CN108562633B - Photoelectrochemical sensor for detecting sulfadimethoxine and detection method thereof - Google Patents
Photoelectrochemical sensor for detecting sulfadimethoxine and detection method thereof Download PDFInfo
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Abstract
The invention discloses a photoelectrochemical sensor for detecting sulfadimethoxine and a detection method thereof, wherein the photoelectrochemical sensor comprises: electrode, WS modified on electrode surface2Nanosheets and DNA aptamers; the DNA aptamer and WS2The nanoplates are in intimate contact. The invention utilizes the specific recognition function of DNA aptamer and sulfadoxine, realizes the specific and sensitive detection of the target compound sulfadoxine by signal isothermal amplification catalyzed by DNase I, and provides a new method for the fields of environmental monitoring, food safety and the like.
Description
Technical Field
The invention relates to the technical field of photoelectrochemical analysis, in particular to a photoelectrochemical sensor for detecting sulfadimethoxine and a detection method thereof.
Background
Sulfadimethoxine belongs to sulfonamide antibiotics, has strong antibacterial and protozoan infection resistance, can be used for upper respiratory tract and urinary tract infection, and is widely used for treating and preventing livestock and poultry diseases at present; in addition, it has the function of promoting the growth of livestock and poultry and is widely applied as a feed additive. Due to abuse and non-compliance with withdrawal periods, the sulfadoxine has certain residue in animal food, and has potential hazard to food sanitation and human health.
The current detection method of the sulfadimethoxine mainly comprises an ultra-high performance liquid chromatography, a liquid chromatography-mass spectrometry combined method, a fluorescence immunoassay method and the like. However, these methods have the disadvantages of low detection sensitivity, low detection specificity, high detection cost, expensive instrument, complex operation, and the need for professional operators, and therefore, it is very important to establish a simple, fast, highly sensitive and highly selective method for clinical diagnosis and food safety analysis of sulfadimethoxine.
The photoelectrochemical process is a charge transfer process in which a substance having photoelectric activity absorbs a photon to place an electron in an excited state. Photoelectrochemical aptamer analysis is a novel analysis method based on photoelectrochemical sensing and a specific recognition technology of an aptamer to a target molecule. At present, no report of detecting sulfadimethoxine by adopting a photoelectrochemical analysis method is seen.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a photoelectrochemical sensor for detecting sulfadoxine and a detection method thereof, so that high specificity and high sensitivity detection of the sulfadoxine are realized, and a new method is provided for the fields of environmental monitoring, food safety and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a photoelectrochemical sensor for the detection of sulfadoxine, comprising: electrode, WS modified on electrode surface2Nanosheets and DNA aptamers;
the DNA aptamer and WS2The nanoplates are in intimate contact.
Preferably, the nucleotide sequence of the DNA aptamer is shown as SEQ ID NO. 1.
In a second aspect of the present invention, there is provided a method for preparing the above-mentioned photoelectrochemical sensor, comprising the steps of:
(1) pretreating the electrode;
(2) let WS be2Modifying the nanosheets to the surface of the electrode;
(3) dropping DNA aptamer onto WS2And standing and reacting the electrode surface modified by the nano-sheets for 1-hour under a humid condition to obtain the nano-sheet modified electrode.
Preferably, in the step (1), the electrode pretreatment method comprises: the electrode is firstly cleaned by mixed liquor of ethanol and sodium hydroxide, then cleaned by acetone and secondary water and dried.
Preferably, in the step (1), the electrode is an ITO electrode.
Preferably, in step (2), WS2The operation of modifying the nanosheet to the surface of the electrode is as follows: let WS be2Adding the nano-sheet into deionized water to prepare WS2Nano meterDropping the water dispersion on the surface of the electrode, and drying by infrared lamp irradiation.
Preferably, in the step (3), the wet condition refers to a humidity of 99%.
In a third aspect of the present invention, there is provided a method for detecting sulfadoxine by using the above photoelectrochemical sensor, comprising the steps of:
(1) dripping sulfadoxine and DNase I with different concentrations on the surface of the photoelectrochemical sensor, incubating, carrying out the combination of the sulfadoxine and the DNA aptamer, and carrying out the circulating combination-hydrolysis reaction by catalyzing and hydrolyzing the DNA aptamer with the DNase I to release the sulfadoxine;
(2) and detecting the sulfadimethoxine by establishing a relation curve of the photocurrent and the concentration of the sulfadimethoxine.
Preferably, in step (1), the incubation conditions are: the reaction was allowed to stand at 37 ℃ and 99% humidity for 2 hours.
Preferably, in the step (2), the relationship between the photocurrent and the sulfadoxine concentration is as follows: 89.49log c +380, where I is photocurrent in nA; c is the concentration of sulfadimethoxine, and the unit is nmol/L; r is 0.9962, linear range is 10 nmol/L-1 pmol/L.
The invention has the beneficial effects that:
(1) the detection method is simple, and the sulfadimethoxine can be detected only by performing three times of modification on the surface of the ITO electrode.
(2) The photoelectrochemical sensor realizes the miniaturization of instruments, is easy to operate and has low detection cost.
(3) The detection sensitivity of the invention is very high, and the detection limit can reach pmol/L; moreover, the invention is based on the specific binding action of the sulfadoxine and the DNA aptamer thereof, and has high detection selectivity.
Drawings
FIG. 1: the principle of the detection method of the present invention is schematically illustrated.
FIG. 2: WS2Comparing the transmission electron microscope spectra before and after stripping; (A) before stripping; (B) and (5) stripping.
FIG. 3: of differently-modified electrodesAn electrochemical impedance spectrum; (a) ITO, (b) WS2/ITO，(c)DNA/WS2/ITO，(d)DNA-C/WS2/ITO。
FIG. 4: photoelectric response curves of different modified electrodes; (a) ITO, (b) WS2/ITO，(c)DNA/WS2/ITO，(d)DNA-C/WS2/ITO。
FIG. 5: photoelectric response curves under different concentrations of sulfadimethoxine; curves a-i represent sulfadoxine at concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10nmol/L, respectively.
FIG. 6: a linear fit curve of photocurrent to sulfadoxine concentration.
FIG. 7: the photoelectric response histogram under different antibiotic conditions, the antibiotic concentration is all 1 nmol/L; Δ I ═ I2-I1。I2Is DNA-C/WS2Photocurrent of/ITO (DNA-C/WS)2ITO means here DNA/WS dripped with different kinds of antibiotics2ITO electrode), I1Is DNA/WS2Photocurrent of/ITO.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, the existing detection method of sulfadimethoxine generally has the defects of low detection sensitivity, weak detection specificity, high detection cost, expensive instruments, complex operation, requirement of professional operators and the like. Based on this, the object of the present invention is to provide a simple, fast, highly sensitive and highly selective method for detecting sulfadimethoxine.
The invention is based on DNA aptamers, WS2The sensitive detection of the sulfadimethoxine is realized by a photoelectrochemical method of isothermal cycle amplification catalyzed by a nanosheet and a DNase. The detection principle of the invention is as follows: firstly, dripping tungsten sulfide nanosheets on the surface of a glassy carbon electrode, and then adsorbing a DNA nucleic acid aptamer on the surface of the electrode. Since DNA aptamers and WS2The close contact of the nano-sheets hinders the DNA hydrolysis of DNase I and WS2The photoelectrochemical activity of the nanosheets has an inhibiting effect, so that the photocurrent of the electrode is reduced. However, when sulfadoxine, which is the target molecule of the aptamer, is present, sulfadoxine binds to the DNA aptamer, causing the DNA aptamer to transform from WS2The surface of the nano-sheet is desorbed, so that the DNA aptamer can be hydrolyzed by DNase I, thereby releasing sulfadimethoxine. The released sulfadimethoxine can be combined with the DNA aptamers on the surface of the electrode, so that a series of desorption-hydrolysis-desorption-hydrolysis processes are triggered, the number of the DNA aptamers on the surface of the electrode is obviously reduced, and the photocurrent is obviously enhanced. The invention utilizes the specific recognition function of DNA aptamer and sulfadoxine, realizes the specific and sensitive detection of the target compound sulfadoxine by DNase I catalyzed signal isothermal amplification (figure 1).
The invention firstly designs a photoelectrochemical sensor for detecting sulfadimethoxine, wherein the photoelectrochemical sensor is formed by modifying the surface of an ITO electrode with WS2Nanosheets and DNA aptamers; the DNA aptamer is a sulfadimethoxine aptamer, can be specifically combined with sulfadimethoxine, and has the following base sequence:
5′-GAGGGCAACGAGTGTTTATAGA-3′；(SEQ ID NO.1)
the photoelectrochemical sensor designed by the invention can be used for detecting sulfadimethoxine, and the detection method comprises the following steps:
(1) uniformly mixing sulfadimethoxine solutions with different concentrations and DNase I in equal volume, and dropwise adding the mixture to a modified electrode (modified with WS)2Nanosheet and DNA aptamer), incubating, performing binding of sulfadoxine and DNA aptamer, and DNase I catalytically hydrolyzing DNA aptamer, releasing sulfadoxine, performing a cyclic binding-hydrolysis reaction.
(2) And performing photoelectrochemical detection, establishing a relation between the photocurrent and the concentration of the sulfadimethoxine, and detecting the content of the sulfadimethoxine in the sample to be detected by using the relation.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available.
Wherein WS2The nano-sheet is prepared by an ultrasonic-assisted chemical stripping method. Precursor WS2Powder material and sulfadimethoxine were purchased from Sigma-Aldrich, usa. Chemical stripping reagents sodium cholate and other antibiotics were purchased from alatin reagent (shanghai) ltd.
Example 1: preparation of photoelectrochemical sensor for detecting sulfadimethoxine
1. Electrode pretreatment:
firstly, the ITO conductive glass is divided into 5 multiplied by 1cm2Ultrasonically cleaning with ethanol/NaOH mixed solution (1:1) for 30min, cleaning with acetone and secondary water for 30min, and air drying at room temperature.
2.WS2Preparing a nano sheet:
preparation of WS by ultrasonic-assisted chemical stripping2Nanosheets. Weighing 1 g of WS2Adding the powder material into 200mL of deionized water, and performing ultrasonic dispersion for 30 minutes to obtain WS2An aqueous dispersion. Then 2 g sodium cholate was added to WS2The aqueous dispersion was stirred for 30 minutes. Subsequently, the dispersion was sonicated for 24 hours. Centrifuging at 3000 rpm for 30min, discarding the precipitate, and collecting the solution. The collected solution was centrifuged at 12000 rpm to collect a precipitate. The precipitate was washed three times with deionized water and dried under vacuum at 60 ℃. WS2The comparative transmission electron microscopy patterns before and after the peeling are shown in FIG. 2.
3.WS2Preparing a nanosheet aqueous dispersion:
weighing 40mg WS2Adding the nanosheet into 10mL of deionized water, and ultrasonically dispersing for 30 minutes to obtain WS with the concentration of 4mg/mL2A dispersion of nanoplatelets.
4.WS2Preparing a modified electrode:
10 microliter of 4mgWS of/mL2And dropwise adding the dispersion liquid of the nanosheets onto the surface of the pretreated ITO electrode, and drying under infrared lamps and the like. After washing with 10mmol/L Tris-HCl (pH7.4) buffer solution, WS is obtained2The ITO electrode modified by the nanosheets is marked as: WS2/ITO。
DNA aptamer immobilization:
using WS2Having the property of adsorbing DNA, 10. mu.l of 1. mu. mol/L DNA aptamer was dropped onto WS2The reaction was carried out on an ITO surface by standing in a humid environment (humidity 99%) at a temperature of 37 ℃ for 2 hours. After washing with 10mmol/L Tris-HCl (pH7.4) buffer solution, the resulting aptamer-modified electrode was labeled: DNA/WS2and/ITO. Namely, the photoelectrochemical sensor for detecting the sulfadimethoxine is prepared.
Example 2: detection of sulfadimethoxine
1. A sulfadoxine and aptamer combination reaction, and a DNase I catalyzed isothermal amplification reaction:
and uniformly mixing the sulfadoxine solution with different concentrations with 100unit/mL DNase I solution in equal volume. Then, 10. mu.l of this mixed solution was added dropwise to DNA/WS2The ITO electrode surface was left to stand and reacted for 2 hours in a humid environment (humidity 99%) at a temperature of 37 ℃. After washing with 10mmol/L Tris-HCl (pH7.4) buffer, the resulting electrode was labeled: DNA-C/WS2/ITO。
2. Photoelectrochemical detection:
an electrochemical workstation is used as a signal acquisition instrument, a 500W xenon lamp is used as a visible light source (an ultraviolet filtering lens is additionally arranged), a saturated calomel electrode is used as a reference electrode, a platinum column electrode is used as a counter electrode, 10mM Tris-HCl (pH7.4) buffer solution is used as detection liquid, a-0.3V voltage is used as a working voltage, an I-t technology is adopted to carry out detection research on an object to be detected, the relation between photocurrent and sulfadimethoxine concentration is established, the linear range is 10 nmol/L-1 pmol/L, the correction curve is I (89.49 logc + 380) (nA, nmol/L, R (0.9962)), and the detection limit is 0.31 pmol/L.
3. Stability test:
using the same method and detection conditions, with DNA-C/WS2ITO as working power1nmol/L of sulfadimethoxine is detected extremely, and the relative standard deviation is 4.38%, which shows that the method has good reproducibility. Using a DNA-C/WS strand2The ITO electrode was tested 10 times in succession, giving a relative standard deviation of 3.28%, indicating a very good reproducibility of the method. DNA-C/WS2The ITO sensor was stored at 4 c for 2 weeks and gave a current response of 94.38% of the original response, indicating a good stability of the method.
4. Detection selectivity experiment:
different antibiotic solutions (the concentration is 1nmol/L) and 100unit/mL DNase I solution are mixed uniformly in equal volume. Then, 10. mu.l of this mixed solution was added dropwise to DNA/WS2The ITO electrode surface was left to stand and reacted for 2 hours in a wet environment at a temperature of 37 ℃. Washing with 10mmol/L Tris-HCl (pH7.4) buffer solution to obtain electrodes (DNA-C/WS) modified with different antibiotics2ITO). And carrying out photoelectrochemical detection, and testing the photoelectric response condition under different antibiotic conditions, wherein the result is shown in figure 7.
As can be seen from FIG. 7, the method can specifically detect sulfadimethoxine, and has good detection selectivity.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> photoelectrochemical sensor for detecting sulfadimethoxine and detection method thereof
<130>2018
<160>1
<170>PatentIn version 3.5
<210>1
<211>22
<212>DNA
<213> DNA aptamer
<400>1
gagggcaacg agtgtttata ga 22
Claims (7)
1. A photoelectrochemical sensor for the detection of sulfadimethoxine, comprising: electrode, WS modified on electrode surface2Nanosheets and DNA aptamers;
the DNA aptamer and WS2The nano sheets are in close contact;
the nucleotide sequence of the DNA aptamer is shown as SEQ ID NO. 1;
the photoelectrochemical sensor is prepared by the following steps:
(1) pretreating the electrode;
(2) let WS be2The nanosheet is modified to the surface of the electrode: let WS be2Adding the nano-sheet into deionized water to prepare WS2The nano-sheet water dispersion is dripped on the surface of an electrode and is irradiated and dried by an infrared lamp;
(3) dropping DNA aptamer onto WS2Standing and reacting the electrode surface modified by the nanosheets for 1-3h under a humid condition to obtain the nano-particles;
the WS2The nano-sheet is prepared by the following method:
weighing WS2Adding the powder material into deionized water, and performing ultrasonic dispersion to obtain WS2An aqueous dispersion; then adding sodium cholate to WS2Stirring in the aqueous dispersion; then, the dispersion is treated by ultrasonic wave, centrifuged, and the precipitate is discarded, and the solution is collected; the collected solution was centrifuged, the precipitate was collected, washed with deionized water, and dried in vacuo.
2. The photoelectrochemical sensor according to claim 1, wherein in the step (1), the electrode is pretreated by: the electrode is firstly cleaned by mixed liquor of ethanol and sodium hydroxide, then cleaned by acetone and secondary water and dried.
3. The photoelectric chemical sensor according to claim 1, wherein in step (1), the electrode is an ITO electrode.
4. The photoelectric chemical sensor according to claim 1, wherein in the step (3), the wet condition is a humidity of 99%.
5. Method for the detection of sulfadoxine with the photoelectrochemical sensor according to any of the claims 1 to 4, characterised in that it comprises the following steps:
(1) dripping sulfadoxine and DNase I with different concentrations on the surface of the photoelectrochemical sensor, incubating, carrying out the combination of the sulfadoxine and the DNA aptamer, and carrying out the circulating combination-hydrolysis reaction by catalyzing and hydrolyzing the DNA aptamer with the DNase I to release the sulfadoxine;
(2) and detecting the sulfadimethoxine by establishing a relation curve of the photocurrent and the concentration of the sulfadimethoxine.
6. The method according to claim 5, wherein in the step (1), the incubation conditions are as follows: standing and reacting for 2 hours at 37 ℃ and 99% humidity.
7. The method of claim 5, wherein: in the step (2), the relationship curve of the photocurrent and the sulfadoxine concentration is as follows: 89.49log c +380, where I is photocurrent in nA; c is the concentration of sulfadimethoxine, and the unit is nmol/L; r is 0.9962, linear range is 10 nmol/L-1 pmol/L.
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| CN109459477B (en) * | 2018-09-27 | 2020-09-15 | 曲阜师范大学 | Assembling method and application of double-signal amplified biosensor |
| CN109828006B (en) * | 2019-02-27 | 2019-12-03 | 山东农业大学 | A kind of the optical electro-chemistry sensor and its detection method of detection methylation RNA |
| CN109752434B (en) * | 2019-03-05 | 2021-04-16 | 江西科技师范大学 | Electrochemical sensor for rapidly and selectively detecting quercetin and preparation method thereof |
| CN115792231B (en) * | 2022-11-04 | 2023-07-25 | 中拓生物有限公司 | DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction |
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