Propetamphos colorimetric aptamer sensor, preparation method and application thereof

Abstract

The application discloses a propetamphos colorimetric aptamer sensor, a preparation method and application thereof, and belongs to the technical field of pesticide residue detection. The nucleic acid aptamer sensor comprises a signal probe and a capture probe. The signal probe is a gold platinum palladium nanoparticle modified nucleic acid aptamer 1, and the sequence of the nucleic acid aptamer 1 is shown as SEQ ID NO:1. The capture probe is a ferroferric oxide nanoparticle modified nucleic acid aptamer 2, and the sequence of the nucleic acid aptamer 2 is shown as SEQ ID NO:2. The nucleic acid aptamer sensor can specifically recognize and combine propetamphos, has high sensitivity, is simple to operate, does not need to perform complex pretreatment on a sample to be detected, has low detection cost, is fast in detection, and has low requirement on a detection instrument.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pesticide residue detection, and particularly relates to a profenofos colorimetric aptamer sensor, a preparation method and application thereof. BACKGROUND

[0002] Organophosphorus pesticides are a class of organic compound pesticides containing phosphorus elements, and are one of the commonly used pesticides in crops. They mainly protect crops and plants from the invasion of pests, diseases and weeds, and improve the yield of crops. However, excessive organophosphorus pesticides will be left in vegetables and fruits, causing food safety problems, and will penetrate into soil and water, causing environmental pollution. Therefore, the detection of organophosphorus pesticides has attracted more and more attention. Traditional organophosphorus detection methods include high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS) and the like, but they need complex pretreatment, large equipment support, and are expensive, which is difficult to meet the current detection requirements. Therefore, it is necessary to establish a rapid and sensitive detection strategy for organophosphorus pesticides. SUMMARY

[0003] The application provides a profenofos colorimetric aptamer sensor, which comprises a signal probe and a capture probe; the signal probe is selected from a gold platinum palladium nanoparticle (AuPtPd NPs) modified nucleic acid aptamer 1, namely AuPtPd-aptamer 1; and the capture probe is selected from a ferroferric oxide nanoparticle (Fe3O4) modified nucleic acid aptamer 2, namely Fe3O4-aptamer 2. The sequence of the nucleic acid aptamer 1 is shown in SEQ ID NO: 1, and the 5' end of the nucleic acid aptamer 1 can be modified with a mercapto group (-SH); the sequence of the nucleic acid aptamer 2 is shown in SEQ ID NO: 2, and the 5' end of the nucleic acid aptamer 2 can be modified with an amino group (-NH2). When the above-mentioned sensor is used for profenofos detection, an "AuPtPd-aptamer 1 / profenofos / Fe3O4-aptamer 2" sandwich structure can be formed in the reaction system.

[0004] The sequence of the nucleic acid aptamer 1 is:

[0005] 5'-AAGCTTGCTTTATAGCCTGCAGCGATTCTTGATCGGAAAAGGCTGAGAGCTACGC-3' (SEQ ID NO: 1)

[0006] The sequence of the nucleic acid aptamer 2 is:

[0007] 5'-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT-3' (SEQ ID NO: 2)

[0008] The AuPtPd-aptamer 1 can be prepared by mixing a gold platinum palladium nanoparticle solution with a nucleic acid aptamer 1 solution, incubating at 37℃, centrifuging, and washing the product to obtain the AuPtPd-aptamer 1. The volume ratio of the gold platinum palladium nanoparticle solution to the nucleic acid aptamer 1 solution is selected from 25-50:1-3.

[0009] The Fe3O4-aptamer 2 can be prepared by mixing a COOH-Fe3O4 solution with a nucleic acid aptamer 2 solution, incubating at 4℃, and magnetically separating to obtain a precipitate, i.e., the Fe3O4-aptamer 2. The volume ratio of the COOH-Fe3O4 solution to the nucleic acid aptamer 2 solution is selected from 5-10:2-4.

[0010] The gold platinum palladium nanoparticle solution can be prepared by suspending and dispersing gold platinum palladium nanoparticles in a PBS solution, and the concentration is selected from 0.01-0.02 mM. The COOH-Fe3O4 solution can be prepared by suspending and dispersing COOH-Fe3O4 in a PBS solution, and the concentration is selected from 60-100 μg / mL. Before preparing the COOH-Fe3O4 solution, the COOH-Fe3O4 can be activated by dissolving COOH-Fe3O4 in deionized water, adding EDS and NHS, and activating at 4℃ for 1 h, and then discarding the supernatant to obtain COOH-Fe3O4 activated by carboxyl. The nucleic acid aptamer 1 solution can be prepared by suspending and dispersing nucleic acid aptamer 1 in a PBS solution, and the concentration is selected from 10 μM. The nucleic acid aptamer 2 solution can be prepared by suspending and dispersing nucleic acid aptamer 2 in a PBS solution, and the concentration is selected from 10 μM. The PBS solution can have a specification of 0.01 M, pH = 7.4.

[0011] The gold platinum palladium nanoparticles can be prepared by the following method:

[0012] The HAuCl4 solution, the K2PtCl4 solution, the K2PdCl4 solution, the HCl solution, and poloxamer are mixed. The poloxamer is completely dissolved by ultrasonic treatment, and an aqueous ascorbic acid solution is added to the mixture and stirred at room temperature. After the reaction is completed, centrifugation and washing are performed to obtain the gold platinum palladium nanoparticles.

[0013] The HAuCl4 solution, the K2PtCl4 solution, the K2PdCl4 solution, and the HCl solution can be prepared by dissolving HAuCl4, K2PtCl4, K2PdCl4, and HCl in water, respectively, and the concentrations can be 0.02 M, 0.02 M, 0.02 M, and 6.0 M, respectively.

[0014] The application provides application of the nucleic acid aptamer sensor in detection of profenofos.

[0015] The application provides a detection method of profenofos, and the steps are as follows:

[0016] The capture probe is added into the sample solution to be detected, and then the signal probe is added to form a sandwich structure; the incubation product is separated by magnetism, and H2O2 solution, TMB solution and NaAc-HAc buffer solution are sequentially added into the product precipitate, and then incubation is carried out at room temperature; if the solution changes from colorless to blue, it indicates that profenofos exists in the sample solution to be detected; the absorbance of the sample solution to be detected is measured, and the concentration of profenofos can be obtained by comparing with a standard curve.

[0017] The use amount of the capture probe, the sample solution to be detected and the signal probe can be selected from the following: for example, when the sample solution to be detected is 200 μL, the addition amount of the capture probe can be selected from 80-150 μL, and the addition amount of the signal probe can be selected from 80-150 μL. In actual application, the use amount of each component can be enlarged or reduced according to the above proportion relationship. However, it should be declared that the implementation of the above technical solution and the obtaining of the technical effect thereof do not completely depend on the above use amount range of each component, for example, when the addition amount of the capture probe is 60 μL, 70 μL or 79 μL, the technical solution of the application can still be implemented, and the technical effect claimed by the application can be achieved.

[0018] The H2O2 solution and the TMB solution can be prepared by dissolving H2O2 and TMB in water respectively, and the concentrations can be preferably 100 mM and 8 mM respectively.

[0019] The standard curve can be constructed by the following method:

[0020] Different concentrations of profenofos sample solutions to be detected are prepared. The capture probe is added into the sample solution to be detected, and then the signal probe is added to form a sandwich structure; the incubation product is separated, and then H2O2, TMB and NaAc-HAc buffer solution are sequentially added; after reaction at room temperature, the absorbance value of the reaction solution at 652 nm is measured. The concentration of profenofos is taken as the abscissa, and the ultraviolet absorbance is taken as the ordinate, so as to construct a standard curve of profenofos concentration-ultraviolet absorbance.

[0021] The application has the following beneficial effects:

[0022] The nucleic acid aptamer sensor of the application can specifically recognize and combine profenofos, has high sensitivity, is simple to operate, does not need to perform complex pretreatment on the sample to be detected, has low detection cost, is fast in detection, and has low requirement on a detection instrument. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The schematic diagram of the detection principle of the nucleic acid aptamer sensor is shown in the figure;

[0024] Figure 2 The color development diagram of the detection results of different concentrations of profenofos is shown in the figure, and the concentrations from left to right are 0.1 μg / L, 25 μg / L, 50 μg / L, 100 μg / L and 200 μg / L;

[0025] Figure 3 The concentration-profenofos-UV absorbance standard curve is shown in the figure;

[0026] Figure 4 The detection result diagram of different organophosphorus pesticides is shown in the figure;

[0027] Figure 5 The color development diagram of the TMB / H2O2 system catalyzed by gold platinum palladium and gold platinum rhodium is shown in the figure, and the color on the left is lighter, and the color on the right is darker. DETAILED DESCRIPTION

[0028] (I) About the nucleic acid aptamer sensor

[0029] The nucleic acid aptamer sensor provided by the application comprises a signal probe and a capture probe; the signal probe is a gold platinum palladium nanoparticle modified nucleic acid aptamer 1, namely AuPtPd-aptamer 1; and the capture probe is a ferroferric oxide nanoparticle (Fe3O4) modified nucleic acid aptamer 2, namely Fe3O4-aptamer 2. In a specific implementation case, the 5' end of the nucleic acid aptamer 1 is modified with a thiol group (-SH), and the 5' end of the nucleic acid aptamer 2 is modified with an amino group (-NH2), and the sequences after modification are as follows:

[0030] The nucleic acid aptamer 1 modified with a thiol group is as follows:

[0031] 5'-SH-AAGCTTGCTTTATAGCCTGCAGCGATTCTTGATCGGAAAAGGCTGAGAGCTACGC-3'

[0032] The nucleic acid aptamer 2 modified with an amino group is as follows:

[0033] 5'-NH2-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT-3'

[0034] The above-mentioned AuPtPd-aptamer 1 is prepared by the following method:

[0035] Mix 500 μL gold platinum palladium nanoparticle solution (concentration 0.01 mM; solvent PBS, 0.01 M, pH = 7.4) with 30 μL aptamer 1 solution (10 μM; solvent PBS, 0.01 M, pH = 7.4), incubate at 37℃ overnight, centrifuge at 8000 r / min for 10 min, wash the precipitate with alcohol, and obtain AuPtPd-aptamer 1.

[0036] The gold platinum palladium nanoparticle is prepared by the following method:

[0037] Mix 0.5 mL HAuCl4 solution (0.02 M, solvent water), 2.5 mL K2PtCl4 solution (0.02 M, solvent water), 2.5 mL K2PdCl4 solution (0.02 M, solvent water), 120 μL HCl solution (6.0 M, solvent water), and 100.8 mg Pluronic F127. Ultrasonically dissolve the Pluronic F127, add 4.0 mL ascorbic acid aqueous solution (0.1 M) to the mixture, and stir at room temperature for 1 h. After the reaction is completed, centrifuge at 8000 r / min for 10 min, and wash / centrifuge with ultrapure water for three times in succession, and obtain the gold platinum palladium nanoparticle.

[0038] The Fe3O4-aptamer 2 is prepared by the following method:

[0039] Mix 100 μL COOH-Fe3O4 solution (concentration 60 μg / mL; solvent PBS, 0.01 M, pH = 7.4) with 40 μL aptamer 2 solution (10 μM; solvent PBS, 0.01 M, pH = 7.4), incubate at 4℃ overnight, magnetically separate the precipitate, and obtain Fe3O4-aptamer 2.

[0040] The COOH-Fe3O4 can be activated before use, and the activation step is as follows:

[0041] Take 100 μL COOH-Fe3O4 and dissolve in 400 mL deionized water, add 100 μL 200 mmol / L EDS and 100 μL 200 mmol / L NHS, activate at 4℃ for 1 h, then discard the supernatant, resuspend in PBS solution to form a COOH-Fe3O4 solution with a concentration of 60-100 μg / mL, and reserve for use. After this step, the carboxyl groups on the Fe3O4 are fully activated.

[0042] (II) Detection principle

[0043] The present application provides the detection principle of the above-mentioned aptamer sensor, as follows:

[0044] Both gold-platinum-palladium aptamer 1 (AuPtPd-aptamer 1) and iron(III) oxide aptamer 2 (Fe3O4-aptamer 2) possess bromophos binding sites, but the binding sites differ. When both are added to the test solution, they both capture the target and form a sandwich structure "Fe3O4-aptamer-2 / bromophos / AuPtPd-aptamer-1". AuPtPd exhibits strong nanozyme activity and catalytic ability, capable of catalyzing the TMB / H2O2 system, causing a color reaction and turning the solution blue, thus amplifying and indicating the signal. Since the amount of AuPtPd NPs-aptamer-1 is directly proportional to the bromophos content, the absorbance value can be detected using a UV spectrophotometer, and the concentration of bromophos in the test solution can be obtained by comparing with a standard curve. Iron(III) oxide is magnetic, facilitating separation and reducing color interference. This invention provides... Figure 1 This is to assist those skilled in the art in understanding the above detection principles.

[0045] (III) On the plotting of standard curves

[0046] Profenofos analyte solutions with concentrations of 0.1 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, and 200 μg / L were prepared. 100 μL of the capture probe was added to the analyte solution, and the mixture was incubated at room temperature for 15 min. Then, 100 μL of the signal probe was added to form a sandwich structure. Utilizing the magnetic properties of Fe3O4, magnetic separation was performed using a magnet. 200 μL of H2O2 (100 mM), 120 μL of TMB (8 mM), and 400 μL of NaAc-HAc buffer (pH = 4.5) were added sequentially to the product precipitate. After reacting at room temperature for 20 min, the absorbance signal of the resulting solution at 652 nm was rapidly recorded using UV-Vis spectroscopy. The detection results for different concentrations of profenofos analyte are shown below. Figure 2 As shown, with the increase of profenofos concentration, the color of the liquid in each tube changed from light blue to dark blue. The absorbance test results are shown in Table 1.

[0047] Table 1

[0048] Prothiofos concentration (pg / L) Absorbance values (a.o.) Deviation 0.1 0.11763 0.01264 25 0.21294 0.01644 50 0.3475 0.01934 100 0.5634 0.02193 200 0.8956 0.01986

[0049] A standard curve of profenofos concentration versus ultraviolet absorbance was constructed with the concentration of profenofos on the x-axis and the ultraviolet absorbance on the y-axis, as follows: Figure 3 As shown. The nucleic acid aptamer sensor of the present invention exhibits a linear range of 0.1–200 μg / L, with a detection limit as low as 0.1 μg / L. The fitting equation is y = 0.0038x + 0.1545, R0 2 =0.9922.

[0050] Other terms used in the present application have meanings generally understood by those of ordinary skill in the art unless otherwise defined. The present application is described in further detail below in conjunction with specific examples and with reference to the data. The following examples are merely illustrative of the present application and do not in any way limit the scope of the present application.

[0051] Example 1

[0052] Verification of the accuracy of the aptamer sensor:

[0053] A solution of profenofos with a concentration of 100 μg / L (solvent: acetone) was prepared as the sample solution to be detected.

[0054] 100 μL of Fe3O4-aptamer 2 was added to 200 μL of the sample solution to be detected, and incubated at room temperature for 15 min, and then 100 μL of AuPtPd-aptamer 1 was added to form a sandwich structure; the incubation product was magnetically separated, and 200 μL of H2O2 (100 mM), 120 μL of TMB (8 mM) and 400 μL of NaAc-HAc buffer (pH = 4.5) were sequentially added to the product precipitate; incubation was carried out at room temperature for 15 min; the solution changed from colorless to blue, indicating the presence of profenofos in the sample solution to be detected. The absorbance at 652 nm was determined by UV-visible spectroscopy, and the absorbance was 0.5267 a.o. The concentration of profenofos was obtained by comparing with the standard curve, which was 97.94 μg / L. The concentration was almost the same as the prepared concentration of the sample to be detected, which indicated that the aptamer sensor of the present application had high accuracy.

[0055] Example 2

[0056] Verification of the specificity of the aptamer sensor:

[0057] The following sample solutions to be detected were prepared: fenthion solution, diazinon solution, chlorpyrifos solution and phoxim solution, each with a concentration of 100 μg / L (solvent: acetone). The method of Example 1 was used to incubate with the aptamer sensor to verify the specificity of the aptamer sensor.

[0058] The test results are shown in Table 2.

[0059] Table 2

[0060] Organophosphates Absorbance values (a.o.) Prothiofos 0.5267 Fenthion 0.181 Diazinon 0.15165 Chlorpyrifos 0.111 Phoxim 0.08245

[0061] As can be seen from Table 2, the absorbance value of profenofos is significantly higher than that of other organophosphorus pesticides. Figure 4 According to the data in Table 2, the absorbance of profenofos is more intuitively displayed. Therefore, the aptamer sensor of the present application has high specificity for profenofos.

[0062] Example 3

[0063] Catalytic effect of nanomaterials:

[0064] 30 μL of 1 μM AuPtPd (gold platinum palladium) and AuPtRh (gold platinum rhodium) were respectively taken and added into 200 μL H2O2 (100 mM), 120 μL TMB (8 mM), 400 μL NaAc-HAc buffer (pH = 4.5); incubated at room temperature for 15 min; the absorbance at 652 nm was determined by UV-visible spectroscopy.

[0065] The determination results are shown in Table 3:

[0066] Table 3

[0067] Nanomaterials Absorbance values (a.o.) Gold platinum palladium 1.1524 Gold platinum rhodium 0.7362

[0068] At a concentration of 1 μM, AuPtPd and AuPtRh have obvious difference in the ability of catalyzing TMB / H2O2, as shown in Table 3, the color development of the reaction system catalyzed by AuPtPd is more obvious, which indicates that the catalytic activity of AuPtPd is much better than that of AuPtRh, thereby improving the detection sensitivity of the aptamer sensor of the present application. Figure 5

[0069] The above description is only the preferred embodiments of the present application, and is not intended to limit the present application in other forms, any skilled person in the art can modify or change the above disclosed technical content into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made on the basis of the technical essence of the present application to the above embodiments, which does not deviate from the technical solution content of the present application, still belongs to the protection scope of the technical solution of the present application.​

Claims

1. A propetamphos colorimetric aptamer sensor, characterized in that, The signal probe is a gold platinum palladium nanoparticle modified nucleic acid aptamer 1, namely AuPtPd-aptamer 1; and the capture probe is a ferroferric oxide nanoparticle modified nucleic acid aptamer 2, namely Fe3O4-aptamer 2; wherein the sequence of the nucleic acid aptamer 1 is shown as SEQ ID NO: 1, and the sequence of the nucleic acid aptamer 2 is shown as SEQ ID NO:

2.

2. The aptamer sensor of claim 1, wherein, The AuPtPd-aptamer 1 is prepared by mixing a gold platinum palladium nanoparticle solution with a nucleic acid aptamer 1 solution, incubating at 37 DEG C, centrifuging, and washing the product to obtain the AuPtPd-aptamer 1.

3. The aptamer sensor of claim 1, wherein, The Fe3O4-aptamer 2 is prepared by mixing a COOH-Fe3O4 solution with a nucleic acid aptamer 2 solution, incubating at 4 DEG C, and magnetically separating to obtain a precipitate, namely the Fe3O4-aptamer 2.

4. The aptamer sensor of claim 2, wherein, The gold platinum palladium nanoparticle is prepared by mixing HAuCl4 solution, K2PtCl4 solution, K2PdCl4 solution, HCl solution and poloxamer, ultrasonically dissolving the poloxamer, adding an ascorbic acid aqueous solution to the mixture, and stirring at room temperature; after the reaction is completed, centrifuging, and washing to obtain the gold platinum palladium nanoparticle. The COOH-Fe3O4 solution is prepared by dissolving COOH-Fe3O4 in deionized water, adding EDS and NHS, activating at 4 DEG C for 1 h, then discarding the supernatant, and resuspending in a PBS solution to form the COOH-Fe3O4 solution.

5. The aptamer sensor of claim 3, wherein, 6. The nucleic acid aptamer sensor according to any one of claims 1-5 for detecting profenofos. The nucleic acid aptamer sensor according to any one of claims 1-5. The steps are as follows:

7. A profenofos test reagent or kit characterized in that, The capture probe is added to the sample solution to be tested, incubated at room temperature, and then the signal probe is added to form a sandwich structure; the incubation product is magnetically separated, and H2O2 solution, TMB solution and NaAc-HAc buffer are sequentially added to the product precipitate, and incubated at room temperature; if the solution changes from colorless to blue, it indicates that profenofos exists in the sample solution to be tested; 8. A method for detecting profenofos using the colorimetric nucleic acid aptamer sensor of claim 1, characterized in that, The absorbance of the sample solution to be tested is measured, and the concentration of profenofos can be obtained by comparing with the standard curve. The standard curve is constructed by: Different concentrations of profenofos test solution are prepared; the capture probe is added to the test solution, mixed and incubated at room temperature, and then the signal probe is added to form a sandwich structure; the incubation product is separated, and H2O2, TMB and NaAc-HAc buffer are sequentially added; after reaction at room temperature, the absorbance value of the reaction solution at 652 nm is measured; the concentration of profenofos is taken as the abscissa, and the ultraviolet absorbance is taken as the ordinate to construct the profenofos concentration-ultraviolet absorbance standard curve.

9. The detection method according to claim 8, characterized in that, ​ ​

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