CN110261453A - A kind of avian influenza virus electrochemical sensor based on screen printing electrode - Google Patents
A kind of avian influenza virus electrochemical sensor based on screen printing electrode Download PDFInfo
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- CN110261453A CN110261453A CN201910418115.5A CN201910418115A CN110261453A CN 110261453 A CN110261453 A CN 110261453A CN 201910418115 A CN201910418115 A CN 201910418115A CN 110261453 A CN110261453 A CN 110261453A
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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
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Abstract
The present invention relates to bio-sensing fields, field of virus detection, specifically disclose a kind of avian influenza virus electrochemical sensor based on screen printing electrode.The present invention by the way of silk-screen printing by working electrode, be printed onto printed base plate surface to electrode, reference electrode, guide rail and insulating layer and be fabricated to screen printing electrode.Pass through the modified nano gold on the working electrode of screen printing electrode and HRP, and avian influenza virus electrochemical immunosensor is constructed by the rabbit polyclonal antibody of the fixed anti-H9N2 avian flu virus hemagglutinin of carrier of HRP, signal amplification is carried out to sensor using the monoclonal antibody of the anti-H9N2 avian flu virus hemagglutinin of Nano carbon balls labelled glucose oxidizing ferment mark, realizes the Sensitive Determination to H9N2 avian influenza virus.
Description
Technical field
The invention belongs to bio-sensing fields, field of virus detection, and in particular to a kind of fowl based on screen printing electrode
Influenza virus electrochemical sensor.
Background technique
Viral infectious all seriously threatens the health and lives property safety of the mankind all the time.It is with bird flu
Example, it is a kind of global birds epidemic infectious diseases, and the bird flu epidemic situation of outburst in such as 2013 is not only caused to the mankind
Serious economic loss also proposes safely unprecedented challenge to the health and lives of the mankind.It is therefore proposed that simple, fast
Fast, highly sensitive method for detecting virus is of great significance for the early diagnosis of disease, the propagation of control virus and infection.
Electrical chemiluminescence immunoassay analysis method is the Immunosensors Technology based on electrochemical principle.It is by immunoassay and electricity
Chemical technology combines, and in addition to having the characteristics that biosensor preparation is simple, analysis is rapid, sensitive, selectivity is good, is also included in
The elements such as portable, micromation, integrated, have greatly pushed it in clinical medicine, biology, chemistry, environment, agricultural, industry
The application and development in equal fields.
Although the electrochemical method of a variety of detection avian influenza virus has been disclosed in the prior art, for how to improve
Detection sensitivity is still the focus of attention of those skilled in the art.The present invention passes through on the working electrode of screen printing electrode
Modified nano gold and HRP, and fowl is constructed by the rabbit polyclonal antibody of the fixed anti-H9N2 avian flu virus hemagglutinin of carrier of HRP
Influenza virus immunization electrochemical sensor utilizes the anti-H9N2 avian flu virus hemagglutinin of Nano carbon balls labelled glucose oxidizing ferment mark
Monoclonal antibody signal amplification is carried out to sensor, realize to the Sensitive Determination of H9N2 avian influenza virus.
Summary of the invention
On the basis of existing technology, inventor is prepared for a kind of avian influenza virus electrochemistry based on screen printing electrode
Sensor establishes the new method of detection avian influenza virus.
Specifically, this invention takes following technical measures:
Firstly, preparing screen printing electrode, its step are as follows:
Printed base plate table is printed by working electrode, to electrode, reference electrode, guide rail and insulating layer by way of silk-screen printing
Face is fabricated to screen printing electrode.The working electrode and to electrode be carbon electrode, reference electrode be silver/silver chloride electrode.
Secondly, preparing a kind of avian influenza virus electrochemical sensor based on screen printing electrode, its step are as follows:
(1) screen printing electrode prepared by the present invention is subjected in chlorauric acid solution cyclic voltammetry scan, obtains nanogold and repairs
The screen printing electrode Au/SPE of decorations, the Au/SPE is put into mercaptoethylmaine solution, and 4 DEG C stand overnight progress sulfydryl from group
Dress；The electrode obtained after sulfydryl self assembly is impregnated into 2h in the phosphate buffer (PH7.2) containing glutaraldehyde, milli-Q water,
After being dried with nitrogen, 5 μ L HRP solution (1mg/mL, 0.1MTris-HCl buffer) are added drop-wise to Au/SPE electrode surface, are placed in
30min is incubated in 37 DEG C of insulating boxs, PBS obtains HRP/Au/SPE electrode after rinsing, and 4 DEG C save backup；
(2) rabbit polyclonal antibody (Ab of the anti-H9N2 avian flu virus hemagglutinin of 5 μ L is taken1) solution (80ng/mL, 0.1 mol/L
PBS, pH7.0) it is added dropwise to HRP/Au/SPE electrode surface and dries, it is crosslinked with glutaraldehyde, then rinsed with phosphate buffer solution,
It dries, obtains Ab1/ HRP/Au/SPE modified electrode；By the Ab1/ HRP/Au/SPE modified electrode is immersed in BSA solution and is reacted
30 min take out, are rinsed, dried with phosphate buffer solution, obtain avian influenza virus immunosensor (BSA/Ab1/HRP/Au/
SPE)；The immunosensor is placed in 4 DEG C of refrigerators and is saved backup.
Again, the signal amplification and detection of a kind of avian influenza virus electrochemical sensor based on screen printing electrode
By BSA/Ab1/ HRP/Au/SPE immunosensor immerses H9N2 avian influenza virus solution (40ng/mL, 0.1 mol/L
PBS, pH7.0) 20 min of middle incubation, are rinsed with phosphate buffer solution, are dried, the oxidation of 5 μ L Nano carbon balls labelled glucoses is added dropwise
Monoclonal antibody solution (the CNS-GOD-Ab of the anti-H9N2 avian flu virus hemagglutinin of enzyme mark2), it is immunized and combines 1h, use phosphoric acid buffer
After solution rinses, dry；In -0.4 ~ 0.4 V potential range, differential pulse voltammetry measurement is carried out, test bottom liquid is 20 μ
The PBS solution (0.1M, pH7.0) of M HQ and 18mM glucose；
Monoclonal antibody solution (the CNS- of the anti-H9N2 avian flu virus hemagglutinin of Nano carbon balls labelled glucose oxidizing ferment mark
GOD-Ab2) the preparation method comprises the following steps: the glucose oxidase mark of 0.5 mL carboxylated Nano carbon balls (CNS) solution and 0.8 mL are anti-
The monoclonal antibody solution GOD-Ab of H9N2 avian flu virus hemagglutinin2Mixing, is stirred overnight at room temperature, and BSA solution room temperature is added
Stirring, is rinsed with phosphate buffer solution, is separated, is obtained CNS-GOD-Ab2。
Compared with prior art, the advantages and beneficial effects of the present invention are:
(1) electrochemical sensor structure prepared by the present invention is simple and convenient to operate and detection sensitivity is high.
(2) present invention combines catalysis with glucose oxidase by using HPR, improves catalysis amplification factor, improves sensing
The sensitivity of device detection, is limited to 0.083ng/mL to the detection of H9N2 avian influenza virus.
(3) present invention prepares electrochemical sensor using screen printing electrode.Compared to glass-carbon electrode, screen printing electrode
Instant throwing, can avoid error caused by manual polishing glass-carbon electrode, and experimental data reproducibility is high.
Detailed description of the invention
Fig. 1 is a kind of avian influenza virus electrochemical sensor preparation process based on screen printing electrode of the present invention
Schematic diagram.
Fig. 2 is a kind of avian influenza virus electrochemical sensor based on screen printing electrode of the present invention to different dense
Spend the H9N2 fowl stream of (0.1ng/mL, 3 ng/mL, 10 ng/mL, 50 ng/mL, 75 ng/mL, 100 ng/mL and 120ng/mL)
The calibration curve of the current-responsive value of Influenza Virus solution.
Fig. 3 be SPE electrode (a) prepared by the present invention, Au/SPE(b), HRP/Au/SPE(c), pAb1/HRP/Au /SPE
(d) and BSA/pAb1/ HRP/Au/SPE(e) AC impedance figure.
Fig. 4 is a kind of avian influenza virus electrochemical sensor specificity inspection based on screen printing electrode of the present invention
Survey result schematic diagram.
Specific embodiment
Below by specific embodiment, technical solution of the present invention is used as and is described in further detail；But it is of the invention
It is not limited to these examples.
Embodiment 1
The preparation of screen printing electrode
Printed base plate table is printed by working electrode, to electrode, reference electrode, guide rail, insulating layer by way of silk-screen printing
Face is fabricated to and prints electrode.The working electrode is conductive carbon paste, is conductive carbon paste to electrode, and reference electrode is silver/silver chlorate
Mixed ink, guide rail are conductive silver paste, and insulating layer is dielectric ink, printed base plate PET.
Embodiment 2
A kind of preparation method of the avian influenza virus electrochemical sensor based on screen printing electrode, as shown in Figure 1:
(1) screen printing electrode for preparing embodiment 1 carries out cyclic voltammetry scan in chlorauric acid solution, obtains nanogold and repairs
The screen printing electrode Au/SPE of decorations, the Au/SPE is put into mercaptoethylmaine solution, and 4 DEG C stand overnight progress sulfydryl from group
Dress；The electrode obtained after sulfydryl self assembly is impregnated into 2h in the phosphate buffer (PH7.2) containing glutaraldehyde, milli-Q water,
After being dried with nitrogen, 5 μ L HRP solution (1mg/mL, 0.1MTris-HCl buffer) are added drop-wise to Au/SPE electrode surface, are placed in
30min is incubated in 37 DEG C of insulating boxs, PBS obtains HRP/Au/SPE electrode after rinsing, and 4 DEG C save backup；
(2) rabbit polyclonal antibody (Ab of the anti-H9N2 avian flu virus hemagglutinin of 5 μ L is taken1) solution (80ng/mL, 0.1 mol/L
PBS, pH7.0) it is added dropwise to HRP/Au/SPE electrode surface and dries, it is crosslinked with glutaraldehyde, then rinsed with phosphate buffer solution,
It dries, obtains Ab1/ HRP/Au/SPE modified electrode；By the Ab1/ HRP/Au/SPE modified electrode immerses in BSA solution and reacts 30
Min takes out, is rinsed, dried to get avian influenza virus immunosensor (BSA/Ab with phosphate buffer solution1/HRP/Au/
SPE)；The immunosensor is placed in 4 DEG C of refrigerators and is saved backup.
Embodiment 3
A kind of amplification of avian influenza virus electrochemical sensor signal and detection based on screen printing electrode
By immunosensor a series of different concentration (0.1ng/mL, 3ng/mL, 10ng/mL, 50ng/mL, 75ng/mL,
100ng/mL and 120ng/mL) H9N2 avian influenza virus solution (0.1 mol/L PBS, PH7.0) 20 min of middle incubation, then at carbon
Monoclonal antibody solution (the CNS-GOD-Ab of the anti-H9N2 avian flu virus hemagglutinin of nanosphere labelled glucose oxidizing ferment mark2) in
It is incubated for 1h, after then being rinsed with phosphate buffer solution, is dried.In -0.4 ~ 0.4 V potential range, differential pulse volt is carried out
The measurement of peace method, test bottom liquid are the PBS solution (0.1M, pH7.0) of 20 μM of HQ and 18mM glucose.Testing principle: grape glycosyloxy
Change enzyme will test the glucose in liquid and be oxidized to gluconic acid, while by oxygen reduction at H2O2, fixed on screen printing electrode
HRP can further be catalyzed H2O2Redox reaction occurs with HQ, to reach double enzyme-linked conjunction catalysis signals, utilizes the signal
Quantitative detection is carried out to H9N2 avian influenza virus.
With immunosensor identification H9N2 avian influenza virus current-responsive value (I) to H9N2 avian influenza virus concentration (c)
Mapping, as shown in Fig. 2, its response current value increases with H9N2 avian influenza virus concentration and linearly increases, 0.1~120.0
Good linear relationship, linear equation are presented in ng/mL concentration range are as follows:I (µA) = 0.420 c (ng/mL) +
7.639 (R=0.995), detection are limited to 0.083 ng/mL.In addition, different batches electrochemica biological prepared by the present invention is passed
Sensor carries out 5 parallel determinations according to method described in the present embodiment, and 3.1% lower standard difference proves invented electrochemistry
Biosensor has good reproducibility.
The monoclonal antibody solution of the anti-H9N2 avian flu virus hemagglutinin of Nano carbon balls labelled glucose oxidizing ferment mark
(CNS-GOD-Ab2) the preparation method comprises the following steps: the glucose oxidase of 0.5 mL carboxylated Nano carbon balls (CNS) solution and 0.8 mL
Mark the monoclonal antibody solution GOD-Ab of anti-H9N2 avian flu virus hemagglutinin2Mixing, is stirred overnight at room temperature, and BSA solution is added
It is stirred at room temperature, is rinsed with phosphate buffer solution, separate, obtain CNS-GOD-Ab2。
Embodiment 4
Electrochemical alternate impedance spectrum (EIS) characterization is carried out to modified electrode obtained in embodiment 3, EIS is to explore chemistry to repair
Adorn one of the effective tool of electrode interface property.Its figure is made of low frequency range and high frequency region two parts, and low frequency range therein is corresponding
In diffusion control zone, and the high frequency region of semi-circular portions then corresponds to dynamics Controlling area, and the diameter of semicircle reflects electrode
Surface charge transfer resistance size.In 5.0mmol/L K3[Fe(CN)6]/K4[Fe(CN) 6] (1:1)+0.1 mol/L PBS(pH
=7.0) AC impedance characterization is carried out in+0.1mol/L KCl solution.The Nyquist of modified electrode is bent in sensor preparation process
Line is as shown in Figure 3, it can be seen that screen printing electrode (semicircle very little of the AC impedance spectroscopy of curve a) in high frequency section；When
Electrochemically after electrode face finish nanogold, half circular diameter of high frequency section reduces (curve b), impedance reduction；
Curve c is AC impedance spectroscopy of the HRP modification to Au/SPE electrode surface, and impedance further increases；Work as Ab1Modify electrode table
Behind face, Ab1Half circular diameter of high frequency section of/HRP/Au/SPE modified electrode increases (curve d), this is because protein is unfavorable
In the large biological molecule of electron transmission, the electron transmission at interface can be hindered.Further, by Ab1/ HRP/Au/SPE modified electrode
After being closed with BSA, BSA/Ab1/ HRP/Au/SPE electrode impedance further increases (curve e), this is because BSA is unfavorable
In the large biological molecule of electron transmission.The above result shows that the present invention after gradually modifying, successfully prepares electrochemical sensing
Device.
Embodiment 5
A kind of specificity of the avian influenza virus electrochemical sensor based on screen printing electrode
In order to detect a kind of specificity of avian influenza virus electrochemical sensor based on screen printing electrode, 6 parts of solution are prepared
(newcastle disease virus (NDV) of the inactivation of H9N2 avian influenza virus and 500 ng/mL containing 10 ng/mL, containing 10 ng/mL's
The foot and mouth disease virus (FMDV) of H9N2 avian influenza virus and 500 ng/mL, containing the H9N2 avian influenza virus of 10 ng/mL and 500
The porcine reproductive and respiratory syndrome virus (PRRSV) of ng/mL, the H9N2 avian influenza virus containing 10 ng/mL and 500 ng/mL
The H5N1 avian influenza virus of infectious bronchitis virus (IB), the H9N2 avian influenza virus containing 10 ng/mL and 500 ng/mL
(H5N1) solution and the H9N2 avian influenza virus containing 10 ng/mL), electrochemistry survey is carried out respectively according to method described in embodiment 3
Examination, experimental result are basically unchanged as shown in figure 4, chaff interferent front and back immunosensor current-responsive difference is added, and illustrate that this is immune
Sensor specificity is good.
Although disclosing the embodiment of the present invention for the purpose of illustration, it will be appreciated by those skilled in the art that: not
Be detached from the present invention and spirit and scope of the appended claims in, various substitutions, changes and modifications be all it is possible, therefore, this
The range of invention is not limited to the embodiment disclosure of that.
Claims (3)
1. a kind of avian influenza virus electrochemical sensor based on screen printing electrode, which is characterized in that its step are as follows:
(1) screen printing electrode is carried out to cyclic voltammetry scan in chlorauric acid solution, obtains the silk-screen printing of decorated by nano-gold
The Au/SPE is put into mercaptoethylmaine solution by electrode A u/SPE, and 4 DEG C stand overnight progress sulfydryl self assembly；Certainly by sulfydryl
The electrode obtained immersion 2h in the phosphate buffer (PH7.2) containing glutaraldehyde after assembling, milli-Q water, after being dried with nitrogen,
5 μ L HRP solution (1mg/mL, 0.1MTris-HCl buffer) are added drop-wise to Au/SPE electrode surface, are placed in 37 DEG C of insulating boxs
It is incubated for 30min and obtains HRP/Au/SPE electrode, 4 DEG C save backup after PBS is rinsed；
The working electrode of the screen printing electrode and to electrode be carbon electrode, reference electrode be silver/silver chloride electrode；
(2) rabbit polyclonal antibody (Ab of the anti-H9N2 avian flu virus hemagglutinin of 5 μ L is taken1) solution (80ng/mL, 0.1 mol/L
PBS, pH7.0) it is added dropwise to HRP/Au/SPE electrode surface and dries, it is crosslinked with glutaraldehyde, then rinsed with phosphate buffer solution,
It dries, obtains Ab1/ HRP/Au/SPE modified electrode；By the Ab1/ HRP/Au/SPE modified electrode is immersed in BSA solution and is reacted
30 min take out, are rinsed, dried with phosphate buffer, obtain avian influenza virus electrochemical immunosensor (BSA/Ab1/HRP/
Au/SPE), the electrochemical immunosensor is placed in 4 DEG C of refrigerators and is saved backup.
2. a kind of signal of avian influenza virus electrochemical sensor based on screen printing electrode amplifies and detection, feature exist
In its step are as follows:
By BSA/Ab1/ HRP/Au/SPE immunosensor immerses H9N2 avian influenza virus solution (40ng/mL, 0.1 mol/L
PBS, pH7.0) 20 min of middle incubation, are rinsed with phosphate buffer solution, are dried, the oxidation of 5 μ L Nano carbon balls labelled glucoses is added dropwise
Monoclonal antibody solution (the CNS-GOD-Ab of the anti-H9N2 avian flu virus hemagglutinin of enzyme mark2), it is immunized and combines 1h, use phosphoric acid buffer
After solution rinses, dry；In -0.4 ~ 0.4 V potential range, differential pulse voltammetry measurement is carried out, test bottom liquid is 20 μ
The PBS solution (0.1M, pH7.0) of M HQ and 18mM glucose；
Monoclonal antibody solution (the CNS- of the anti-H9N2 avian flu virus hemagglutinin of Nano carbon balls labelled glucose oxidizing ferment mark
GOD-Ab2) the preparation method comprises the following steps: the glucose oxidase mark of 0.5 mL carboxylated Nano carbon balls (CNS) solution and 0.8 mL are anti-
The monoclonal antibody solution GOD-Ab of H9N2 avian flu virus hemagglutinin2Mixing, is stirred overnight at room temperature, and BSA solution room temperature is added
Stirring, is rinsed with phosphate buffer solution, is separated, is obtained CNS-GOD-Ab2。
3. a kind of avian influenza virus electrochemical sensor based on screen printing electrode, which is characterized in that it is for detecting
H9N2 avian influenza virus.
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