of microarray, the spirit for providing cell growth substrate and hydrogen peroxide
Quick detection；James Tour seminars of Rice Univ USA are produced using the cheap polyimide plastic of laser combustion based on stone
The flexible sensor of black alkene foam, can be used for wrist pulse signal detection；King Abdullah University of Science and Technology of Saudi Arabia Pranati
Nayak is prepared for the flexible sensor based on graphene using laser writing technology in PI substrates, can be used for ascorbic acid etc.
Molecular Detection.
Currently, the research of flexible biosensor is still in infancy, domestic and international correlative study is not also very much, be there is no
The report of flexible immunosensor is prepared using polylactic acid and graphene composite material.
Invention content
It is can be mass-produced and have excellent performance the purpose of the present invention is to provide a kind of processing is simple, low-cost
Flexible electrode immunosensor, be used as can disposal type sensor, to solve, existing immunosensor is of high cost, biomolecule
The processing of probe reproducibility is cumbersome, use scope is narrow, reuses the deficiencies of causing accuracy to decline.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of immunosensor based on flexible electrode, including flexible electrode, the flexible electrode include flexible electrode base
Bottom and the probe biomolecule for being fixed on flexible electrode substrate surface, the composition material of the flexible electrode substrate includes polylactic acid
And graphene.
The present invention prepares flexible electrode substrate using polylactic acid and graphene composite material, and poly-lactic acid in high molecular weight is base
Body, excellent plasticity can be made it is variously-shaped, graphene uniform disperse wherein, due to graphene have big specific surface
Product, electric conductivity, high Young's modulus of elasticity can effectively improve the electric conductivity and ductility of polylactic acid.
It can be used for immobilized bio-molecule probes containing abundant carboxyl on polylactic acid molecule, the probe biomolecule is
There is one kind the substance of free amine group, amino and the carboxyl condensation reaction on electrode basement surface to form amido bond so that biology point
Sub- probe is fixed on electrode basement surface.Polylactic acid good biocompatibility can effectively keep the bioactivity of probe biomolecule.
The flexible electrode substrate can be made into linear or thin film shape, polylactic acid and graphene composite material and pass through extrusion
Linear or thin film shape electrode basement is prepared in machine or processing film equipment.
Preferably, a diameter of 0.01-3mm of wire electrode, length are unlimited；The thickness of membrane electrode is 50-500 μm.
Graphene and the mass ratio of polylactic acid are 0.5-10 in the flexible electrode substrate:100, the stone of Effective Doping amount
Black alkene is ensureing the electric conductivity and flexibility of flexible electrode.
Probe biomolecule of the present invention is a kind of substance specifically bound with target substance, preferably,
The probe biomolecule is any one in antibody, antigen, DNA, aptamer etc., containing free in molecular structure
Amino.Preferably, the antigen is protein.
The preparation method of the flexible electrode, includes the following steps：
(1) graphene powder and particles of polylactic acid are mixed, heating fusing post-processing is at linear or film-form flexible electrical
Pole substrate；
(2) flexible electrode substrate is put into containing n-hydroxysuccinimide and 1- (3- dimethylamino-propyls) -3- ethyls
It is activated in the phosphate buffer of carbodiimide hydrochloride；
(3) the flexible electrode substrate after activation is dipped into the solution containing probe biomolecule；Or life will be contained
Flexible electrode substrate surface after the solution dropwise addition of object molecular probe or spray printing to activation, reacted rear probe biomolecule are fixed
In flexible electrode substrate surface, the flexible electrode is made；
The probe biomolecule has free amino, reacts to form amido bond with the carboxyl of polylactic acid.
Preferably, in step (1), the mass ratio of graphene powder and particles of polylactic acid is 1:20-30.
The particles of polylactic acid is the mixture of l-polylactic acid, D- polylactic acid or l-polylactic acid and D- polylactic acid.
The graphene powder can be the graphene of graphene oxide, reduction-state graphene oxide or element doping,
The graphene of middle element doping is the graphene of the element dopings such as nitrogen, phosphorus or sulphur, and doped chemical includes but not limited to above-mentioned several.
Preferably, in step (1), the temperature of heating is 170-230 DEG C.It will be answered using extruder or processing film equipment
Condensation material is processed into linear or thin film shape.
In step (2), a large amount of carboxyls on electrode basement surface can by coupling agent n-hydroxysuccinimide (NHS) with
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) activate.
Preferably, in step (2), n-hydroxysuccinimide is sub- with 1- (3- dimethylamino-propyls) -3- ethyls carbon two
The molar ratio of amine hydrochlorate is 0.1-10:1.
More preferably, the molar concentration of NHS is 0.08mol/L in buffer solution, the molar concentration rate of EDC is 0.05mol/L.
Preferably, in step (2), the time of activation is 3-5h.
Preferably, before activation, first flexible electrode substrate is immersed in n,N-Dimethylformamide (DMF) and handles 5-
120min is conducive to the roughness and permeability that increase polylactic acid for dissolving the polylactic acid for the melting that electrode surface adheres to.
More preferably, flexible electrode substrate is immersed in n,N-Dimethylformamide and handles 10min.
After DMF is handled well, flexible electrode surface is cleaned successively with ethyl alcohol, clear water, activation step is carried out after dry.
In step (3), after the activated carboxylic on electrode basement surface, is reacted with the amino of probe biomolecule and generate amide
Key so that probe biomolecule is fixed on electrode basement surface.
Preferably, in step (3), the condition of the reaction is to place 5min-24h at room temperature.More preferably, when placing
Between be 15h.
The operation principle of flexible electrode immunosensor provided by the invention is：The probe biomolecule of electrode surface with wait for
Survey after target substance specifically binds, electrode impedance changes, and target substance concentration is bigger, and impedance is bigger, variable quantity and
Concentration is in a linear relationship, and the variable quantity of impedance can be measured by direct impedance mensuration and can calculate the dense of target substance to be measured indirectly
Degree.
Specifically, first flexible electrode is placed in solution to be measured so that target substance is attached to the biology on flexible electrode surface
On molecular probe, then the both ends for the flexible electrode for combining target substance are connected respectively on LCR measuring instruments, measure impedance, root
The concentration of target substance to be measured is calculated according to calibration curve.
Immunosensor provided by the invention based on flexible electrode can also use three-electrode system or two electrode bodies
System, the flexible electrode form three-electrode system as working electrode, with auxiliary electrode and reference electrode；It can also be with auxiliary electricity
Pole forms two electrode systems, is measured by electrochemical measuring method.Since impedance variations occur after flexible electrode combination target substance,
Respective change can also occur for electric current, measure impedance variations by detecting curent change, and then calculate the dense of target substance to be measured
Degree.The auxiliary electrode uses but is not limited to platinum electrode or carbon electrode, and the reference electrode uses but is not limited to Ag/AgCl electrodes
Or saturated calomel electrode.
Specifically, first flexible electrode is placed in solution to be measured so that target substance is attached to the biology on flexible electrode surface
On molecular probe, recomposition three-electrode system or two electrode systems are measured by electrochemical measuring method.
The present invention provides the immunosensors in detecting liquid or in gas and the biology on flexible electrode
Application in the concentration of the target substance of molecular probe specific binding.
Immunosensor provided by the invention can be used for detecting can be special by the fixed probe biomolecule in flexible electrode surface
The target substance that the opposite sex combines, is chloramphenicol antibody as fixed on flexible electrode, can specifically bind chloramphenicol, then this is immune
Sensor can be applied to detection chloramphenicol；It is cortisol antibody as fixed on flexible electrode, cortisol can be specifically bound, that
The immunosensor can be applied to detection cortisol；It, can be special such as fixed Listeria monocytogenes aptamer on flexible electrode
The opposite sex combines Listeria monocytogenes nucleic acid, then the immunosensor can be applied to detection Listeria monocytogenes；The present invention's is soft
Property electrode, the fixed probe biomolecule of institute includes but not limited to above-mentioned several.
The advantageous effect that the present invention has：
(1) present invention uses electrode basement prepared by polylactic acid and graphene composite material for flexible material, in the flexibility
Electrode basement surface immobilized bio-molecule probes prepare high performance immunosensor, and polylactic acid has good biocompatibility, can
The advantages that biodegradable, while can be used to immobilized bio-molecule probes containing abundant carboxylic group on its molecule and keeping its life
Object activity；The graphene-doped electric conductivity and flexibility for improving electrode material, greatly promotes the property of sensor in polylactic acid matrix
Energy.
(2) immunosensor based on flexible electrode of the invention can be mass-produced, of low cost, can be used as to abandon
Type immunosensor, and it is flexible, occupy little space, widened application scenario and application field significantly.
Description of the drawings
Fig. 1 is the preparation flow figure of the flexible electrode of embodiment 1.
Fig. 2 is 1 flexible electrode structure composition schematic diagram of embodiment.
Fig. 3 is the chloramphenicol immunosensor of unused DMF processing prepared by embodiment 1 to various concentration chloromycetin solution
Electrochemical impedance response curve.
Fig. 4 is the preparation flow figure of the flexible electrode of embodiment 2.
Fig. 5 is the chloramphenicol immunosensor of the preparation of embodiment 2 handled using DMF to various concentration chloromycetin solution
The linear matched curve of impedance.
Fig. 6 is the preparation flow figure of the flexible electrode of embodiment 3.
Fig. 7 is 3 flexible electrode structure composition schematic diagram of embodiment.
Fig. 8 is the cortisol immunosensor of unused DMF processing prepared by embodiment 3 to various concentration cortex alcoholic solution
Electrochemical impedance linear fit curve.
Fig. 9 is the preparation flow figure of the flexible electrode of embodiment 4.
Figure 10 is 4 flexible electrode structure composition schematic diagram of embodiment.
Figure 11 is the Listeria monocytogenes immunosensor of unused DMF processing prepared by embodiment 4 to various concentration list
Increase the linear matched curve of impedance of Listeria solution.
Specific implementation mode
It elaborates below in conjunction with the accompanying drawings to the embodiment of the present invention：It is carrying out based on the technical solution of the present invention in fact
It applies, gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following embodiments.
Immunosensor includes flexible electrode, and flexible electrode is by flexible electrode substrate and is fixed in flexible electrode substrate
Probe biomolecule forms, and the composition material of flexible electrode substrate is polylactic acid and graphene, and shape can be linear or film
Shape.
Immunosensor is used for the specific detection of corresponding target substance according to fixed probe biomolecule, passes through electrochemistry
Measurement method or the variable quantity of direct impedance mensuration measurement impedance can calculate the concentration of target substance to be measured indirectly.
Using graphene powder, particles of polylactic acid, chloramphenicol antibody, cortisol antibody, the list used in following embodiments
It is the material bought on the market to increase Listeria aptamer.
Embodiment 1
A kind of immunosensor based on flexible electrode provided in this embodiment is for detecting chloramphenicol concentration.
In the present embodiment, the preparation method of flexible electrode includes the following steps (Fig. 1)：
S1, polylactic acid and graphene flexibility wire electrode are prepared using double screw extruder.
Graphene powder and l-polylactic acid particle are pressed 1:30 mass ratioes mix, and the charging box of double screw extruder is added
In, it is 180 degrees Celsius to adjust double screw extruder temperature, starts to squeeze out flexible wire electrode after 60min, setting hauling speed is
10cm/min。
S2, prepared flexible electrode substrate is cleaned using ethyl alcohol, pure water.
By the polylactic acid-graphene flexibility wire electrode prepared using double screw extruder extrusion molding respectively in ethyl alcohol and pure
It is dry after ultrasound 20min in water.
S3, chloramphenicol antibody is fixed by EDC/NHS activation in flexible linear electrode surface.
A certain amount of NHS and EDC are dissolved in phosphate buffer, prepare the EDC's of NHS and 0.05M containing 0.08M
The flexible wire electrode cleaned is immersed in EDC/NHS mixed liquors and carries out activation 3h by mixed liquor；
Later, the flexible wire electrode after activation is dipped into the solution containing 50mg/mL chloramphenicol antibodies, is placed
The present embodiment flexible electrode is made, as shown in Figure 2 in 15h.
After the flexible electrode of the present embodiment completes, by the chloromycetin solution of configuration standard concentration, to making
Sensor is demarcated, and specific scaling method includes the following steps：
F1, by Na2HPO4(0.2M) and NaH2PO4(0.2M) is mixed in a certain ratio, and it is 7.2 to adjust pH, and not homogeneity is added
The chloramphenicol of amount is configured to the chloromycetin solution of 7 concentration gradients.
F2, prepared flexible electrode is immersed in a minimum concentration chloromycetin solution, is waited 30 minutes so that chlorine
Mycin molecule is attached on the fixed chloramphenicol antibody molecule in flexible electrode surface.
F3, the above-mentioned flexible electrode for combining chloramphenicol molecule is placed into the potassium ferricyanide containing 5mM and 0.1M potassium chloride
In phosphate buffer (pH7.4), using three-electrode electro Chemical measurement method, using the flexible electrode of the present embodiment as work electricity
Pole, using Ag/AgCl electrodes as reference electrode, then passes through Electrode with Electrochemical Impedance Spectroscopy measurement sensor pair using Pt pieces as auxiliary electrode
The response of the concentration chloromycetin solution.
F4, the Electrode with Electrochemical Impedance Spectroscopy design parameter of the present embodiment are as follows：Range of scanned frequencies is 0.1-105Hz, setting electricity
Pressure is open-circuit voltage.
F5, according to the above method, measure the electricity of the flexible electrode immunosensor to other concentration chloramphenicol of the present embodiment
Chemical impedance responds numerical value.
After calibration, the immunosensor of DMF processing is not used to various concentration chloramphenicol in obtained the present embodiment
Electrochemical impedance response curve detects as shown in figure 3, as a result realizing the chloromycetin solution that concentration range is 0.1-10ng/mL.
Embodiment 2
A kind of immunosensor based on flexible electrode of the present embodiment is detected for chloramphenicol concentration.
In the present embodiment, the preparation method of flexible electrode immunosensor includes the following steps (Fig. 4)：
Flexible electrode substrate, the mass ratio of graphene oxide and D- polylactic acid are made using graphene oxide and D- polylactic acid
It is 1：25, processing is prepared as the S1 steps of embodiment 1.
It is further preferred that following step, such as the S2 steps of embodiment 1, prepared flexible electrode substrate is first soaked
Bubble handles 10min in DMF, then ultrasound 10min and drying in ethyl alcohol and pure water respectively, is further continued for carrying out embodiment 1
The flexible electrode immunosensor of the present embodiment is made in step S3.
By the chloromycetin solution of configuration standard concentration, the sensor of making is demarcated, specific scaling method packet
Include following steps：
F1, by Na2HPO4(0.2M) and NaH2PO4(0.2M) is mixed in a certain ratio, and it is 7.2 to adjust pH, and not homogeneity is added
The chloramphenicol of amount is configured to the chloromycetin solution of 6 concentration gradients.
F2, prepared flexible electrode immunosensor is immersed in a minimum concentration chloromycetin solution, waiting 30
Minute so that chloramphenicol molecule is attached on the fixed chloramphenicol antibody molecule in flexible electrode surface.
F3, the both ends of the obtained flexible electrode immunosensor for combining chloramphenicol molecule are connected respectively to hand-held
LCR instruments (such as Keysight U1733C) choose one grade of impedance measurement, and setting scan frequency is 100kHz, until the resistance of display
When anti-numerical value is the number that one stablizes, impedance response of the numerical value as sensor to corresponding chloramphenicol concentration is write down.
According to step F2, F3 method, impedance response of the measurement sensor to other concentration chloromycetin solutions.
F4, after calibration, obtained the present embodiment is using the immunosensor of DMF processing to various concentration chloramphenicol
Impedance response linear fit curve, as shown in figure 5, abscissa is the Ln concentration of chloramphenicol, ordinate is flexible electrode combined chloride
Impedance value after mycin realizes the detection of chloramphenicol concentration range 0.05-10ng/mL.
Embodiment 3
A kind of immunosensor based on flexible electrode of the present embodiment is for detecting concentration of cortisol.
In the present embodiment, the preparation method of flexible electrode includes the following steps (Fig. 6)：
S1, polylactic acid and graphene flexibility wire electrode are prepared using double screw extruder.
Graphene powder and l-polylactic acid particle are pressed 1:40 mass ratioes mix, and the charging box of double screw extruder is added
In, it is 180 degrees Celsius to adjust double screw extruder temperature, starts to squeeze out flexible wire electrode after 60min, setting hauling speed is
10cm/min。
S2, prepared flexible electrode substrate is cleaned using ethyl alcohol, pure water.
By the polylactic acid-graphene flexibility wire electrode prepared using double screw extruder extrusion molding respectively in ethyl alcohol and pure
It is dry after ultrasound 20min in water.
S3, cortisol antibody is fixed by EDC/NHS activation in flexible linear electrode surface.
A certain amount of NHS and EDC are dissolved in phosphate buffer, prepare the EDC's of NHS and 0.05M containing 0.08M
The flexible wire electrode cleaned is immersed in EDC/NHS mixed liquors and activates 3h by mixed liquor；
Later, the flexible wire electrode after activation is dipped into the cortisol antibody-solutions containing 50mg/mL, is placed
The present embodiment flexible electrode is made, as shown in Figure 7 in 15h.
By the cortex alcoholic solution of configuration standard concentration, the sensor of making is demarcated, specific scaling method packet
Include following steps：
F1, by Na2HPO4(0.2M) and NaH2PO4(0.2M) is mixed in a certain ratio, and it is 7.2 to adjust pH, and not homogeneity is added
The cortisol of amount is configured to the cortex alcoholic solution of 5 concentration gradients.
F2, prepared immunosensor is immersed in a minimum concentration cortex alcoholic solution, is waited 40 minutes so that
Cortisol molecule is attached on the fixed cortisol antibody molecule in flexible electrode surface.
F3, the above-mentioned flexible electrode for combining cortisol molecule is placed into the potassium ferricyanide containing 5mM and 0.1M potassium chloride
In phosphate buffer (pH7.4), three-electrode electro Chemical measurement method is then used, wherein the flexible electrode with the present embodiment is
Working electrode, using Ag/AgCl electrodes as reference electrode, is then measured by Electrode with Electrochemical Impedance Spectroscopy and is passed using Pt pieces as auxiliary electrode
Response of the sensor to the concentration cortex alcoholic solution.
F4, the Electrode with Electrochemical Impedance Spectroscopy design parameter of the present embodiment are as follows：Range of scanned frequencies is 0.1-105Hz, setting electricity
Pressure is open-circuit voltage.
F5, according to the above method, measure the electricity of the flexible electrode immunosensor to other concentration cortisols of the present embodiment
Chemical impedance responds numerical value.
After calibration, the immunosensor of DMF processing is not used to various concentration cortisol in obtained the present embodiment
Electrochemical impedance linear fit curve, as shown in figure 8, it is 10-100ng mL as a result to realize concentration range-1Cortex alcoholic solution
Detection.
Embodiment 4
A kind of immunosensor based on flexible electrode of the present embodiment is for detecting Listeria monocytogenes concentration.
In the present embodiment, the preparation method of flexible electrode includes the following steps (Fig. 9)：
S1, polylactic acid and graphene flexibility wire electrode are prepared using double screw extruder
Nitrogen-doped graphene powder and l-polylactic acid particle are pressed 1:20 mass ratioes mix, and adding for double screw extruder is added
In hopper, it is 180 degrees Celsius to adjust double screw extruder extrusion temperature, extruding pelletization, then is blown by Single screw extrusion inflation film manufacturing machine
It is moulded into film, blown film temperature is 180 DEG C, and blown film film bubble is passed through air by extruder head makes its inflation cool down, and then film passes through people
Letter stencil is shaped, and carry-over pinch rolls traction is finally wound by wrap-up flattening-out, obtains thin film shape flexible electrode substrate of the present invention, thin
Film thickness is 200 μm.
S2, flexible electrode substrate is handled using DMF, ethyl alcohol, pure water is used in combination to be respectively washed prepared flexible electrode substrate.
The polylactic acid of above-mentioned preparation-graphene fexible film shape electrode is cut to the band of long 5cm, width 3cm, then is taken
One of band flexible electrode, which is first immersed in DMF, handles 10min, and then ultrasound 10min is simultaneously in ethyl alcohol and pure water respectively
It is dry,
S3, the Listeria monocytogenes nucleic acid with amino is fixed by EDC/NHS activation in flexible linear electrode surface
Aptamer.
A certain amount of NHS and EDC are dissolved in phosphate buffer, prepare the EDC's of NHS and 0.05M containing 0.08M
The fexible film shape electrode cleaned is immersed in EDC/NHS mixed liquors, is sealed 3h by mixed liquor；
Later, the flexible wire electrode after activation is dipped into the solution containing Listeria monocytogenes aptamer, is put
15h is set, the present embodiment flexible electrode is made, as shown in Figure 10.
By the Listeria monocytogenes solution of configuration standard concentration, the sensor of making is demarcated, it is specific to demarcate
Method includes the following steps：
F1, by Na2HPO4(0.2M) and NaH2PO4(0.2M) is mixed in a certain ratio, and it is 7.4 to adjust pH, and different numbers are added
The Listeria monocytogenes of amount are configured to the Listeria monocytogenes solution of 6 concentration gradients.
F2, prepared flexible electrode is immersed in a minimum concentration Listeria monocytogenes solution, is waited 40 minutes,
So that Listeria monocytogenes are attached on the fixed Listeria monocytogenes Nucleic acid aptamer molecules in immunosensor surface.
F3, the phosphate-buffered that the above-mentioned flexible electrode for combining Listeria monocytogenes is placed into the 2.5mM potassium ferricyanides
In liquid (pH7.4), three-electrode electro Chemical measurement method is then used, wherein using the flexible electrode of the present embodiment as working electrode,
It is then dense to this by Electrode with Electrochemical Impedance Spectroscopy measurement sensor using Ag/AgCl electrodes as reference electrode with Pt for auxiliary electrode
Spend the response of Listeria monocytogenes solution.
F4, the Electrode with Electrochemical Impedance Spectroscopy design parameter of the present embodiment are as follows：Range of scanned frequencies is 0.1-105Hz, setting electricity
Pressure is 0.24V.
F5, according to the above method, the flexible electrode immunosensor for measuring the present embodiment increases Liszt to other concentration lists
The electrochemical impedance of bacterium responds numerical value.
After calibration, the immunosensor of DMF processing is not used to various concentration list increasing Li Si in obtained the present embodiment
The electrochemical impedance linear fit curve of special bacterium, as shown in figure 11, it is 1-1 × 10 as a result to realize concentration range5CFU/mL's
Listeria monocytogenes solution detects, and abscissa is the Lg concentration of Listeria monocytogenes, and ordinate is that flexible electrode combines difference
Impedance value after concentration Listeria monocytogenes.
Claims (10)
1. a kind of immunosensor based on flexible electrode, including flexible electrode, which is characterized in that the flexible electrode includes soft
Property electrode basement and be fixed on the probe biomolecule of flexible electrode substrate surface, the composition material packet of the flexible electrode substrate
Include polylactic acid and graphene.
2. immunosensor as described in claim 1, which is characterized in that graphene and poly- breast in the flexible electrode substrate
The mass ratio of acid is 0.5-10:100.
3. immunosensor as described in claim 1, which is characterized in that the flexible electrode substrate is a diameter of 0.01-
The film that the linear or thickness of 3mm is 50-500 μm.
4. immunosensor as described in claim 1, which is characterized in that the probe biomolecule be antibody, antigen,
Any one in DNA, aptamer.
5. immunosensor as described in claim 1, which is characterized in that the preparation method of the flexible electrode, including it is following
Step：
(1) graphene powder and particles of polylactic acid are mixed, heating fusing post-processing is at linear or film-form flexible electrode base
Bottom；
(2) flexible electrode substrate is put into containing n-hydroxysuccinimide and 1- (3- dimethylamino-propyls) -3- ethyls carbon two
It is activated in the phosphate buffer of inferior amine salt hydrochlorate；
(3) the flexible electrode substrate after activation is dipped into the solution containing probe biomolecule；Or biology point will be contained
Flexible electrode substrate surface after the solution dropwise addition of sub- probe or spray printing to activation, reacted rear probe biomolecule are fixed on soft
Property electrode basement surface, be made the flexible electrode；
The probe biomolecule has free amino, reacts to form amido bond with the carboxyl of polylactic acid.
6. immunosensor as claimed in claim 5, which is characterized in that in step (1), the temperature of heating is 170-230 DEG C.
7. immunosensor as claimed in claim 5, which is characterized in that in step (2), n-hydroxysuccinimide and 1-
The molar ratio of (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides is 0.1-10:1.
8. immunosensor as claimed in claim 5, which is characterized in that in step (2), before activation, first by flexible electrode base
Bottom, which is immersed in N,N-dimethylformamide, handles 5-120min.
9. immunosensor as described in claim 1, which is characterized in that further include auxiliary electrode, the flexible electrode conduct
Working electrode.
10. immunosensor as described in claim 1, which is characterized in that the polylactic acid is l-polylactic acid, D- polylactic acid
Or both mixture；The graphene is graphene oxide, reduction-state graphene oxide, the stone of nitrogen, phosphorus or element sulphur doping
Any one in black alkene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810668237.5A CN108593719B (en) | 2018-06-26 | 2018-06-26 | Immunosensor based on flexible electrode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810668237.5A CN108593719B (en) | 2018-06-26 | 2018-06-26 | Immunosensor based on flexible electrode |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108593719A true CN108593719A (en) | 2018-09-28 |
| CN108593719B CN108593719B (en) | 2020-06-19 |
Family
ID=63633898
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810668237.5A Active CN108593719B (en) | 2018-06-26 | 2018-06-26 | Immunosensor based on flexible electrode |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108593719B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113138211A (en) * | 2020-01-20 | 2021-07-20 | 安徽省麦米医疗科技有限公司 | Sweat cortisol monitoring patch and preparation method thereof |
| CN113866237A (en) * | 2021-09-16 | 2021-12-31 | 湖南大学 | Flexible photoelectrochemical aptamer sensor and preparation method and application thereof |
| CN114137040A (en) * | 2021-11-26 | 2022-03-04 | 南京农业大学 | Foldable passive NFC flexible sensing device and manufacturing method and using method thereof |
| CN114324514A (en) * | 2021-11-30 | 2022-04-12 | 南通大学 | PET-based flexible electrode and preparation method and application thereof |
| CN114577874A (en) * | 2022-03-04 | 2022-06-03 | 清华大学 | Electrode and preparation method thereof, sensor and preparation method and application thereof |
| US12055513B2 (en) | 2019-12-14 | 2024-08-06 | Lansion Biotechnology Co., Ltd. | Method for preparing immunoelectrode system |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003040688A2 (en) * | 2001-11-06 | 2003-05-15 | North Carolina State University | 2d/3d chemical sensors and methods of fabricating and operating the same |
| CN102943315A (en) * | 2012-12-11 | 2013-02-27 | 江南大学 | PLA (polylactic acid) conductive fiber and preparation method thereof |
| CN103302910A (en) * | 2013-06-25 | 2013-09-18 | 电子科技大学 | Biodegradable flexible conductive base plate and preparation method thereof |
| CN104634847A (en) * | 2013-11-14 | 2015-05-20 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Electrochemical immunosensing method for detecting Listeria monocytogenes |
| CN104931693A (en) * | 2015-06-11 | 2015-09-23 | 上海海洋大学 | Electrochemical immunosensor and preparation method thereof |
| CN105548318A (en) * | 2016-01-25 | 2016-05-04 | 深圳大学 | Wearable electrochemical sensor electrode and wearable electrochemical sensor |
| CN106645320A (en) * | 2016-11-15 | 2017-05-10 | 中国科学院大学 | Flexible electrode based on biocompatible materials, preparation method of flexible electrode and application |
-
2018
- 2018-06-26 CN CN201810668237.5A patent/CN108593719B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003040688A2 (en) * | 2001-11-06 | 2003-05-15 | North Carolina State University | 2d/3d chemical sensors and methods of fabricating and operating the same |
| CN102943315A (en) * | 2012-12-11 | 2013-02-27 | 江南大学 | PLA (polylactic acid) conductive fiber and preparation method thereof |
| CN103302910A (en) * | 2013-06-25 | 2013-09-18 | 电子科技大学 | Biodegradable flexible conductive base plate and preparation method thereof |
| CN104634847A (en) * | 2013-11-14 | 2015-05-20 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Electrochemical immunosensing method for detecting Listeria monocytogenes |
| CN104931693A (en) * | 2015-06-11 | 2015-09-23 | 上海海洋大学 | Electrochemical immunosensor and preparation method thereof |
| CN105548318A (en) * | 2016-01-25 | 2016-05-04 | 深圳大学 | Wearable electrochemical sensor electrode and wearable electrochemical sensor |
| CN106645320A (en) * | 2016-11-15 | 2017-05-10 | 中国科学院大学 | Flexible electrode based on biocompatible materials, preparation method of flexible electrode and application |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US12055513B2 (en) | 2019-12-14 | 2024-08-06 | Lansion Biotechnology Co., Ltd. | Method for preparing immunoelectrode system |
| CN113138211A (en) * | 2020-01-20 | 2021-07-20 | 安徽省麦米医疗科技有限公司 | Sweat cortisol monitoring patch and preparation method thereof |
| CN113866237A (en) * | 2021-09-16 | 2021-12-31 | 湖南大学 | Flexible photoelectrochemical aptamer sensor and preparation method and application thereof |
| CN113866237B (en) * | 2021-09-16 | 2023-09-12 | 湖南大学 | Flexible photoelectrochemical aptamer sensor and preparation method and application thereof |
| CN114137040A (en) * | 2021-11-26 | 2022-03-04 | 南京农业大学 | Foldable passive NFC flexible sensing device and manufacturing method and using method thereof |
| CN114137040B (en) * | 2021-11-26 | 2023-09-01 | 南京农业大学 | Foldable passive NFC flexible sensing device and manufacturing method and using method thereof |
| CN114324514A (en) * | 2021-11-30 | 2022-04-12 | 南通大学 | PET-based flexible electrode and preparation method and application thereof |
| CN114577874A (en) * | 2022-03-04 | 2022-06-03 | 清华大学 | Electrode and preparation method thereof, sensor and preparation method and application thereof |
| CN114577874B (en) * | 2022-03-04 | 2022-11-18 | 清华大学 | Electrode and preparation method thereof, sensor and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108593719B (en) | 2020-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108593719A (en) | A kind of immunosensor based on flexible electrode | |
| Pundir et al. | Determination of urea with special emphasis on biosensors: A review | |
| Ayankojo et al. | Molecularly imprinted polymer based electrochemical sensor for quantitative detection of SARS-CoV-2 spike protein | |
| CN104142361B (en) | A kind of protein molecule engram poly ion liquid membrane electrochemical sensor | |
| Yao et al. | Flexible freestanding graphene paper-based potentiometric enzymatic aptasensor for ultrasensitive wireless detection of kanamycin | |
| Karunakaran et al. | Introduction to biosensors | |
| KR101159012B1 (en) | Electrode for electrochemical biosensor and biosensor including the same | |
| Wang et al. | Electrochemical biosensors based on antibody, nucleic acid and enzyme functionalized graphene for the detection of disease-related biomolecules | |
| Sagadevan et al. | Recent trends in nanobiosensors and their applications-a review | |
| Wasilewski et al. | Prospects of ionic liquids application in electronic and bioelectronic nose instruments | |
| CN101126734B (en) | Biosensor based on aptamer modified conducting polymer and its preparation method and uses | |
| Power et al. | Electroanalytical sensor technology | |
| Tiwari et al. | RETRACTED: Detection of p53 gene point mutation using sequence-specific molecularly imprinted PoPD electrode | |
| CN108645903A (en) | The preparation and application of molecular engram sensor based on the chitosan-modified glass-carbon electrode of carbon dots- | |
| Jumpathong et al. | A sensitive and disposable graphene oxide electrochemical immunosensor for label-free detection of human immunoglobulin G | |
| CN103063715A (en) | Method for detecting surviving gene based on graphene-gold composite material electrochemical DNA (Deoxyribose Nucleic Acid) biosensor | |
| Cui et al. | An antifouling electrochemical biosensor based on a protein imprinted hydrogel for human immunoglobulin G recognition in complex biological media | |
| Ekabutr et al. | Electrospinning: a carbonized gold/graphene/PAN nanofiber for high performance biosensing | |
| Brodowski et al. | Highly selective impedimetric determination of Haemophilus influenzae protein D using maze-like boron-doped carbon nanowall electrodes | |
| CN109060915A (en) | A kind of enzyme biologic sensor based on flexible electrode | |
| Yin et al. | Ultra-sensitive electrochemical detection of ampicillin with hollow tubular mesoporous In2O3@ Au@ Apta nanofibers as electrode materials | |
| CN116203092A (en) | Preparation method and detection method of electrochemical sensor for detecting di (2-ethylhexyl) phthalate | |
| Arris et al. | Characterization of electrochemical transducers for biosensor applications | |
| Hu et al. | A novel composite of covalent organic framework and molecularly imprinted polymer with 1, 1′-bis (allylcarbamoyl) ferrocene as cross-linker for ratiometric electrochemical detection of 5-hydroxytryptamine | |
| CN201522473U (en) | Nano-synergy glycosylated functional molecular imprint membrane electrode for detecting bacterial toxins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |