Construction of a long-afterglow-based aptamer sensor and its application in carbendazim detection

Abstract

The application discloses a kind of long afterglow base ratio type aptamer sensor construction and carbendazim detection application, belong to spectral analysis technical field.The application is based on zinc germanate manganese-doped long afterglow material (ZGM) and the methylene blue with fluorescent characteristic constructs the ratio type aptamer sensor without complementary chain, using the overlapping characteristics of ultraviolet absorption spectrum and ZGM phosphorescence emission spectrum of methylene blue, through resonance energy transfer mechanism, realize the ZGM phosphorescence quenching and methylene blue fluorescence enhancement of probe, and then construct the long afterglow ratio type aptamer sensor without complementary chain.The characteristic emission peak of the long afterglow ratio type aptamer sensor is at the green phosphorescence emission of 542nm and the fluorescence emission of 700nm, the probe has the advantages of without in-situ excitation, without background interference and exogenous interference, high specificity, can sensitively, accurately and specifically detect CBZ residue in food sample.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of spectral analysis, and relates to construction of a long-afterglow aptamer sensor and application of the long-afterglow aptamer sensor in carbendazim detection. BACKGROUND

[0002] Carbendazim (CBZ) is a widely used broad-spectrum fungicide in the prevention of agricultural fungal diseases. However, the benzimidazole ring in the molecular structure of carbendazim has high stability and is prone to long-term residue in the environment. Excessive use of carbendazim can lead to accumulation of carbendazim in agricultural products and the ecological environment, and enrichment through the food chain, which poses a potential risk to human health and ecological safety, such as causing spermatogenic disorders, testicular deformities, kidney toxicity, and embryonic toxicity in mammals. The World Health Organization has listed carbendazim as a dangerous chemical, and the European Union has identified it as an endocrine disruptor. Many countries have implemented restriction measures: the European Union has banned carbendazim from being used in the ninth category of biological fungicide products and has limited its application in the fields of grains and feed; Brazil has completely banned the use of carbendazim. Therefore, it is of great significance to establish a rapid, sensitive, and accurate method for detecting carbendazim residues to protect food safety and ecological environment safety.

[0003] At present, the detection techniques for carbendazim mainly include high-performance liquid chromatography, gas chromatography / mass spectrometry, fluorescence immunoassay, surface-enhanced Raman spectroscopy, and electrochemical method. Among them, the fluorescence analysis method is favored due to its simple operation, rapid response, and high sensitivity. Especially, the introduction of specific recognition groups and the detection strategy of reading ratio self-calibration can effectively improve the detection specificity and reduce the influence of factors such as probe concentration fluctuation, instrument difference, operation error, and environmental interference on the detection results, significantly improving the detection accuracy. However, the existing fluorescence detection methods generally rely on external light source excitation, which is difficult to avoid the interference of autofluorescence generated by complex sample matrix. Although the fluorescence aptamer sensing system has shown outstanding advantages in specific detection, it mostly needs the joint action of aptamer complementary chains, which is complicated to prepare and increases the cost. Therefore, it is of great significance to design a simple construction strategy and develop a new method for detecting carbendazim that has flexibility, selectivity, and high sensitivity, and can effectively avoid autofluorescence and external interference for complex sample analysis. SUMMARY

[0004] [TECHNICAL PROBLEM]

[0005] Most fluorescence analysis methods are difficult to avoid the self-fluorescence interference of complex sample matrix due to the need for continuous excitation light, resulting in lower accuracy of detection results. When using long afterglow materials for detection, the characteristics of not needing continuous excitation can effectively avoid matrix interference and improve signal-to-noise ratio. Current long afterglow detection probes mostly rely on single luminescence signal, and the detection results are easily affected by factors such as instrument parameters, detection time and probe concentration, resulting in errors. Although the long afterglow probe based on double signals shows the advantage of self-calibration reading, it still needs to be constructed by complementary strands, and there are problems such as high preparation cost and complex method.

[0006] [Technical scheme]

[0007] In order to solve the above problems, the present application provides a long afterglow ratio type aptamer probe without complementary chain, which can realize the luminescence "turn off" at 542nm and the luminescence "turn on" at 700nm at the same time, detect carbendazim through the significant change of luminescence intensity ratio at two wavelengths, and effectively remove the background fluorescence of the sample and external interference. The ratio type sensor constructed by the present application has the advantages of good stability, no self-fluorescence interference of the sample, no influence of external conditions, high sensitivity, good selectivity and the like, and has wide prospect in the detection of food carbendazim.

[0008] The first object of the present application is to construct a method for long afterglow ratio type aptamer sensor without complementary chain, comprising the following steps:

[0009] (1) Preparation of long afterglow nanoparticles ZGM: Dissolve zinc salt and manganese salt in water, then add acid, Na2GeO3 solution, mix, and then add CTAB to perform hydrothermal reaction; after the reaction is completed, cool, centrifuge to collect the precipitate, wash, dry, grind, and obtain long afterglow nanomaterial ZGM;

[0010] (2) Preparation of amino-functionalized long afterglow nanoparticles ZGM: Disperse the long afterglow nanomaterial ZGM obtained in step (1) in an alkali solution, stir and react, then centrifuge to collect the precipitate, wash, dry, disperse in N,N-dimethylformamide solution, slowly add 3-aminopropyltriethoxysilane, heat and react, then centrifuge to collect the precipitate, wash, dry, and obtain amino-functionalized ZGM;

[0011] (3) Modification of surface carbendazim aptamer: Disperse the amino-functionalized ZGM obtained in step (2) and Sulfo-SMCC reagent in HEPES buffer, slowly shake and incubate at room temperature, then wash with HEPES buffer, solid-liquid separation, and collect the precipitate; then add the precipitate to a carbendazim aptamer solution, mix and incubate for a period of time, then centrifuge, collect the solid precipitate, wash, and obtain ZGM-apt;

[0012] (4) adding the ZGM-apt obtained in step (3) into a methylene blue (MB) solution, mixing to form a mixed solution, incubating for a period of time, centrifuging and collecting the precipitate after the incubation to obtain a long-afterglow ratio type aptamer sensor, denoted as ZGM-apt-MB.

[0013] In an embodiment of the present application, in step (1), the concentration of the Na2GeO3 solution is 0.2-0.5 mol / L; specifically, 0.4 mol / L can be selected.

[0014] In an embodiment of the present application, in step (1), the molar ratio of the zinc salt, Na2GeO3 and the manganese salt is (1-5):1:(0.002-0.005). Specifically, 2:1:0.005 can be selected.

[0015] In an embodiment of the present application, in step (1), the zinc salt is a soluble salt of zinc, specifically, Zn(NO3)2 can be selected.

[0016] In an embodiment of the present application, in step (1), the manganese salt is a soluble salt of manganese, specifically, Mn(NO3)2 can be selected.

[0017] In an embodiment of the present application, in step (1), the acid specifically can be concentrated nitric acid.

[0018] In an embodiment of the present application, in step (1), the pH is adjusted to 8.5-10.5 for the hydrothermal reaction.

[0019] In an embodiment of the present application, in step (1), the hydrothermal reaction is performed at 200-220 ℃ for 3-6 h.

[0020] In an embodiment of the present application, in step (1), the molar ratio of CTAB to Na2GeO3 is (0.04-0.05):1.

[0021] In an embodiment of the present application, in step (2), the alkali solution specifically can be a 4-6 mmol / L NaOH solution.

[0022] In an embodiment of the present application, in step (2), the amount of the alkali solution relative to the long-afterglow nanomaterial ZGM is 1 mL / mg.

[0023] In an embodiment of the present application, in step (2), the amount of N,N-dimethylformamide relative to the long-afterglow nanomaterial ZGM is 0.5-1.0 mL / mg; specifically, 0.8 mL / mg can be selected.

[0024] In an embodiment of the present application, in step (2), the volume ratio of N,N-dimethylformamide solution to 3-aminopropyl triethoxysilane APTES reagent is 1:0.005.

[0025] In an embodiment of the present application, in step (2), the mass ratio of 3-aminopropyl triethoxysilane to long afterglow nanomaterial ZGM is (3-5):1.

[0026] In an embodiment of the present application, in step (2), the heating reaction condition is refluxing at 80°C for 24h.

[0027] In an embodiment of the present application, in step (3), the mass ratio of long afterglow nanomaterial ZGM to Sulfo-SMCC reagent is 1:(0.2-0.5); specifically, 1:0.3 can be selected.

[0028] In an embodiment of the present application, in step (3), the concentration of HEPES buffer is 10mM, and the pH is 7.2-8.5.

[0029] In an embodiment of the present application, in step (3), the carbendazim aptamer solution is a PBS buffer solution containing a concentration of 0.5nmol / mL-3.0nmol / mL carbendazim aptamer. Specifically, 2.5nmol / mL can be selected as the concentration of carbendazim aptamer.

[0030] In an embodiment of the present application, in step (3), the use amount of carbendazim aptamer relative to amino-functionalized ZGM is 0.5nmol / mg-3.0nmol / mg. Specifically, 2.5nmol / mg can be selected.

[0031] In an embodiment of the present application, in step (3), the sequence of the aptamer of carbendazim is 5'-SH-GGGCACACAACAACCGATGGTCCAGCCACCCGAATGACCAGCCCACCCGCCACCCCGCG.

[0032] In an embodiment of the present application, in step (3), the incubation is performed at 30-37°C with shaking for 3-18h.

[0033] In an embodiment of the present application, in step (4), the methylene blue (MB) solution is an aqueous solution of methylene blue.

[0034] In an embodiment of the present application, in step (4), the final concentration of MB in the mixed solution is 10μg / mL-20μg / mL.

[0035] In an embodiment of the present application, in step (4), the final concentration of ZGM-apt in the mixed solution is 1mg / mL.

[0036] In one embodiment of the present application, in step (4), the MB is first dissolved in pure water with pH 7.0-8.5, and then added to the ZGM-apt.

[0037] In one embodiment of the present application, in step (4), the mass ratio of ZGM-apt to MB is 1:(0.01-0.02).

[0038] In one embodiment of the present application, in step (4), the oscillation incubation is carried out at 30-37℃ for 2-8h.

[0039] In one embodiment of the present application, in step (4), the centrifugation speed is 8000-12000rpm; and the washing time of the precipitate is 5-15min.

[0040] In one embodiment of the present application, the method specifically comprises:

[0041] (1) Preparation of long-afterglow nanoparticles ZGM and modification of surface with carbendazim aptamer:

[0042] ① Preparation of long-afterglow nanoparticles ZGM: Zn(NO3)2 and Mn(NO3)2 were dissolved in ultrapure water, and concentrated nitric acid and Na2GeO3 solution were added under stirring, and CTAB was added, and the pH was adjusted to 9.0 with ammonia water; stirring for 0.5-1h, then transferred to a polytetrafluoroethylene-lined stainless steel hydrothermal reactor, heated at 220℃ for 6h; after cooling to room temperature, the precipitate was collected by centrifugation, and the precipitate was washed with anhydrous ethanol and ultrapure water for 2-3 times, freeze-dried, and the solid was ground with a mortar to obtain ZGM long-afterglow nanomaterial;

[0043] ② Amino functionalization of long-afterglow nanoparticles ZGM: the ZGM powder obtained above was ultrasonically dispersed in NaOH solution, and stirred vigorously at room temperature for 12-24h, and after the reaction was completed, the precipitate was collected by centrifugation, washed with ultrapure water for 2-3 times, and dried, and then dispersed in N,N-dimethylformamide solution, and 3-aminopropyltriethoxysilane reagent was slowly added, and heated to reflux and stirred at 70-90℃ oil bath for 12-24h, and after the reaction was completed, the precipitate was collected by centrifugation, washed with anhydrous ethanol for 2-3 times, and vacuum dried to obtain amino-functionalized ZGM solid powder;

[0044] ③ Modification of surface carbendazim aptamer: disperse amino-functionalized ZGM and Sulfo-SMCC reagent in HEPES buffer, slowly shake and incubate at room temperature for 2-3 h, then wash with HEPES buffer for three times, and finally collect the precipitate to obtain the precipitate; mix the precipitate with the PBS buffer of carbendazim aptamer, shake and incubate at 30-37℃ for 3-18 h, collect the solid precipitate after reaction, wash with HEPES buffer for three times, and finally collect the precipitate to obtain the ZGM-apt.

[0045] ④ Mix the ZGM-apt obtained in step ③ with MB solution, shake well, and shake and incubate at 30-37℃ for 2-8 h, and then collect the precipitate by centrifugation to obtain the detection probe ZGM-apt-MB.

[0046] The second object of the application is the long-afterglow ratio type aptamer sensor ZGM-apt-MB prepared by the method.

[0047] The third object of the application is to provide a carbendazim ratio type spectral analysis method, which uses the long-afterglow ratio type aptamer sensor ZGM-apt-MB without complementary chain modification as a detection probe.

[0048] The fourth object of the application is to provide a method for quantitatively detecting carbendazim, which comprises the following processes:

[0049] Mix a series of known concentration of carbendazim standard solution with the long-afterglow ratio type aptamer sensor ZGM-apt-MB and incubate for a period of time, and then measure the luminescence intensity at 542±5 nm and 700±5 nm by phosphorescence and fluorescence modes on a fluorescence spectrometer, to obtain luminescence intensity I1 and I2, and calculate the luminescence intensity ratio I1 / I2; and construct a linear quantitative model with the concentration of the carbendazim standard sample and the luminescence intensity ratio I1 / I2.

[0050] In an embodiment of the application, the ZGM-apt-M is dispersed in pure water to obtain a ZGM-apt-MB solution; and the ZGM-apt-MB solution is added into the carbendazim standard solution in equal volume and mixed for incubation.

[0051] In an embodiment of the application, the concentration of the ZGM-apt-MB solution is 1-4 mg / mL -1 , and specifically 2 mg / mL -1 .

[0052] Specifically, the following steps are included:

[0053] ZGM-apt-MB solution was added into the carbendazim solution with different concentration gradient, and incubated at 30-37℃ for 40-60 min. Then the phosphorescence emission spectrum of each group of solution under 254 nm excitation and the fluorescence emission spectrum under 650 nm excitation were determined by fluorescence spectrometer, and the standard curve was established with the concentration of carbendazim as the horizontal coordinate and the ratio of the luminescence intensity of the detection probe ZGM-apt-MB at 542 nm and at 700 nm as the vertical coordinate.

[0054] In an embodiment of the present application, the conditions of mixed incubation are preferably: pH 7.5, 37℃, and incubation for 55 min with shaking.

[0055] In an embodiment of the present application, the concentration of the carbendazim standard solution is 0-500 ng / mL. Specifically, 0 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 400 ng / mL, or 500 ng / mL can be selected.

[0056] Long afterglow nanomaterials have unique energy storage properties. After absorbing excitation light, they can store energy and continuously release phosphorescence after the excitation source is removed. This property enables in-situ excitation-free detection. By avoiding continuous irradiation of external light sources, the material can effectively suppress the interference of autofluorescence generated by complex sample matrix, significantly improving the signal-to-noise ratio of the detection signal. However, current detection probes based on long afterglow materials mostly use single signal output mode. This detection method is easily affected by factors such as instrument performance fluctuations and probe concentration differences, leading to unstable detection results and the need for strict control of complex experimental conditions. Therefore, developing a long afterglow ratio sensor with low cost, simple preparation process, simplified operation process, and stable optical performance has become a key direction to break through the bottleneck of existing technology. Based on zinc germanate manganese-doped long afterglow material (ZGM) and methylene blue with fluorescence properties, the present application constructs a complementary strand-free ratio-type aptamer sensor. By utilizing the overlapping characteristics of the ultraviolet absorption spectrum of methylene blue and the phosphorescence emission spectrum of ZGM, and through the resonance energy transfer (FRET) mechanism, the phosphorescence quenching of the probe ZGM and the fluorescence enhancement of methylene blue are realized. Then, a complementary strand-free long afterglow ratio-type aptamer sensor is constructed for high-sensitivity detection of carbendazim (CBZ) residues in food, belonging to the field of spectral analysis technology. The characteristic emission peaks of the constructed long afterglow ratio-type aptamer sensor are green phosphorescence emission at 542 nm and fluorescence emission at 700 nm. This probe has the advantages of no in-situ excitation, no background and exogenous interference, and high specificity, and can accurately and specifically detect CBZ residues in food samples. It shows good application potential in food carbendazim detection. This research not only provides a new idea for the design of ratio-type fluorescent aptamer sensors, but also provides an innovative technical solution for on-site rapid detection of pollutants.

[0057] [Advantages]

[0058] (1) The sensor probe can realize ratio detection of CBZ at two wavelengths of 542 nm phosphorescence emission and 700 nm fluorescence emission, combined with specific aptamer, which can effectively avoid the influence of external factors and probe concentration during detection, and improve the accuracy of detection results. In addition, the sensor also has high affinity and selectivity of aptamer to CBZ, which further improves the sensitivity and accuracy of detection.

[0059] (2) The method has strong scalability, and can realize ratio detection of different target objects combined with different aptamers.

[0060] (3) The sensor does not need multiple luminophores, does not need to regulate the proportion of signal emitting groups, and does not need complementary chains, so that the sensor construction is more simple and the cost is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 It is the principle diagram of the long afterglow ratio aptamer sensor for detecting carbendazim in Example 1;

[0062] Figure 2 It is the luminescence spectrum of the long afterglow ratio aptamer sensor prepared in Example 1;

[0063] Figure 3 It is the zeta potential diagram of ZGM, ZGM-OH, ZGM-NH2, ZGM-apt, MB, and ZGM-apt-MB prepared in Example 1;

[0064] Figure 4 It is the phosphorescence luminescence spectrum (A), fluorescence luminescence spectrum (B) and standard curve (C) of the long afterglow ratio aptamer sensor under different concentrations of carbendazim in Example 2;

[0065] Figure 5 It is the specificity analysis diagram of the ratio phosphorescence sensor for different pesticides and cations in Example 3.

[0066] Figure 6 It is the influence of different methylene blue dosages selected in Example 5 on the luminescence of ZGM-apt-MB.

[0067] Figure 7 It is the influence of different pH conditions selected in Example 5 on the luminescence of ZGM-apt-MB.

[0068] Figure 8 It is the influence of pH and reaction time under the detection conditions of Example 6 on the luminescence of the aptamer sensor. 542 / I 700Effects of the application of the application. DETAILED DESCRIPTION

[0069] The following describes preferred embodiments of the application, and it should be understood that the embodiments are for better explaining the application and are not intended to limit the application.

[0070] Example 1

[0071] A preparation method of a long-afterglow ratio aptamer sensor probe for detecting carbendazim, specifically comprising the following steps:

[0072] (1) Synthesis of ZGM long-afterglow nanomaterials:

[0073] 8 mL of 0.5 mol / L Zn(NO3)2 aqueous solution and 0.232 mL of 0.043 mol / L Mn(NO3)2 aqueous solution, 22 mL of ultrapure water were mixed to form a colorless transparent solution 1. Under vigorous stirring, 600 μL of concentrated nitric acid and 5 mL of 0.4 mol / L Na2GeO3 aqueous solution were slowly added dropwise to the solution 1 to form a colorless transparent solution 2 (wherein the molar ratio of zinc salt, Na2GeO3, and manganese salt is 2:1:0.005); 32 mg (0.088 mmol) of cetyltrimethylammonium bromide (CTAB) was added to the solution 2, and the pH of the solution was adjusted to 9.0 by adding ammonia water, and the solution gradually changed from colorless transparent and clear to white turbidity. After stirring at room temperature for 60 min, it was poured into a polytetrafluoroethylene-lined hydrothermal reactor, and reacted at 220℃ for 6 h. After the reaction was completed and cooled, the mixture was centrifuged and the solid product was collected. The solid product was washed with ethanol and water alternately for three times, and freeze-dried overnight. After sufficient drying, the solid product was ground into powder using a agate mortar to obtain ZGM long-afterglow nanomaterials.

[0074] (2) Amino functionalization of ZGM:

[0075] 10 mg of the ZGM long-afterglow nanomaterials obtained in step (1) was dispersed in 10 mL of 5 mmol / L NaOH aqueous solution, ultrasonicated for 30 min, and stirred vigorously at room temperature for 24 h, and the lower precipitate was collected by centrifugation, washed with ultrapure water for three times, and dried, and then dispersed in 8 mL of N,N-dimethylformamide solution (dispersion concentration of 1.25 mg / mL), and 40 μL (37.84 mg) of 3-aminopropyltriethoxysilane reagent was slowly added dropwise, and heated to reflux in an 80℃ oil bath for 24 h, and after the reaction was completed, the product was collected by centrifugation, washed with N,N-dimethylformamide solution once, and then washed with anhydrous ethanol twice, and dried to obtain the amino-functionalized ZGM long-afterglow nanomaterials.

[0076] (3) Modification of surface carbendazim aptamer:

[0077] The amino-functionalized ZGM long afterglow nanomaterial obtained in step (2) (10 mg) was dispersed in 10 mL of HEPES buffer, 3.0 mg of Sulfo-SMCC reagent was added, and incubated at 37°C for 2 h. After the reaction was completed, the lower precipitate was collected by centrifugation, and the precipitate was washed with HEPES buffer (10 mM in concentration, pH 7.2-8.5) three times. Finally, the precipitate was collected by centrifugation to obtain precipitate 1. 10 mL of 2.5 nmol / mL of a carbendazim aptamer (25 nmol) in PBS buffer (the sequence structure of the carbendazim aptamer is: 5'-SH-GGGCACACAACAACCGATGGTCCAGCCACCCGAATGACCAGCCCACCCGCCACCCCGCG) was added to the precipitate 1 to obtain a mixed solution 2. The mixed solution 2 was incubated at 37°C for 12 h. After the reaction was completed, the lower precipitate was collected by centrifugation, and the lower precipitate was washed with HEPES buffer three times. Finally, the precipitate was collected by centrifugation to obtain precipitate 2, which was ZGM-apt.

[0078] (4) The ZGM-apt obtained in step (3) was added to 10 mL of 20 μg / mL MB aqueous solution, mixed, and the final concentration of ZGM-apt was 1 mg / mL, the final concentration of MB was 20 μg / mL, the pH was 7.5, and the mixture was fully shaken and incubated at 37°C for 6 h. The precipitate was collected by centrifugation to obtain a long afterglow ratio aptamer sensor probe for detecting carbendazim, denoted as ZGM-apt-MB. The concentration of the ZGM-apt-MB was 2 mg / mL. -1 The ZGM-apt-MB was resuspended in pure water to obtain a detection probe ZGM-apt-MB solution for subsequent detection of carbendazim.

[0079] The performance of the obtained long afterglow ratio aptamer sensor probe (ZGM-apt-MB) was tested, and the test results are as follows:

[0080] Figure 2 The long afterglow ratio aptamer sensor probe (ZGM-apt-MB) was obtained; from Figure 2 It can be seen that the phosphorescence emission peak of the synthesized long afterglow nanomaterial at 542 nm under 254 nm excitation is in a quenching state, and there is a fluorescence emission peak at 700 nm under 650 nm excitation.

[0081] Figure 3 The zeta potential diagrams of ZGM-NH2, ZGM-apt, MB and ZGM-apt-MB are shown in Figure 2; from Figure 3 It can be seen that the zeta potential of the material gradually changes, verifying the successful synthesis of the material.

[0082] Example 2 Application of probe ZGM-apt-MB to detect carbendazim

[0083] A long afterglow ratio type aptamer spectral analysis method for detecting carbendazim without complementary chain is a ratio type detection method based on two materials of green light phosphorescence emission (542 nm) long afterglow nanomaterial ZGM and red light fluorescence emission (700 nm) methylene blue, which realizes ratio detection of carbendazim in food.

[0084] Construction of the long afterglow ratio type aptamer spectral analysis method for carbendazim: a series of standard solutions of carbendazim with different concentrations were added to the ZGM-apt-MB solution obtained in Example 1, and the mixture was incubated at 37℃ under oscillation for 55 min at pH 7.5, and then the phosphorescence spectrum and fluorescence spectrum of each group of solutions were tested by a fluorescence spectrometer (F7000), and each group of solutions was tested three times, so as to obtain the standard curve between the ratio of the recovered phosphorescence intensity at 542 nm and the quenched fluorescence intensity at 700 nm of the detection probe and the concentration of carbendazim.

[0085] Specific test process of the long afterglow ratio type aptamer sensor probe (ZGM-apt-MB) for detecting carbendazim:

[0086] 50 μL of CBZ standard solution (0 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL) was mixed with 250 μL of ZGM-apt-MB (2 mg mL -1 ) obtained in Example 1, and Tris-HCl buffer (pH=7.5) was added to make up to 500 μL. The mixture was incubated at 37℃ under gentle oscillation for 55 min. Then, the obtained solution was uniformly dispersed, and the luminescence intensity of the solution at 542 nm and 700 nm was measured respectively in phosphorescence and fluorescence modes on an F-7000 fluorescence spectrometer, so as to construct the linear relationship with the concentration of CBZ standard solution as the abscissa and the luminescence intensity ratio (I 542 / I 700 ) as the ordinate.

[0087] Figure 4 The luminescence spectrum and standard curve of the long afterglow ratio type aptamer sensor for different concentrations of carbendazim; from Figure 4 it can be seen that: the ratio of the recovered luminescence intensity I 542 at 542 nm from ZGM-apt-MB and the quenched luminescence intensity (I 700 ) at 700 nm and the concentration of CBZ present a good linear relationship in the concentration range of 2.27 ng / mL to 500 ng / mL, and the detection limit is 0.68 ng / mL.

[0088] Specificity study of Example 3

[0089] The ZGM-apt-MB solution obtained in Example 1 was mixed with CBZ, methyl parathion (MPP), azoxystrobin (AZ), pyrimethanil (PMT), hexaconazole (HEX), temephos (TEM), bromophos (BRO), propamocarb (PRO), and tebuconazole (Teb), respectively. After mixing, the final concentration of ZGM-apt-MB was 1 mg / mL, and the final concentration of CBZ, methyl parathion (MPP), azoxystrobin (AZ), pyrimethanil (PMT), hexaconazole (HEX), temephos (TEM), bromophos (BRO), propamocarb (PRO), and tebuconazole (Teb) was 100 ng / mL. The mixture was incubated at 37°C for 55 min, and then the luminescence spectrum and luminescence intensity of each group of solutions were tested by a fluorescence spectrometer (F7000).

[0090] Figure 5 The specificity analysis chart of different pesticides and cations; from Figure 5 It can be seen that: only in the solution where the target substance carbendazim exists, I 542 / I 700 changed significantly, in other groups of solutions without the target substance, I 542 / I 700 There was no change, which indicated that this method had high specificity and was suitable for specific detection of CBZ.

[0091] Accuracy study of Example 4

[0092] After the actual food sample was pretreated, the sample extract was obtained. The same concentration of CBZ standard was prepared in different sample extracts, mixed with the ZGM-apt-MB obtained in Example 1, and incubated at 37°C for 55 min. Then the luminescence spectrum and luminescence intensity of each group of solutions were tested by a fluorescence spectrometer (F7000).

[0093] Table 1 shows the accuracy study, and the recovery rate of CBZ in five actual food samples is in the range of 93.3%-107.1%. This result indicates that this method has high accuracy and can be applied to sensitive detection of CBZ in actual samples.

[0094] Table 1 Determination and analysis of CBZ in food samples

[0095]

[0096] Selection of methylene blue dosage of Example 5

[0097] Adjusting the final concentration of MB in step (4) in Example 1 to 10 μg / mL, 15 μg / mL, pH 7.0, and keeping other conditions the same as in Example 1, the corresponding ZGM-apt-MB solution was obtained.

[0098] Figure 6 For the influence of methylene blue concentration on the luminescence of ZGM-apt-MB, it can be seen that 20 μg / mL MB after reaction can have the best phosphorescence quenching and the strongest fluorescence, and the luminescence intensity ratio is the smallest, so the condition of 20 μg / mL is selected for reaction.

[0099] Adjusting the pH to 7.5 and 8.0 in step (4) in Example 1, and keeping other conditions the same as in Example 1, the corresponding ZGM-apt-MB solution was obtained.

[0100] Figure 7 For the influence of pH on the luminescence of ZGM-apt-MB, it can be seen that the reaction condition of pH 7.5 can obtain the best phosphorescence quenching and the strongest fluorescence, and the luminescence intensity ratio is the smallest, so the condition of pH 7.5 and 20 μg / mL MB is selected for reaction.

[0101] Example 6: Selection of pH and reaction time

[0102] Adjusting the pH of Tris-HCl used for detection in Example 2 to 7.2 and 8.0, and keeping other conditions the same as in Example 2, the detection results of ZGM-apt-MB on CBZ were as follows:

[0103] Figure 8 For the influence of pH on the I 542 / I 700 of the aptamer sensor; from Figure 8 it can be seen that: changing the detection pH to 7.2 and 8.0, the detection results I 542 / I 700 obtained are less than I 542 / I 700 obtained when the pH is 7.5. It can be seen that when the pH of Tris-HCl used for detection is 7.5, the detection is more sensitive.

[0104] Adjusting the incubation time in Example 2 to 15 min, 35 min, and 75 min, and keeping other conditions the same as in Example 2, the detection results of ZGM-apt-MB on CBZ were as follows:

[0105] Figure 8 For the influence of reaction time on the I 542 / I 700 of the aptamer sensor; from Figure 8It can be seen that: when the reaction time is changed to 15, 35, 75 min, the detection results I 542 / I 700 are all less than I 542 / I 700 obtained when the reaction time is 55 min. It can be seen that the detection is more sensitive when incubated for 55 minutes.

[0106] The above provided examples are not intended to limit the scope covered by the present application, and the described steps are not intended to limit the execution order. The improvements of the present application made by those skilled in the art in combination with the existing common knowledge are also within the protection scope defined by the claims of the present application.

Claims

1. A method for preparing a long-persistence ratio-type aptamer sensor for detecting carbendazim, characterized in that, Includes the following steps: (1) Preparation of long afterglow nanoparticles ZGM: Zinc salt and manganese salt were dissolved in water, followed by acid and Na2GeO3 solution, mixed well, and then CTAB was added for hydrothermal reaction; after the reaction was completed, the mixture was cooled, centrifuged to collect the precipitate, washed, dried, and ground to obtain long afterglow nanomaterial ZGM. (2) Preparation of amino-functionalized long afterglow nanoparticles ZGM: The long afterglow nanomaterial ZGM obtained in step (1) was dispersed in an alkaline solution, stirred and reacted, then centrifuged to collect the precipitate, washed and dried, and then dispersed in N,N-dimethylformamide solution. 3-aminopropyltriethoxysilane was slowly added and heated to react. After the reaction was completed, the precipitate was centrifuged to collect the precipitate, washed and dried to obtain amino-functionalized ZGM. (3) Modification of surface carbendazim aptamer: The amino-functionalized ZGM obtained in step (2) was dispersed in HEPES buffer and incubated with slow shaking at room temperature. After the incubation, the mixture was washed with HEPES buffer, and the solid and liquid were separated and the precipitate was collected. The precipitate was then added to the carbendazim aptamer solution, mixed and incubated for a period of time. After incubation, the solid precipitate was collected by centrifugation, washed, and ZGM-apt was obtained. (4) Add the ZGM-apt obtained in step (3) to the methylene blue solution, mix, form a mixture, incubate for a period of time, centrifuge after incubation, collect the precipitate, and obtain the long afterglow ratio aptamer sensor.

2. The method according to claim 1, characterized in that, In step (1), the concentration of the Na2GeO3 solution is 0.2-0.5 mol / L; the molar ratio of zinc salt, Na2GeO3, and manganese salt is (1-5):1:(0.002-0.005); and the molar ratio of CTAB to Na2GeO3 is (0.04-0.05):

1.

3. The method according to claim 1, characterized in that, In step (2), the dosage of N,N-dimethylformamide relative to long-afterglow nanomaterial ZGM is 0.5-1.0 mL / mg; the mass ratio of 3-aminopropyltriethoxysilane to long-afterglow nanomaterial ZGM is (3-5):

1.

4. The method according to claim 1, characterized in that, In step (3), the mass ratio of long afterglow nanomaterial ZGM to Sulfo-SMCC reagent is 1:0.2-0.

5.

5. The method according to claim 1, characterized in that, In step (3), the dosage of carbendazim aptamer relative to amino-functionalized ZGM is 0.5 nmol / mg-3.0 nmol / mg.

6. The method according to claim 1, characterized in that, In step (4), the final concentration of methylene blue in the mixture is 10 μg / mL-20 μg / mL, and the mass ratio of ZGM-apt to methylene blue is 1:(0.01-0.02).

7. The long afterglow ratio aptamer sensor prepared by the method according to any one of claims 1-6.

8. A carbendazim ratio-based spectral analysis method, characterized in that, The method uses the long afterglow ratio aptamer sensor as described in claim 7 as a detection probe.

9. A method for quantitative detection of carbendazim, characterized in that, The process includes the following: A series of carbendazim standard solutions of known concentrations were mixed with a long-afterglow ratiometric aptamer sensor ZGM-apt-MB and incubated for a period of time. Then, the luminescence intensity at 542±5nm and 700±5nm was measured on a fluorescence spectrometer in phosphorescence and fluorescence modes, respectively, and the luminescence intensities I1 and I2 were obtained accordingly. The luminescence intensity ratio I1 / I2 was calculated. A linear quantitative model was constructed using the concentration of carbendazim standard samples and the luminescence intensity ratio I1 / I2.

10. The method according to claim 9, characterized in that, ZGM-apt-MB was dispersed in pure water to obtain concentrations of 1-4 mg / mL. -1 ZGM-apt-MB solution; Add an equal volume of ZGM-apt-MB solution to the carbendazim standard solution and incubate.

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