Histidine functionalized N-SnS2 QDs fluorescence sensor and its preparation method and application

Abstract

The present invention belongs to the field of fluorescence analysis and detection technology, and specifically provides a histidine functionalized N-SnS2 QDs fluorescence sensor and its preparation method and application, including histidine functionalized N-SnS2 QDs (L-His-N-SnS2 QDs) as a donor in fluorescence energy resonance transfer, manganese dioxide nanosheets (MnO2) as an acceptor in fluorescence energy resonance transfer, L-His-N-SnS2 QDs and MnO2 are connected by an aptamer; L-His-N-SnS2 QDs are prepared by compounding histidine and N-SnS2 QD. The fluorescence sensor based on fluorescence resonance energy transfer of the present invention has high detection sensitivity, and the control method is simple. In the practical application of detecting acetamiprid, it has the advantages of low cost, high sensitivity and strong specificity.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence analysis and detection, and relates to a nucleic acid aptamer sensor based on fluorescence resonance energy transfer, in particular to a histidine functionalized N-SnS2 QDs fluorescence sensor and a preparation method and application thereof. Background Art

[0002] Acetamiprid, also known as acetamiprid, is a new type of nicotinic insecticide that has been widely used in pest control of agricultural products. Acetamiprid acts on the nicotinic acetylcholine receptors at the synaptic sites of the insect nervous system, interfering with the stimulation conduction of the insect nervous system, causing blockage of the nervous system pathways, and causing the accumulation of neurotransmitters at the acetylcholine synaptic sites, thereby causing insect paralysis and eventual death. It has contact and stomach poisoning effects, as well as strong penetration, good rapidity and long-lasting effect. However, it may be potentially toxic to the nervous and reproductive systems of animals. What is more serious is that long-term consumption of fruits and vegetables containing acetamiprid residues will have great adverse effects on human health. Therefore, it is very necessary to determine the residual amount of acetamiprid in food or actual water samples.

[0003] Fluorescence detection based on nucleic acid aptamers is a relatively new detection method in recent years. It has the advantages of high sensitivity, simple operation, and fast response speed, which has attracted widespread attention. Among them, fluorescence resonance energy transfer (FRET) technology is an earlier developed technology. With the development of green fluorescent protein application technology, FRET has become a powerful tool for detecting nanoscale distances and nanoscale distance changes of biological macromolecules in living bodies. It has a wide range of applications in biological macromolecular interaction analysis, cell physiological research, and immune analysis. A Chinese patent document (application number 201711305898.3) discloses a fluorescence detection method for acetamiprid, comprising the following steps: S1: activating carbon quantum dots, and then dividing the carbon quantum dots into two equal parts, adding a nucleic acid aptamer solution that has a specific recognition effect on acetamiprid to one part, and adding a nucleic acid aptamer complementary chain solution to the other part, and after oscillation coupling, mixing the two solutions to obtain a carbon quantum dot mixed solution; S2: using an electrophoresis method to separate the remaining nucleic acid aptamer and its complementary chain in the carbon quantum dot mixed solution obtained in S1 to obtain a carbon quantum dot solution modified only with the aptamer and the complementary chain; S3: adding the acetamiprid sample to be tested to the carbon quantum dot mixed solution described in S1, shaking to react, and then measuring the fluorescence intensity of the solution with a molecular fluorescence photometer. However, this method has the following technical defects: (1) The detection limit range of acetamiprid is relatively narrow (10ng / L-100ng / L), and the minimum detection limit (10ng / L) is relatively high; (2) The complementary cDNA chain of the acetamiprid aptamer is used, which increases the material cost; (3) The electrophoresis method used in this patent is damaging to DNA molecules and has a certain interference with subsequent experiments. The obtained carbon quantum dot solution contains only aptamers and complementary chain modifications. The aptamer and complementary chain are separated in the later stage. The steps are complicated and there is a situation where the separation cannot be carried out and there are residues, which affects the subsequent experiments. Summary of the invention

[0004] In order to determine the residual amount of acetamiprid in food or actual water samples with high sensitivity and selectivity, the present invention proposes a histidine functionalized N-SnS2 QDs fluorescent sensor and its preparation method and application. Based on the fluorescence resonance energy transfer between L-His-N-SnS2 QDs-aptamer and MnO2 nanosheets, and the selection of aptamers, the selectivity and sensitivity of the target detection object are greatly improved. Based on the fluorescent nucleic acid aptamer sensor of the present invention, it is possible to achieve rapid detection of acetamiprid content, the method of use is simple, and it has high use value.

[0005] In order to achieve the above-mentioned object, the present invention provides a histidine functionalized N-SnS2 QDs fluorescence sensor, comprising a L-His-N-SnS2 QDs (histidine functionalized nitrogen-doped tin sulfide quantum dots) fluorescent composite material as a donor in fluorescence resonance energy transfer, a MnO2 nanosheet as an acceptor in fluorescence resonance energy transfer, and a acetamiprid aptamer for connecting the donor and the acceptor.

[0006] The specific acetamiprid aptamer nucleotide sequence is: 5'-COOH TGT AAT TTG TCT GCA GCG GTTCTT GAT CGC TGA CAC CAT ATT ATG AAG A-3'. The present invention also provides a method for preparing the above-mentioned histidine functionalized N-SnS2QDs fluorescence sensor, comprising the following steps:

[0007] The aptamer solution is dropped into a mixed aqueous solution of EDC and NHS to obtain an activated aptamer solution; then the L-His-N-SnS2 QDs solution is added to obtain the L-His-N-SnS2 QDs-aptamer solution; finally, MnO2 nanosheets are added and stirred thoroughly to obtain the highly sensitive and selective fluorescence sensor based on histidine functionalized N-SnS2 QDs.

[0008] L-His-N-SnS2 QDs are prepared by compounding histidine and N-SnS2 QDs. Furthermore, the preparation method of L-His-N-SnS2QDs includes: activating nitrogen-doped tin sulfide quantum dots (N-SnS2QDs) solution with EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and then adding L-His (L-histidine) solution to obtain L-His-N-SnS2 QDs composite material.

[0009] More specifically, 40 μL of N-SnS2 QDs solution was mixed with 100 μL of 4 g / L EDC and 2 g / L NHS aqueous solution at room temperature for 0.5 h to obtain activated N-SnS2 QDs. Then 100 μL of 10 mM L-His (L-histidine) was added to the above solution.

[0010] Furthermore, the preparation of N-SnS2 QDs: SnCl4·5H2O, L-cysteine ​​and ethylenediamine were added into water and mixed to form a clear and transparent precursor solution, and then the precursor solution was subjected to hydrothermal reaction in an autoclave, and the yellow supernatant obtained by centrifugation was the N-SnS2 QDs solution.

[0011] Specifically, SnCl4·5H2O (6 mM), L-cysteine ​​(20 mM) and ethylenediamine (1.5 mL) were added to 25 mL of water to form a clear and transparent precursor solution, and then the mixture was heated at 180°C for 6 h in a 50 mL autoclave and centrifuged to obtain a yellow supernatant.

[0012] Furthermore, the preparation method of MnO2 nanosheets includes adding ethanol and sodium dodecylbenzene sulfonate into water and mixing, heating to the reaction temperature (the reaction temperature is preferably 90°C), slowly adding KMnO4 solution under stirring, and after the reaction is completed (the reaction time is preferably 30 minutes), cooling and separating the MnO2 nanosheets.

[0013] Specifically, the separation method is: separate the solid from the reaction system, wash it with ultrapure water and ethanol, and then add it into a BSA aqueous solution for ultrasonic treatment to remove the unstripped MnO2.

[0014] The present invention also provides an application of a histidine functionalized N-SnS2 QDs fluorescent sensor in detecting acetamiprid, comprising the following steps:

[0015] Step 1, prepare acetamiprid ethanol solution, add a preset amount of acetamiprid ethanol solution into a PBS buffer solution with a pH of 7.4 to obtain a series of acetamiprid standard solutions with different concentrations, ranging from 1.0×10 -14 mol / L~1.0×10 -5 mol / L, the concentration of PBS buffer solution is 0.1 mol / L;

[0016] Step 2, using the histidine functionalized N-SnS2 QDs fluorescence sensor as a liquid sample for fluorescence testing, scanning the emission spectrum, controlling the excitation wavelength to 310 nm, the slit width to 5 nm, the starting scanning wavelength to 320 nm, and the ending scanning wavelength to 700 nm, to obtain a series of concentration-fluorescence intensity correspondences, and then obtain the standard curve of acetamiprid, establish a linear relationship between the fluorescence intensity after adding acetamiprid and the logarithm of the acetamiprid concentration, and obtain the corresponding linear regression equation;

[0017] Step 3, sample testing, after filtering and removing impurities from the sample, adjust the pH to 7.4, test according to the above steps and obtain the fluorescence intensity, and calculate the obtained fluorescence intensity using the linear regression equation obtained in step 2 to obtain the concentration of acetamiprid in the sample.

[0018] Preferably, the incubation time of the fluorescence resonance energy transfer-based nucleic acid aptamer sensor in step 2 is 50 minutes, which can ensure better binding between the sensor and the detection object.

[0019] The significant advantage of the present invention is that a histidine functionalized N-SnS2 QDs fluorescence sensor for detecting acetamiprid and a preparation method thereof are developed, and a resonance energy transfer mechanism is used to perform an "on-off-on" experiment on L-His-N-SnS2 QDs to achieve the purpose of detection. Compared with ordinary fluorescence sensors, the present invention has the following two significant advantages:

[0020] (1) The present invention uses histidine-functionalized N-SnS2 QDs as a fluorescence donor and manganese dioxide nanosheets as a quencher. The addition of MnO2 nanosheets quenches the fluorescence intensity of histidine-functionalized N-SnS2 QDs to a certain extent; the addition of the aptamer enables the sensor to specifically detect acetamiprid and the preparation of the donor-receptor is more conducive to improving the sensitivity of acetamiprid detection;

[0021] (2) The introduction of the fluorescence resonance energy transfer system makes the aptamer sensor more sensitive and specific, and the detection limit of acetamiprid is 1.0×10 -14 mol / L~1.0×10 -5 mol / L, the minimum detection limit is 1.08×10 - 15 mol / L, with the advantages of wide linear range and low detection line. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a brief flow chart of the preparation of the histidine functionalized N-SnS2 QDs fluorescence sensor and the detection of acetamiprid in the examples of the present invention.

[0023] Figure 2 The fluorescence emission spectra of different concentrations of acetamiprid are shown in Figure 2. The concentrations of acetamiprid are as follows from top to bottom according to the peak values ​​of the curve: 1.0×10 -5 mol / L(a), 1.0×10 -6 mol / L(b), 1.0×10 -7 mol / L(c), 1.0×10 -8 mol / L(d), 1.0×10 -9 mol / L(e), 1.0×10 -10 mol / L(f), 1.0×10 -11 mol / L(g), 1.0×10 -12 mol / L(h), 1.0×10 -13 mol / L(i), and 1.0×10 -14 mol / L(j).

[0024] Figure 3 It is a standard curve of the difference in fluorescence intensity before and after the addition of acetamiprid and the logarithm of the acetamiprid concentration.

[0025] Figure 4 The interference performance test results (the mixture in the figure is a mixture of five test substances, and the concentration is 1.0×10 -13 mol / L). DETAILED DESCRIPTION

[0026] The present invention is not limited to the following specific embodiments. A person skilled in the art can implement the present invention in various other specific embodiments according to the contents disclosed in the present invention, or any simple changes or modifications made to the design structure and ideas of the present invention fall within the protection scope of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0027] In the embodiment of the present invention, L-His-N-SnS2 QDs are used as donors in fluorescence resonance energy transfer, and MnO2 nanosheets are used as acceptors in fluorescence resonance energy transfer. The donor and the acceptor are connected by an aptamer, thereby constructing a L-His-N-SnS2 QDs-aptamer / MnO2 nucleic acid aptamer sensor in the solution for the detection of acetamiprid. The test results show that the nucleic acid aptamer sensor has high sensitivity, low detection line and simple operation.

[0028] The present invention is further described in detail below in conjunction with embodiments:

[0029] Embodiment 1:

[0030] (1) Preparation of L-His-N-SnS2 QDs composite materials:

[0031] 40 μL of N-SnS2 QDs solution was mixed with 100 μL of 4 g / L EDC and 2 g / L NHS aqueous solution at room temperature for 0.5 h to obtain activated N-SnS2 QDs. Then 100 μL of 10 mM L-His was added to the above solution for subsequent experiments;

[0032] (2) Preparation of L-His-N-SnS2 QDs-aptamer mixed solution:

[0033] 30 μL of aptamer was added to 100 μL of 4 mg / mL·EDC·2 mg / mL·NHS and shaken for 30 min, and then the L-His-N-SnS2 QDs solution in step (1) was added and shaken for 2 h to obtain a L-His-N-SnS2QDs-aptamer mixed solution;

[0034] (3) Preparation of MnO2 nanosheets:

[0035] Mix 3.3mL ethanol, 0.5M, 2.5mL sodium dodecylbenzene sulfonate (SDBS) and 0.1M, 0.2mL H2SO4 in 22mL ultrapure water. Heat the solution to 90°C, then slowly add 0.05M, 2mL KMnO4 solution to the mixed solution and keep stirring for 30min. After cooling to room temperature, centrifuge the resulting precipitate and wash it with ultrapure water and ethanol.

[0036] The MnO2 solid prepared above was added into 10 mL, 1 mg / mL BSA aqueous solution and ultrasonicated for 12 h. The suspension was then centrifuged for 20 min to remove the unstripped MnO2.

[0037] (4) Preparation of highly sensitive and selective fluorescence sensors based on histidine-functionalized N-SnS2 QDs:

[0038] 30 μL of 1 μM aptamer was dropped into an aqueous solution containing 100 μL of 4 g / L EDC and 2 g / L NHS to obtain an activated aptamer solution; then, the L-His-N-SnS2 QDs solution prepared in step (1) was added to the aptamer to obtain a L-His-N-SnS2 QDs-aptamer solution; then, manganese dioxide nanosheets were added to the L-His-N-SnS2 QDs-aptamer solution to obtain the highly sensitive and selective fluorescence sensor based on histidine functionalized N-SnS2QDs.

[0039] In the above sensor, the aptamer nucleotide sequence is as follows: (Ordered from Sangon Biotech (Shanghai) Co., Ltd.)

[0040] Aptamer: 5′-COOH TGT AAT TTG TCT GCA GCG GTT CTT GAT CGC TGA CAC CATATT ATG AAG A-3′.

[0041] (5) Drawing of standard curve

[0042] Prepare a standard solution of acetamiprid, add a preset amount of acetamiprid ethanol solution into a PBS buffer solution (specifically, a 0.1 mol / L PBS buffer solution with a pH of 7.4) to obtain a series of ethanol solution standard solutions of different concentrations;

[0043] The highly sensitive and selective fluorescence sensor L-His-N-SnS2QDs-aptamer / MnO2 solution based on histidine functionalized N-SnS2 QDs was used as a liquid sample for fluorescence test, and the emission spectrum was scanned to detect acetamiprid. The incubation time of the highly sensitive and selective fluorescence sensor based on histidine functionalized N-SnS2 QDs was 50 minutes.

[0044] The sensor was placed in a series of acetamiprid concentrations (1.0×10 -5 mol / L、1.0×10 -6 mol / L、1.0×10 - 7 mol / L、1.0×10 -8 mol / L、1.0×10 -9 mol / L、1.0×10 -10 mol / L、1.0×10 -11 mol / L、1.0×10 - 12 mol / L(h), 1.0×10 -13 mol / L(i), and 1.0×10 -14 mol / L(j)) in a buffer solution containing 0.1mol / L PBS with a pH of 7.4, the excitation wavelength was controlled to be 310nm, the slit width was 5nm, the starting scanning wavelength was 320nm, and the ending scanning wavelength was 700nm, and a series of concentration-fluorescence intensity correspondences were obtained, and then the standard curve of acetamiprid was obtained. The linear relationship between the fluorescence intensity after adding acetamiprid and the logarithm of acetamiprid concentration was established, and the corresponding linear regression equation was obtained: I = 0.01983 + 0.97161LogC (mol / L), and the correlation coefficient (R) was 0.9959. The detection range of the linear regression equation is 1.0×10 -14 ~1.0×10 -5 mol / L, the minimum detection limit is 1.08×10 -15 mol / L.

[0045] (6) Sample testing

[0046] A certain amount of filtered river water was added to a 0.1 mol / L PBS buffer solution with a pH of 7.4 for fluorescence spectrum detection. The concentration of acetamiprid in the sample to be tested was calculated according to the linear regression equation corresponding to the above step (5). The results are listed in Table 1.

[0047] (7) Interference performance test:

[0048] The concentration of acetamiprid in the test object is 1.0x10 -11 mol / , and other interfering substances were DDVP, Chlorpyrifos, NaF, and Di-n-butylphthalate, all at a concentration of 1.0x10 -13 mol / L, see Figure 4 .

[0049] Comparative Example 1

[0050] (1) Preparation of L-His-N-SnS2 QDs-aptamer mixed solution:

[0051] 30 μL of aptamer was added to 100 μL of 4 mg / mL·EDC·2 mg / mL·NHS and shaken for 30 min, and then the L-His-N-SnS2 QDs solution in step (1) was added and shaken for 2 h to obtain a L-His-N-SnS2QDs-aptamer mixed solution;

[0052] (2) Drawing of standard curve

[0053] The L-His-N-SnS2 QDs-aptamer mixed solution was used as the liquid sample for fluorescence spectrum test and was used to detect a series of acetamiprid standard solutions with different concentrations. The detection method was the same as that in Example 1.

[0054] The results are listed in Table 1.

[0055] Comparative Example 2:

[0056] (1) Preparation of MnO2 nanosheet solution

[0057] The cuvette was washed with anhydrous ethanol and deionized water in turn, and dried naturally for later use. 2 mL of MnO2 nanosheet aqueous solution (the preparation method of MnO2 nanosheet aqueous solution is as shown in Example 1) was removed by a microinjector and added to a clean cuvette to obtain a MnO2 nanosheet liquid sample.

[0058] (2) Drawing of standard curve

[0059] The MnO2 nanosheet aqueous solution was used as the liquid sample for fluorescence spectrum test to detect a series of acetamiprid standard solutions with different concentrations. The detection method was the same as that in Example 1.

[0060] The results are listed in Table 1.

[0061] Table 1 Determination results of acetamiprid in river water

[0062]

[0063]

[0064] Note: a is the average of three measurements.

[0065] As shown in Table 1, the sample was tested three times in parallel, the relative standard deviation was less than 5%, and the spike recovery rate was 94% to 102%. The above results show that acetamiprid cannot be detected by using L-His-N-SnS2QDs-aptamer or MnO2 liquid sample alone without using L-His-N-SnS2QDs-aptamer / MnO2 liquid sample, and the present invention is feasible for detecting acetamiprid in river water.

[0066] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A histidine-functionalized N-SnS2 QDs fluorescence sensor for the detection of acetamiprid, characterized in that: The invention comprises L-His-N-SnS2 QDs as a donor in fluorescence resonance energy transfer, MnO2 nanosheets as an acceptor in fluorescence resonance energy transfer, and an acetamiprid aptamer for connecting the donor and the acceptor; The nucleotide sequence of the acetamiprid aptamer: 5′-COOH TGT AAT TTG TCT GCA GCG GTT CTT GAT CGCTGA CAC CAT ATT ATG AAG A -3′; The preparation method of L-His-N-SnS2 QDs includes the following steps: Nitrogen-doped tin sulfide quantum dots were activated with EDC and NHS, and then L-histidine L-His solution was added and stirred thoroughly to obtain L-His-N-SnS2 QDs composite materials.

2. The method for preparing the histidine functionalized N-SnS2 QDs fluorescence sensor according to claim 1, characterized in that: The method comprises the following steps: dropping an acetamiprid aptamer solution into a mixed aqueous solution of EDC and NHS to obtain an activated aptamer solution; subsequently adding an L-His-N-SnS2 QDs solution to obtain an L-His-N-SnS2 QDs-aptamer solution; and finally adding MnO2 nanosheets and stirring the mixture thoroughly to obtain a histidine-functionalized N-SnS2 QDs fluorescent sensor.

3. The method for preparing the histidine functionalized N-SnS2 QDs fluorescence sensor according to claim 2, characterized in that: The preparation method of nitrogen-doped tin sulfide quantum dots comprises the following steps: preparing a mixed aqueous solution containing SnCl4, L-cysteine ​​and ethylenediamine, stirring until clear and transparent to obtain a precursor solution, then subjecting the precursor solution to a hydrothermal reaction in an autoclave, and centrifuging out the supernatant to obtain a nitrogen-doped tin sulfide quantum dot solution.

4. The method for preparing the histidine functionalized N-SnS2 QDs fluorescence sensor according to claim 2, characterized in that: The preparation method of MnO2 nanosheets includes: mixing ethanol, sodium dodecylbenzene sulfonate and H2SO4 into water, mixing, heating to a reaction temperature, slowly dropping a KMnO4 solution under stirring, cooling after the reaction is completed and separating the MnO2 nanosheets.

5. The method for preparing the histidine functionalized N-SnS2 QDs fluorescence sensor according to claim 4, characterized in that: The method for separating MnO2 nanosheets is as follows: separating the solid from the reaction system, washing it with ultrapure water and ethanol, and then adding it to a BSA aqueous solution for ultrasonic treatment to remove the unstripped MnO2.

6. The use of the histidine functionalized N-SnS2 QDs fluorescent sensor according to claim 1 in detecting acetamiprid, characterized in that: The steps include: (1) Prepare 0.1 mol / L PBS buffer solution with a pH of 7.4; (2) Preparation of acetamiprid at different concentrations and PBS buffer solution at pH = 7.4; Prepare acetamiprid solution with deionized water, and then add a certain amount of acetamiprid solution to the PBS buffer solution in step (1) to obtain a series of acetamiprid standard solutions with different concentrations, ranging from 1.0×10 -14 mol / L~1.0×10 -5 mol / L; (3) Drawing of standard curve A series of acetamiprid standard solutions of different concentrations in step (2) were added dropwise to the L-His-N-SnS2 QDs-aptamer / MnO2 solution, and the solution was fully incubated at room temperature. The obtained solution was recorded as L-His-N-SnS2 QDs-aptamer / MnO2+acetamiprid; L-His-N-SnS2 QDs-aptamer / MnO2 / + acetamiprid solution was used as the liquid sample for fluorescence test, and the emission spectrum was scanned. The excitation wavelength was controlled to be 310 nm, the slit width was 5 nm, the starting scanning wavelength was 320 nm, and the ending scanning wavelength was 700 nm. A series of concentration-fluorescence intensity correspondences were obtained, and then the standard curve of acetamiprid was obtained. The linear relationship between the fluorescence intensity after adding acetamiprid and the logarithm of acetamiprid concentration was established, and the corresponding linear regression equation was obtained. (4) Actual sample testing After filtering and removing impurities from the sample, the pH was adjusted to 7.4, and the test was performed according to the above steps to obtain the fluorescence intensity. The obtained fluorescence intensity was calculated using the linear regression equation obtained in step 2 to obtain the concentration of acetamiprid in the sample.

7. The use of the histidine functionalized N-SnS2 QDs fluorescence sensor according to claim 6 in detecting acetamiprid, characterized in that: The incubation time of the tested L-His-N-SnS2 QDs-aptamer / MnO2 and acetamiprid solution was 50 min.

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