A fluorescent method for on-site detection of acetamiprid based on nanomagnetic beads

Abstract

The application discloses a kind of fluorescence methods for detecting acetamiprid on site based on nano magnetic beads, belong to analytical chemistry and biotechnology field.The method will be modified with biotin acetamiprid specific aptamer Apt probe fixed on the surface of modified with streptavidin magnetic beads MBs, when acetamiprid ACE exists, Apt and acetamiprid are combined to form stable MBs-Apt-ACE complex.Subsequently, labeled with fluorescent quantum dots QD cDNA is added, Apt that has been combined with acetamiprid cannot be combined with cDNA again, at this time, magnet is used to remove MBs, and the supernatant is detected by fluorescence.The higher the concentration of acetamiprid, the greater the fluorescence intensity of supernatant, so as to realize the detection of acetamiprid.

Description

Technical Field

[0001] This invention belongs to the fields of analytical chemistry and biotechnology, and specifically relates to a fluorescence method for on-site detection of acetamiprid based on magnetic nanobeads. Background Technology

[0002] Acetamiprid (ACE) is a neonicotinoid insecticide with the chemical formula C0. 10 H 11 ClN4, due to its advantages such as high efficiency, broad spectrum, low toxicity, and low cost, has been used to replace conventional highly toxic pesticides such as organophosphates and organochlorines in the treatment of fruits, vegetables, and other agricultural products. However, acetamiprid can accumulate in the body through the food chain over a long period of time, threatening human health and causing irreversible damage to the central nervous system. Therefore, it is essential to develop analytical methods for rapid on-site detection of acetamiprid.

[0003] Currently, the main methods for detecting pesticide residues in Chinese medicinal herbs fall into two categories. The first category is chromatography-mass spectrometry (GC-MS), such as liquid chromatography-tandem mass spectrometry (LC-MS), ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS), and ultra-fast liquid chromatography-time-of-flight mass spectrometry (UHPLC-ToF-MS). These methods offer high sensitivity and accuracy, but the equipment requires demanding operating conditions and specialized technicians, thus limiting their application. The second category is immunoassay, with enzyme-linked immunosorbent assay (ELISA) being the most commonly used. This method offers high sensitivity, strong specificity, and portability; however, due to the presence of antibodies and the bioenzyme HRP in its system, the cost is relatively high. Furthermore, the bioenzyme is susceptible to environmental factors such as temperature, and antibody preparation can exhibit batch-to-batch variations, which to some extent limits the practical application and promotion of ELISA.

[0004] In this method, nucleic acid aptamers are used as recognition elements. They have advantages such as high specificity, low cost, high affinity, and ease of chemical modification, and have been widely used in the field of rapid detection.

[0005] The magnetic nanobeads used in this method possess characteristics such as good fluidity, small size, strong attraction, and good biocompatibility. By modifying the surface of the magnetic beads with groups such as streptavidin and amino groups, and utilizing the binding principles of streptavidin-biotin and amino-carboxyl groups, the magnetic beads can capture target molecules such as nucleic acids, proteins, or peptides. More importantly, the aptamer-modified magnetic nanobeads can rapidly capture target analytes and separate them from the original detection system under the action of an external magnetic field, thereby reducing interference from other substances in the complex detection environment.

[0006] Quantum dots possess inherent fluorescence and offer advantages such as large specific surface area, high fluorescence quantum yield, and high photochemical stability. Furthermore, they are visually identifiable under a fluorescence microscope and have been widely applied in fields such as biosensing, in vivo and in vitro imaging, detection of heavy metal ions in the environment, and food safety testing. Combining quantum dots with magnetic beads to construct fluorescent aptamer sensors can effectively improve the sensor's specificity in identifying targets, making it suitable for the detection and analysis of acetamiprid. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a fluorescence-based method for the on-site detection of acetamiprid based on magnetic nanobeads. This method exhibits strong anti-interference capabilities, a simple analytical process, high specificity, and good stability, and can be used for rapid on-site detection of acetamiprid.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A fluorescence method for on-site detection of acetamiprid based on nanomagnetic beads: Biotin-modified acetamiprid-specific nucleic acid aptamer (Apt) probes are immobilized on the surface of streptavidin-modified magnetic beads (MBs). In the presence of acetamiprid (ACE), the nucleic acid aptamer binds to acetamiprid to form a stable magnetic bead-aptamer-acetamiprid complex. Subsequently, cDNA labeled with fluorescent quantum dots (QD) is added. The nucleic acid aptamer (Apt) that has already bound to acetamiprid can no longer bind to the cDNA. At this point, the magnetic beads (MBs) are removed using a magnet, and the fluorescence spectrum of the supernatant is measured, thus enabling the detection of different concentrations of acetamiprid.

[0010] A fluorescence method for on-site detection of acetamiprid based on magnetic nanobeads includes the following specific steps:

[0011] (1) Preparation of fluorescent quantum dot labeled cDNA complex: Take 50 μL of 100 nM biotin-modified cDNA and 50 μL of 100 nM streptavidin-modified QD solution and place them in a centrifuge tube. Incubate at 25℃ for 1 h to obtain fluorescent quantum dot labeled cDNA complex QD-cDNA.

[0012] (2) Preparation of magnetic bead-nucleic acid aptamer complex: Take 200 μL of WB buffer at pH 7.5 and wash streptavidin-modified magnetic beads MBs three times, and resuspend them in 100 μL of 10 mM PBST buffer at pH 7.4 to make the final concentration of magnetic beads 150 μg / mL; then add 50 μL of biotin-modified acetamiprid-specific nucleic acid aptamer Apt at a concentration of 1 μM, vortex for 30 s, and incubate at 25℃ for 1 h. At this time, the biotin modified on Apt combines with the streptavidin modified on MBs to form magnetic bead-nucleic acid aptamer MBs-Apt complex. After magnetic separation to remove the supernatant, add 200 μL of PBS-T buffer, wash three times to remove excess Apt in the supernatant, and resuspend in 100 μL of PBST buffer to obtain MBs-Apt solution.

[0013] (3) Fluorescence detection of acetamiprid: Take 100 μL of MBs-Apt solution prepared in step (2) and 20 μL of acetamiprid solution diluted with PBS buffer at pH 7.4 and place them in a centrifuge tube. Vortex for 10 s and incubate at 30℃ for 60 min. Then add 50 μL of QD-cDNA complex prepared in step (1), vortex to mix, and incubate at 25℃ for 60 min. Then separate MBs from supernatant with a magnet. Place the supernatant in a 96-well black microplate and use a multi-functional microplate reader to detect the fluorescence intensity of the supernatant.

[0014] The nucleotide sequence of the cDNA mentioned in step (1) above is: 5'-Biotin-TCTTCATAATATGGTGTCAG-3'.

[0015] The nucleotide sequence of the acetamiprid aptamer Apt mentioned in step (2) above is: 5'-Biotin-CTGACACCATATTATGAAGA-3'.

[0016] In the above methods:

[0017] The WB buffer (pH 7.5) formula is as follows: Weigh 1.2114g Tris, dissolve it in ultrapure water, add dilute HCl to adjust the pH to 7.5, then add 0.3722g EDTA and 0.117g NaCl, sonicate for 30s, and add ultrapure water to make up to 1 L.

[0018] The PBS buffer (pH 7.4) is formulated as follows: Weigh 8.0000 g of NaCl, 0.2013 g of KCl, 3.5814 g of Na2HPO4 and 0.2722 g of KH2PO4 and add ultrapure water to a final volume of 1 L.

[0019] The PBS-T buffer formulation is as follows: PBS buffer at pH 7.4 + 0.5 mL Tween-20.

[0020] The concentration of the acetamiprid solution mentioned in step (3) above is 20~55 μg / mL.

[0021] The above-mentioned fluorescence method for detecting acetamiprid based on magnetic nanobeads is applied to the detection of acetamiprid.

[0022] The technical principle of this invention is as follows:

[0023] This invention immobilizes a biotin-modified acetamiprid-specific aptamer, Apt, onto the surface of immobilized biotin-modified immobilized protein molecules (MBs). In the presence of acetamiprid ACE, Apt binds to acetamiprid to form a stable MBs-Apt-ACE complex. Subsequently, cDNA labeled with fluorescent quantum dots (QDs) is added. Apt, already bound to acetamiprid, can no longer bind to cDNA, at which point the MBs are removed using a magnet, and the supernatant is subjected to fluorescence detection. Higher acetamiprid concentrations result in greater fluorescence intensity in the supernatant, thus enabling the detection of acetamiprid. A schematic diagram of the above principle is shown below. Figure 1 .

[0024] In this method, nucleic acid aptamers are used as recognition elements, which not only ensures specific recognition of acetamiprid during detection but also reduces detection costs. The introduced magnetic nanobeads possess characteristics such as good fluidity, small size, strong adsorption, and good biocompatibility, making the operation simpler and faster. Furthermore, their large specific surface area can amplify the signal to a certain extent, improving the method's sensitivity. More importantly, their use significantly enhances the method's anti-interference capability. The quantum dots themselves exhibit fluorescence, possessing advantages such as large specific surface area, high fluorescence quantum yield, and high photochemical stability, which can improve the method's stability and sensitivity.

[0025] The advantages of this invention are:

[0026] 1. This invention utilizes acetamiprid-specific aptamers as biorecognition elements, ensuring the stability and selectivity of the detection method.

[0027] 2. This invention introduces magnetic beads and quantum dots to construct a fluorescent aptamer sensor, which can effectively improve the ease of operation, sensitivity and anti-interference ability of the method.

[0028] 3. This invention achieves highly specific detection of acetamiprid, is easy to operate, has strong anti-interference ability, and is suitable for on-site detection of acetamiprid.

[0029] 4. This method is a "signal-on" detection method, which can avoid the "false positive" results of "signal-off" methods. Attached Figure Description

[0030] Figure 1 A schematic diagram of the detection of acetamiprid using a fluorescence biosensing method based on magnetic nanobeads.

[0031] Figure 2 The present invention describes the fluorescence detection results of different concentrations of acetamiprid and establishes a standard working curve.

[0032] Figure 3 Verification of the specificity of the method of the present invention for detecting acetamiprid. Detailed Implementation

[0033] To make the content of this invention easier to understand, the technical solution of this invention will be further described below in conjunction with specific embodiments. However, the following examples are merely examples of this invention and do not represent the scope of protection of this invention. The scope of protection of this invention is determined by the claims.

[0034] Example 1

[0035] A fluorescence method for on-site detection of acetamiprid based on magnetic nanobeads includes the following specific steps:

[0036] (1) Preparation of QD-cDNA complex: Take 50 μL of 100 nM biotin-modified cDNA and 50 μL of 100 nM streptavidin-modified QD solution and place them in a centrifuge tube. Incubate at 25℃ for 1 h to obtain the complex QD-cDNA.

[0037] (2) Preparation of magnetic bead-nucleic acid aptamer complex: Take 200 μL of WB buffer at pH 7.5 and wash streptavidin-modified magnetic beads MBs three times, and resuspend them in 100 μL of 10 mM PBST buffer at pH 7.4 to make the final concentration of magnetic beads 150 μg / mL; then add 50 μL of biotin-modified acetamiprid-specific nucleic acid aptamer Apt at a concentration of 1 μM, vortex for 30 s, and incubate at 25℃ for 1 h. At this time, the biotin modified on Apt combines with the streptavidin modified on MBs to form magnetic bead-nucleic acid aptamer MBs-Apt complex. After magnetic separation to remove the supernatant, add 200 μL of PBS-T buffer, wash three times to remove excess Apt in the supernatant, and resuspend in 100 μL of PBST buffer to obtain MBs-Apt solution.

[0038] (3) Fluorescence detection of acetamiprid: Take 100 μL of MBs-Apt solution prepared in step (2) and 20 μL of acetamiprid solution diluted with PBS buffer at pH 7.4 and place them in a centrifuge tube. Vortex for 10 s and incubate at 30℃ for 60 min. Then add 50 μL of QD-cDNA complex prepared in step (1), vortex to mix, and incubate at 25℃ for 60 min. Then separate MBs from supernatant with a magnet. Place the collected supernatant in a 96-well black microplate and use a multi-functional microplate reader to detect the fluorescence intensity of the supernatant.

[0039] The nucleotide sequence of the cDNA mentioned in step (1) above is: 5'-Biotin-TCTTCATAATATGGTGTCAG-3'.

[0040] The nucleotide sequence of the acetamiprid aptamer Apt mentioned in step (2) above is: 5'-Biotin-CTGACACCATATTATGAAGA-3'.

[0041] In the above methods:

[0042] The WB buffer (pH 7.5) formula is as follows: Weigh 1.2114g Tris, dissolve it in ultrapure water, add dilute HCl to adjust the pH to 7.5, then add 0.3722g EDTA and 0.117g NaCl, sonicate for 30s, and add ultrapure water to make up to 1 L.

[0043] The PBS buffer (pH 7.4) is formulated as follows: Weigh 8.0000 g of NaCl, 0.2013 g of KCl, 3.5814 g of Na2HPO4 and 0.2722 g of KH2PO4 and add ultrapure water to a final volume of 1 L.

[0044] The PBS-T buffer formulation is as follows: PBS buffer at pH 7.4 + 0.5 mL Tween-20.

[0045] Example 2

[0046] In Example 1, step (3) involved adding acetamiprid solution at concentrations of 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, and 55 μg / mL, respectively. The remaining steps were the same as in Example 1. Fluorescence detection of different concentrations of acetamiprid was achieved. The detection results are shown below. Figure 2 It can be seen that as the concentration of acetamiprid increases, the fluorescence intensity value F of the system also increases.

[0047] A standard curve was established by analyzing the relationship between the fluorescence intensity F and the concentration of acetamiprid. Figure 2This method enables the quantitative detection of acetamiprid. The limit of detection (LOD) of this method was calculated using 3σ / S (where σ represents the standard deviation of the signal obtained from multiple measurements of the blank test solution, and S is the slope of the fitted linear equation), yielding a theoretical detection limit of 0.90 μg / mL.

[0048] Example 3 Specificity Verification

[0049] In Example 1, step (3) of the acetamiprid solution was replaced with solutions of five common pesticide residues—atrazine, phoxim, thiamethoxam, chlorpyrifos, and imidacloprid—each at a concentration of 200 μg / mL. The acetamiprid concentration was 20 μg / mL. This verified the specificity of the method of the present invention. The detection results are shown in […]. Figure 3 It can be seen that even when the concentration of interfering pesticide residues is 10 times that of acetamiprid, their fluorescence intensity changes are still significantly lower than those of acetamiprid. The results indicate that the method established in this application has good specificity for the detection of acetamiprid.

[0050] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A fluorescence method for on-site detection of acetamiprid based on magnetic nanobeads, characterized in that: Biotin-modified acetamiprid-specific nucleic acid aptamer probes were immobilized on the surface of streptavidin-modified magnetic beads. When the target analyte acetamiprid was present, the nucleic acid aptamer specifically bound to acetamiprid, forming a stable magnetic bead-aptamer-acetamiprid complex. Subsequently, cDNA labeled with fluorescent quantum dots was added. The nucleic acid aptamer that had already bound acetamiprid could not bind to the cDNA. At this point, the magnetic beads were removed using a magnet, and the supernatant was subjected to fluorescence spectroscopy to detect acetamiprid. The fluorescence method includes the following specific steps: (1) Preparation of fluorescent quantum dot labeled cDNA complex: 50 μL of 100 nM biotin-modified cDNA and 50 μL of 100 nM streptavidin-modified fluorescent quantum dot QD solution were placed in a centrifuge tube and incubated at 25 °C for 1 h to obtain fluorescent quantum dot labeled cDNA complex QD-cDNA. (2) Preparation of magnetic bead-nucleic acid aptamer complex: Take 200 μL of WB buffer at pH 7.5 and wash the streptavidin-modified magnetic beads three times, and resuspend them in 100 μL of 10 mM PBST buffer at pH 7.4 to make the final concentration of magnetic beads 150 μg / mL; then add 50 μL of biotin-modified acetamiprid-specific nucleic acid aptamer Apt at a concentration of 1 μM, vortex for 30 s, and incubate at 25 °C for 1 h. At this time, the biotin modified on Apt combines with the streptavidin modified on the magnetic beads to form the magnetic bead-nucleic acid aptamer complex MBs-Apt. After magnetic separation to remove the supernatant, add 200 μL of PBST buffer and wash three times to remove excess Apt in the supernatant. Resuspend in 100 μL of PBST buffer to obtain the magnetic bead-nucleic acid aptamer complex MBs-Apt solution. (3) Fluorescence detection of acetamiprid: Take 100 μL of MBs-Apt solution prepared in step (2) and 20 μL of acetamiprid solution diluted with PBS buffer at pH 7.4 and place it in a centrifuge tube. Vortex for 10 s and incubate at 30 °C for 1 h. Then add 50 μL of fluorescent quantum dot labeled cDNA complex QD-cDNA prepared in step (1), vortex to mix, and incubate at 25 °C for 1 h. Then use a magnet to separate the magnetic beads from the supernatant, collect the supernatant, place it in a 96-well black microplate, and use a multi-functional microplate reader to detect the fluorescence intensity of the supernatant. The nucleotide sequence of the biotin-modified cDNA mentioned in step (1) is: 5'-Biotin-TCTTCATAATATGGTGTCAG-3'; The nucleotide sequence of the biotin-modified acetamiprid-specific nucleic acid aptamer Apt mentioned in step (2) is: 5'-Biotin-CTGACACCATATTATGAAGA-3'.

2. The fluorescence method for on-site detection of acetamiprid based on magnetic nanobeads according to claim 1, characterized in that: The concentration of the acetamiprid solution in step (3) is 20~55 μg / mL.

3. The application of the fluorescence method for on-site detection of acetamiprid based on nanomagnetic beads as described in claim 1 in the detection of acetamiprid.

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