CN108562633A - A kind of optical electro-chemistry sensor and its detection method for detecting sulfadimethoxine - Google Patents
A kind of optical electro-chemistry sensor and its detection method for detecting sulfadimethoxine Download PDFInfo
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- CN108562633A CN108562633A CN201810353675.2A CN201810353675A CN108562633A CN 108562633 A CN108562633 A CN 108562633A CN 201810353675 A CN201810353675 A CN 201810353675A CN 108562633 A CN108562633 A CN 108562633A
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- G—PHYSICS
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- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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Abstract
The invention discloses a kind of optical electro-chemistry sensor and its detection method for detecting sulfadimethoxine, the optical electro-chemistry sensor includes：Electrode modifies the WS in electrode surface2Nanometer sheet and DNA aptamers；The DNA aptamers and WS2Nanometer sheet is in close contact.The present invention is acted on using the specific recognition of DNA aptamers and sulfadimethoxine, with the signal isothermal duplication of DNase I catalysis, specificity and the sensitivity detection to target compound sulfadimethoxine are realized, new method is provided for fields such as environmental monitoring, food securities.
Description
Technical field
The present invention relates to photoelectrochemical assay technical fields, and in particular to a kind of for detecting the photoelectrochemical of sulfadimethoxine
Learn sensor and its detection method.
Background technology
Sulfadimethoxine belongs to sulfa antibiotics, there is very strong antibacterium and protozoan infection, can be used for exhaling
Road and urinary tract infections are inhaled, the treatment and prevention of livestock and poultry are now widely used in；In addition, it has facilitation to growth of animals or poultry,
It is widely used again as feed addictive.Due to there is abuse and not abiding by the use of off-drug period, sulfadimethoxine is caused
There is certain residual in animal food, there is potential risk to food hygiene and health.
The current detection method of sulfadimethoxine is mainly ultra-performance liquid chromatography, high performance liquid chromatography, liquid phase color
Spectrum-Mass Spectrometry, fluoroimmunoassay etc..But these methods have detection sensitivity it is low, detection specificity it is not strong, inspection
The shortcomings of surveying of high cost, expensive equipment and complicated for operation, needing professional operator, therefore, establish it is simple, quick, there is height
Sensitivity and highly selective method, the clinical diagnosis for sulfadimethoxine and Food Safety Analysis, are vital.
Photoelectrochemical process refers to the material absorbing photon for having photoelectric activity, the electricity for making electronics be generated in excitation state
Lotus transmittance process.The analysis of optical electro-chemistry aptamers is built upon specific recognition skill of the optical electro-chemistry sensing with aptamers to target molecule
A kind of novel analysis method on the basis of art.It yet there are no and sulfadimethoxine is examined using photoelectrochemical assay method
The report of survey.
Invention content
For the above-mentioned prior art, the optical electro-chemistry biography that the object of the present invention is to provide a kind of for detecting sulfadimethoxine
Sensor and its detection method realize high specific and the high sensitivity detection to sulfadimethoxine, are environmental monitoring, food peace
Congruent field provides new method.
To achieve the above object, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides a kind of optical electro-chemistry sensor for detecting sulfadimethoxine, including：Electricity
The WS in electrode surface is modified in pole2Nanometer sheet and DNA aptamers；
The DNA aptamers and WS2Nanometer sheet is in close contact.
Preferably, the nucleotide sequence of the DNA aptamers is as shown in SEQ ID NO.1.
The second aspect of the present invention provides the preparation method of above-mentioned optical electro-chemistry sensor, includes the following steps：
(1) electrode is pre-processed；
(2) by WS2Nanometer sheet is modified to electrode surface；
(3) DNA aptamers are added drop-wise to WS2The electrode surface of nanometer sheet modification, reaction 1- hours are stood under wet condition,
To obtain the final product.
Preferably, in step (1), the pretreated method of electrode is：Electrode is first used to the mixed liquor of ethyl alcohol and sodium hydroxide
It is cleaned by ultrasonic, then is cleaned with acetone and secondary water, is dried.
Preferably, in step (1), the electrode is ITO electrode.
Preferably, in step (2), by WS2Nanometer sheet is modified to the operation of electrode surface：By WS2Nanometer sheet is added to
In ionized water, WS is made2Nanometer sheet aqueous dispersions, drop coating is in electrode surface, infrared light irradiation drying.
Preferably, in step (3), the wet condition refers to that humidity is 99%.
The third aspect of the present invention provides a kind of method detecting sulfadimethoxine using above-mentioned optical electro-chemistry sensor,
Include the following steps：
(1) sulfadimethoxine of various concentration and DNase I are added drop-wise to optical electro-chemistry sensor surface, are incubated, carried out
The combination of sulfadimethoxine and DNA aptamers and DNase I catalyzing hydrolysis DNA aptamers discharge sulfadimethoxine, carry out
Combination-hydrolysis of cycle；
(2) sulfadimethoxine is detected by establishing the relation curve of photoelectric current and sulfadimethoxine concentration.
Preferably, in step (1), incubation conditions are：Reaction 2 hours are stood under 37 DEG C and 99% damp condition.
Preferably, in step (2), the relation curve of photoelectric current and sulfadimethoxine concentration is：I=89.49logc+380,
Wherein, I is photoelectric current, unit nA；C is sulfadimethoxine concentration, unit nmol/L；R=0.9962, the range of linearity are
10nmol/L–1pmol/L。
Beneficial effects of the present invention：
(1) detection method of the invention is simple, is only modified three times in ITO electrode surface, you can with carrying out sulfanilamide (SN) rope
Pungent detection.
(2) optical electro-chemistry sensor of the invention realizes instrument miniaturization, and easily operated, and testing cost is low.
(3) the very high detection sensitivity of detection of the invention, detection limit is up to pmol/L；Moreover, the present invention is to be based on
The combination of the specificity of sulfadimethoxine and its DNA aptamers, has very high detecting and selecting property.
Description of the drawings
Fig. 1：The principle schematic of detection method.
Fig. 2：WS2The front and back transmission electron microscope of stripping compares collection of illustrative plates；(A) before removing；(B) after removing.
Fig. 3：The electrochemical impedance spectrogram of different modifying electrode；(a) ITO, (b) WS2/ ITO, (c) DNA/WS2/ ITO, (d)
DNA-C/WS2/ITO。
Fig. 4：The photoelectric respone curve of different modifying electrode；(a) ITO, (b) WS2/ ITO, (c) DNA/WS2/ ITO, (d)
DNA-C/WS2/ITO。
Fig. 5：Photoelectric respone curve under different sulfadimethoxine concentration；The concentration that curve a-i is represented is respectively 0.001,
0.005, the sulfadimethoxine of 0.01,0.05,0.1,0.5,1,5,10nmol/L.
Fig. 6：The linear fit curve of photoelectric current and sulfadimethoxine concentration.
Fig. 7：The block diagram of photoelectric respone under the conditions of different antibiotic, antibiotic concentration are 1nmol/L；Δ I=I2-
I1。I2For DNA-C/WS2Photoelectric current (the DNA-C/WS of/ITO2/ ITO has the DNA/ of variety classes antibiotic referred to herein as being added dropwise
WS2/ ITO electrode), I1For DNA/WS2The photoelectric current of/ITO.
Specific implementation mode
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology is introduced, the detection method generally existing detection sensitivity of sulfadimethoxine is low at present, inspection
It is not strong to survey specificity, testing cost is high, expensive equipment and complicated for operation, the shortcomings of needing professional operator.Based on this, this hair
Bright purpose be to provide it is a kind of it is simple, quick, with high sensitivity and highly selective sulfadimethoxine detection method.
The present invention is based on DNA aptamers, WS2Nanometer sheet and the isothermal circulation of DNA enzymatic catalysis expand photoelectrochemical
Method realizes the Sensitive Detection to sulfadimethoxine.The present invention testing principle be：First by tungsten sulfide nanometer sheet drop coating in glass
Carbon electrodes, then DNA aptamers are adsorbed in electrode surface.Due to DNA aptamers and WS2Nanometer sheet is in close contact,
Hydrolysis of the DNase I to DNA is hindered, and DNA is to WS2The optical electro-chemistry activity of nanometer sheet is inhibited so that electrode
Photoelectric current reduce.But when there are the target molecule sulfadimethoxine of aptamer, sulfadimethoxine and DNA aptamers
In conjunction with making DNA aptamers from WS2Nanometer sheet surface desorption is attached so that DNA aptamers can be hydrolyzed by DNase I, to discharge
Sulfadimethoxine.And the sulfadimethoxine discharged can be combined with the DNA aptamers of electrode surface, it is a series of to trigger
Desorption-hydrolysis-desorption-hydrolytic process makes the DNA aptamers quantity of electrode surface substantially reduce, and photoelectric current is remarkably reinforced.This hair
It is bright to be acted on using the specific recognition of DNA aptamers and sulfadimethoxine, with the signal isothermal duplication of DNase I catalysis,
It realizes and (Fig. 1) is detected to the specificity and sensitivity of target compound sulfadimethoxine.
The present invention devises an optical electro-chemistry sensor for sulfadimethoxine detection first, and the optical electro-chemistry passes
Sensor is to be modified with WS in ITO electrode surface2Nanometer sheet and DNA aptamers；Wherein, DNA aptamers are the core of sulfadimethoxine
Sour aptamers, can and sulfadimethoxine specificity combination, base sequence is as follows：
5′-GAGGGCAACGAGTGTTTATAGA-3′；(SEQ ID NO.1)
The optical electro-chemistry sensor designed using the present invention can be detected sulfadimethoxine, and detection method is as follows：
(1) the sulfadimethoxine solution of various concentration and DNase I are uniformly mixed in equal volume, are added drop-wise to modified electrode
(it is modified with WS2Nanometer sheet and DNA aptamers) surface, it is incubated, carries out the combination of sulfadimethoxine and DNA aptamers, and
DNase I catalyzing hydrolysis DNA aptamers discharge sulfadimethoxine, the combination-hydrolysis recycled.
(2) optical electro-chemistry detection is carried out, the relationship between photoelectric current and sulfadimethoxine concentration is established, utilizes the relational expression
The content of sulfadimethoxine in sample to be tested is detected.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
The test material that test material is this field routine is not specifically described used in the embodiment of the present invention,
It can be commercially available by commercial channel.
Wherein, WS2Nanometer sheet is prepared using ultrasound assisted chemical stripping method.Presoma WS2Powder body material and sulfadimethoxine
Purchased from Sigma-Aldrich.Chemical stripping reagent sodium taurocholate and other antibiotic be purchased from Aladdin reagent (on
Sea) Co., Ltd.
Embodiment 1：Preparation for the optical electro-chemistry sensor for detecting sulfadimethoxine
1. electrode pre-processes：
ITO electro-conductive glass is divided into 5 × 1cm first2, ethyl alcohol/NaOH mixed liquors (1 are used in combination:1) it is cleaned by ultrasonic 30 points
Clock is finally respectively washed 30min with acetone and secondary water again, and dries at room temperature again, for use.
2.WS2The preparation of nanometer sheet：
WS is prepared using ultrasound assisted chemical stripping method2Nanometer sheet.Weigh 1 gram of WS2Powder body material, be added to 200mL go from
In sub- water, ultrasonic disperse 30 minutes obtains WS2Aqueous dispersions.Then 2 grams of sodium taurocholates are added to WS2In aqueous dispersions, stirring
30 minutes.Then, by the dispersion liquid ultrasonication 24 hours.It is centrifuged 30 minutes under the conditions of 3000 revs/min, discards precipitation,
Collect solution.The solution of collection is centrifuged under the conditions of 12000 revs/min, collects precipitation.Precipitation is washed with deionized three
It is secondary, in 60 DEG C of vacuum drying.WS2The front and back transmission electron microscope comparison collection of illustrative plates of stripping is as shown in Figure 2.
3.WS2It is prepared by nanometer sheet aqueous dispersions：
Weigh 40mg WS2Nanometer sheet is added in 10mL deionized waters, and ultrasonic disperse 30 minutes obtains a concentration of 4mg/
The WS of mL2The dispersion liquid of nanometer sheet.
4.WS2The preparation of modified electrode：
By the WS of 10 microlitres of 4mg/mL2The dispersant liquid drop of nanometer sheet is added to the ITO electrode surface pre-processed in infrared etc.
It is dry under lamp.After being washed with Tris-HCl (pH7.4) buffer solution of 10mmol/L, WS is obtained2The ITO electricity of nanometer sheet modification
Pole is labeled as：WS2/ITO。
5.DNA aptamers are fixed：
Utilize WS2The DNA aptamers of 10 microlitres of 1 μm of ol/L are dripped to WS by the property with adsorption of DNA2The surfaces /ITO,
Reaction 2 hours are stood at a temperature of 37 DEG C in moist environment (humidity 99%).It is slow with the Tris-HCl (pH7.4) of 10mmol/L
After rushing solution washing, it is by the electrode designations that obtained aptamers are modified：DNA/WS2/ITO.It is prepared for detecting sulphur
Amine Suo Xin optical electro-chemistry sensor.
Embodiment 2：The detection of sulfadimethoxine
1. sulfadimethoxine and aptamers association reaction and the isothermal amplification of DNase I catalysis：
The sulfadimethoxine solution of various concentration is uniformly mixed in equal volume with the DNase I solution of 100unit/mL.So
Afterwards, by 10 microlitres, the mixed solution is added drop-wise to DNA/WS2/ ITO electrode surface, in moist environment (humidity at a temperature of 37 DEG C
99%) reaction 2 hours are stood in.After being washed with Tris-HCl (pH 7.4) buffer solution of 10mmol/L, the electrode that will obtain
It is labeled as：DNA-C/WS2/ITO。
2. optical electro-chemistry detects：
Using electrochemical workstation as signal acquisition instrument, 500W xenon lamps are that visible light source (installs the mirror of filtering ultraviolet additional
Piece), saturated calomel electrode is reference electrode, and platinum column electrode is to electrode, and 10mM Tris-HCl (pH 7.4) buffer solution is detection
Liquid, using -0.3V voltages as operating voltage, the detection that determinand is carried out using i-t technologies is studied, and establishes photoelectric current with sulfanilamide (SN) rope
Relationship between pungent concentration, the range of linearity be 10nmol/L -1pmol/L, calibration curve be I=89.49logc+380 (nA,
Nmol/L, R=0.9962), detection is limited to 0.31pmol/L.
3. stability experiment：
Using identical method and testing conditions, with DNA-C/WS2/ ITO is the sulfanilamide (SN) that working electrode detects 1nmol/L
Ground Suo Xin, obtain relatively just quasi- deviation be 4.38%, illustrate that this method has good reproducibility.With a DNA-C/WS2/
ITO electrode is carried out continuously 10 detections, and obtained relative standard deviation is 3.28%, illustrates that this method has and reappears well
Property.By DNA-C/WS2/ ITO sensors are stored 2 weeks at 4 DEG C, and it is original corresponding 94.38% to obtain current-responsive, it is meant that
This method has good stability.
4. detecting and selecting property is tested：
Respectively by the DNase I solution of different types of antibiotic solution (concentration is 1nmol/L) and 100unit/mL
It is isometric to be uniformly mixed.Then, by 10 microlitres, the mixed solution is added drop-wise to DNA/WS2/ ITO electrode surface, in 37 DEG C of temperature
Under in moist environment stand reaction 2 hours.After being washed with Tris-HCl (pH 7.4) buffer solution of 10mmol/L, obtain
It is modified with the electrode (DNA-C/WS of variety classes antibiotic2/ITO).Optical electro-chemistry detection is carried out, different antibiotic conditions are tested
Under photoelectric respone situation, the results are shown in Figure 7.
As seen from Figure 7, the detection sulfadimethoxine that the present invention can be specific has good detecting and selecting property.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>A kind of optical electro-chemistry sensor and its detection method for detecting sulfadimethoxine
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>DNA aptamers
<400> 1
gagggcaacg agtgtttata ga 22
Claims (10)
1. a kind of optical electro-chemistry sensor for detecting sulfadimethoxine, which is characterized in that including：Electrode is modified in electrode
The WS on surface2Nanometer sheet and DNA aptamers；
The DNA aptamers and WS2Nanometer sheet is in close contact.
2. optical electro-chemistry sensor according to claim 1, which is characterized in that the nucleotide sequence of the DNA aptamers
As shown in SEQ ID NO.1.
3. the preparation method of optical electro-chemistry sensor as claimed in claim 1 or 2, which is characterized in that include the following steps：
(1) electrode is pre-processed；
(2) by WS2Nanometer sheet is modified to electrode surface；
(3) DNA aptamers are added drop-wise to WS2The electrode surface of nanometer sheet modification, stand under wet condition reaction 1-3h to get.
4. preparation method according to claim 3, which is characterized in that in step (1), the pretreated method of electrode is：It will
Electrode first uses the mixed liquor of ethyl alcohol and sodium hydroxide to be cleaned by ultrasonic, then is cleaned with acetone and secondary water, dries.
5. preparation method according to claim 3 or 4, it is characterised in that in step (1), the electrode is ITO electrode.
6. preparation method according to claim 3, which is characterized in that in step (2), by WS2Nanometer sheet is modified to electrode table
The operation in face is：By WS2Nanometer sheet is add to deionized water, and WS is made2Nanometer sheet aqueous dispersions, drop coating are red in electrode surface
Outer light irradiation drying.
7. preparation method according to claim 3, which is characterized in that in step (3), the wet condition refers to that humidity is
99%.
8. utilizing the method for the optical electro-chemistry sensor detection sulfadimethoxine described in claims 1 or 22, which is characterized in that packet
Include following steps：
(1) sulfadimethoxine of various concentration and DNase I are added drop-wise to optical electro-chemistry sensor surface, are incubated, carry out sulfanilamide (SN)
The combination of ground Suo Xin and DNA aptamers and DNase I catalyzing hydrolysis DNA aptamers discharge sulfadimethoxine, are recycled
Combination-hydrolysis；
(2) sulfadimethoxine is detected by establishing the relation curve of photoelectric current and sulfadimethoxine concentration.
9. according to the method described in claim 8, it is characterized in that, in step (1), incubation conditions are：37 DEG C and 99% humidity
Under the conditions of stand reaction 2h.
10. according to the method described in claim 8, it is characterized in that：In step (2), photoelectric current and sulfadimethoxine concentration
Relation curve is：I=89.49logc+380, wherein I is photoelectric current, unit nA；C is sulfadimethoxine concentration, unit nmol/
L；R=0.9962, the range of linearity are 10nmol/L -1pmol/L.
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