Fluorescence sensor for detecting sulfamethoxazole medicine and preparation method of fluorescence sensor

Abstract

The invention discloses a fluorescence sensor for detecting sulfamethoxazole medicine and a preparation method thereof, the method comprises the following steps: 1, uniformly mixing CdTe quantum dots, cyclohexane, hexyl alcohol and triton X-100, adding ammonia water, a phthalic acid diethylene glycol diacrylate solution and tetraethyl orthosilicate, reacting, adding acetone for demulsification, centrifuging, washing and vacuum drying to obtain R-CdTe coated SiO2; 2, taking the R-CdTe coated SiO2, gamma-methacryloxy propyl trimethoxy silane and methylbenzene, uniformly mixing the R-CdTe coated SiO2, the gamma-methacryloxy propyl trimethoxy silane and the methylbenzene, reacting, centrifuging, washing and drying to obtain KH-570-R-CdTe coated SiO2; 3, mixing the fluorescein aqueous solution and the sulfamethoxazole aqueous solution, and reacting to obtain a sulfamethoxazole derivative solution; and 4, adding acetonitrile and methacrylic acid into the sulfamethoxazole derivative solution, self-assembling, adding azodiisobutyronitrile, ethylene glycol dimethacrylate and KH-570-R-CdTe coated SiO2, charging nitrogen, heating, reacting, centrifuging and washing to obtain the fluorescent sensor with high specific response to the sulfamethoxazole derivative, and indirectly realizing sensitive detection of sulfamethoxazole.

Description

Technical Field

[0001] The invention relates to an antibiotic drug detection technology, in particular to a fluorescent sensor for detecting sulfamethoxazole drugs and a preparation method thereof. Background Art

[0002] Sulfadimethoxazole is a commonly used sulfonamide antibacterial drug with good chemical stability, broad antibacterial spectrum, and low cost and easy availability. It is widely used in the breeding industry and can effectively reduce diseases and increase animal production. However, long-term excessive use of sulfonamides will cause them to accumulate in animals and be transferred to the environment and human body through metabolism and bioaccumulation, which will eventually cause great harm to the environment and human body, such as the emergence of super bacteria or decreased human immunity, vomiting, and allergic reactions. Therefore, timely and accurate detection of sulfadimethoxazole can help to understand the drug residues in animals in real time, thereby reducing environmental and food safety risks.

[0003] At present, the common methods for detecting sulfamethoxazole mainly include instrumental detection methods such as liquid chromatography, liquid chromatography-mass spectrometry, gel chromatography, and rapid detection methods based on enzyme-linked immunosorbent assay technology. Among them: the instrumental detection method has complicated sample pretreatment, large amounts of organic reagents, low detection efficiency, expensive equipment, and requires professional technicians, and the detection site is limited; the advantage of enzyme-linked immunosorbent assay is that it can be detected on-site in real time and does not rely on large equipment, but the key antibodies need to be obtained through cultivation, extraction and purification steps, and the preparation cost is high. At the same time, antibodies from organisms are easily inactivated by factors such as temperature and environment, and in practical applications, they have high requirements for storage and detection environment.

[0004] Molecular imprinting polymers, known as "artificial antibodies", are high-molecular compounds with specific recognition capabilities. They are low-cost, resistant to high temperatures, acids and alkalis, resistant to organic reagents, and can be prepared in a directional manner. Therefore, molecular imprinting fluorescent sensors prepared by combining molecular imprinting polymers with fluorescence sensing technology have great application potential in the field of rapid real-time detection of sulfamethoxazole. However, only a small number of fluorescent sensors for the detection of sulfonamide drugs have been reported, and they mainly use single-emission fluorescent sensors for detection. This type of single-emission fluorescent sensor has problems such as insufficient specificity and a high false positive rate in actual detection. In addition, the fluorescent signal is easily interfered by factors such as the sample matrix and the stability of the excitation light source, thereby affecting the accuracy of the test results. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a fluorescent sensor for detecting sulfamethoxazole drugs and a preparation method thereof. The prepared fluorescent sensor has a fluorescent response to sulfamethoxazole derivatives and has the advantages of high specificity, good sensitivity and strong anti-interference.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs comprises the following steps:

[0008] Step 1, take 1.5-2mL water-soluble red fluorescent CdTe quantum dots, 10-20mL cyclohexane, 3-4mL n-hexanol and 3-4mL Triton X-100 and add them into a round-bottom flask, stir, then add 100-200μL of 25% ammonia water and 100-150μL of 0.075% diethylene glycol diacrylate solution by volume, continue stirring, then add 100-300μL tetraethyl orthosilicate, continue stirring for 16-24h, after the reaction is completed, add 100-200μL acetone, demulsify, centrifuge, wash the precipitate, and dry it at low temperature under vacuum conditions to obtain silica-coated R-CdTe nanospheres, recorded as R-CdTe@SiO2;

[0009] Step 2, weigh 0.5-1 g R-CdTe@SiO2 into a round-bottom flask, add 1.0-3.0 mL γ-methacryloxypropyltrimethoxysilane and 40-60 mL anhydrous toluene, mix well, react at 70-90° C. for 8-12 h, centrifuge, wash the precipitate, and dry it at low temperature under vacuum to obtain vinyl-modified R-CdTe@SiO2, recorded as KH-570-R-CdTe@SiO2;

[0010] Step 3, taking 5-10 mL of a 1 mmol / mL aqueous solution of fluorescent amine and 5-10 mL of a 1 mmol / mL aqueous solution of sulfamethoxazole, mixing them, standing at 20-35° C. for 7-10 min to allow a derivatization reaction to occur, and obtaining a sulfamethoxazole derivative solution having green fluorescence;

[0011] Step 4, take 3-6 mL of sulfamethoxazole derivative solution and add it to 25 mL of acetonitrile, then add 0.6 μmol of methacrylic acid, ultrasonicate, self-assemble at 4-6°C for 4-8 hours, then add 0.02-0.06 g of azobisisobutyronitrile, 0.5-1.5 mL of ethylene glycol dimethacrylate and 0.5-2.5 mg of KH-570-R-CdTe@SiO2 in sequence to obtain a mixed solution A, first fill the mixed solution A with nitrogen to deoxygenate, then transfer it to a constant temperature oil bath at 60-80°C, react for 16-24 hours, after the reaction is completed, centrifuge, and wash the precipitate repeatedly several times until the template molecule can no longer be detected in the elution solution to obtain a ratiometric fluorescent sensor.

[0012] Furthermore, the water-soluble red fluorescent CdTe quantum dots in step 1 are prepared by the following method:

[0013] Step 1.1, weigh 35-40 mg of tellurium powder, 20-60 mg of KBH4 and 2 mL of ultrapure water, put them into a round-bottom flask, introduce nitrogen into the round-bottom flask to remove oxygen in the flask, react at 60-80° C. to form a transparent solution, which is the precursor NaHTe solution;

[0014] Step 1.2: first take 30-60 mg CdCl2·2.5H2O and add it into a three-necked flask, then add 60-80 mL ultrapure water, stir well, then add 50-75 μL thioglycolic acid, mix well, adjust the pH value to alkaline, fill with nitrogen to remove oxygen for 15 minutes, then quickly add 2 mL of precursor NaHTe solution, stir at 100-120°C for 16 hours to obtain water-soluble red fluorescent CdTe quantum dots.

[0015] Furthermore, in step 1.2, a NaOH solution with a concentration of 0.2 mol / L is used to adjust the pH to 9-11.

[0016] Furthermore, the stirring time of step 1 is 20 to 40 minutes.

[0017] Furthermore, the stirring time in step 1 is 10 to 40 minutes.

[0018] Furthermore, the washing in step 1 and step 2 is performed using anhydrous ethanol.

[0019] Furthermore, the ultrasonication time in step 4 is 5 to 10 minutes.

[0020] Furthermore, the time for filling with nitrogen to remove oxygen in step 4 is 15 to 30 minutes.

[0021] Furthermore, the washing in step 4 is performed by repeatedly washing several times with a 1.5% by mass acetic acid ethanol solution.

[0022] A fluorescent sensor for detecting sulfamethoxazole drugs, characterized in that it has a fluorescent response to sulfamethoxazole derivatives.

[0023] Compared with the prior art, the present invention has the following technical effects:

[0024] 1) The present invention adopts a derivatization reaction to convert non-fluorescent sulfamethoxazole into a sulfamethoxazole derivative with green fluorescence through a derivatization reaction with fluorescent amine, thereby greatly reducing the interference of other small molecules and analogs. Then, the sulfamethoxazole derivative is used as a template molecule and the silicon dioxide-coated R-CdTe is used as a reference fluorescence to prepare a ratiometric fluorescence sensor for detecting sulfamethoxazole drugs. Then, the template molecule is removed by solvent elution, and an imprinted cavity complementary to the shape, size and chemical group of the sulfamethoxazole derivative is formed on the sensor surface. The imprinted cavity can specifically adsorb the sulfamethoxazole derivative. As the adsorption amount of the sulfamethoxazole derivative increases, the green fluorescence of the sensor is enhanced, while the red reference fluorescence remains unchanged. A linear relationship is established between the ratio of the green fluorescence intensity to the red fluorescence intensity and the concentration of the sulfamethoxazole derivative, thereby indirectly realizing the detection of sulfamethoxazole in environmental and food samples, and reducing the signal interference caused by temperature and sample matrix factors, thereby having higher detection accuracy.

[0025] 2) The fluorescent sensor prepared by the present invention has low cost, good stability and fast response speed. It can realize on-site real-time detection in combination with small equipment, and has broad prospects in the field of rapid screening of sulfamethoxazole drugs in a large number of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : The change of fluorescence intensity over time after the fluorescamine aqueous solution and the sulfamethoxazole aqueous solution were mixed;

[0027] Figure 2 : Photos of sulfamethoxazole aqueous solution, fluorescein aqueous solution and sulfamethoxazole derivative solution under fluorescent light and ultraviolet light;

[0028] Figure 3 : The curve of the change of fluorescence intensity over time during the process of detecting sulfamethoxazole derivatives by the fluorescence sensor prepared by the present invention;

[0029] Figure 4 The response results of the fluorescent sensor prepared in Example 1 of the present invention to different antibiotics derivatized with fluorescent amine;

[0030] Figure 5 The fluorescence spectra of the fluorescence sensor prepared in Example 1 of the present invention in response to different concentrations of sulfamethoxazole derivatives;

[0031] Figure 6 This is the fitting curve of the fluorescence response of the fluorescence sensor prepared in Example 1 of the present invention to different concentrations of sulfamethoxazole derivatives. DETAILED DESCRIPTION

[0032] The specific contents of the present invention are further explained in detail below in conjunction with embodiments.

[0033] Example 1

[0034] Step 1: Preparation of water-soluble red fluorescent CdTe quantum dots

[0035] Step 1.1, weigh 35 mg of tellurium powder, 20 mg of KBH4 and 2 mL of ultrapure water, put them into a round-bottom flask, introduce nitrogen into the round-bottom flask to remove oxygen in the flask, and react at 60°C until a transparent solution is formed, which is the precursor NaHTe solution;

[0036] Step 1.2, first take 30mg CdCl2·2.5H2O and add it to a three-necked flask, then add 60mL ultrapure water, stir well, then add 50μL thioglycolic acid, mix well, use 0.2mol / L NaOH solution to adjust the pH to 9, fill with nitrogen to remove oxygen for 15min, then quickly add 2mL precursor NaHTe solution, stir at 100℃ for 16h, and obtain water-soluble red fluorescent CdTe quantum dots, recorded as R-CdTe;

[0037] Step 2: Preparation of R-CdTe@SiO2

[0038] 1.5 mL of water-soluble red fluorescent CdTe quantum dots, 10 mL of cyclohexane, 3 mL of n-hexanol and 3 mL of Triton X-100 were added to a 50 mL round-bottom flask and stirred for 20 min. Then, 100 μL of 25% ammonia water and 100 μL of 0.075% diethylene glycol diacrylate solution of phthalate were added and stirred for 10 min. Then, 100 μL of tetraethyl orthosilicate was added and stirred for 16 h. After the reaction was completed, 100 μL of acetone was added to break the emulsion, and the supernatant was removed by centrifugation. The precipitate was washed with anhydrous ethanol and dried at low temperature under vacuum to obtain silica-coated R-CdTe nanospheres, recorded as R-CdTe@SiO2.

[0039] Step 3: Preparation of KH-570-R-CdTe@SiO2

[0040] Weigh 0.5 g of R-CdTe@SiO2 into a round-bottom flask, add 1.0 mL of γ-methacryloxypropyltrimethoxysilane and 40 mL of anhydrous toluene, mix well, react at 70 °C for 8 h, centrifuge, wash the precipitate with anhydrous ethanol, and dry it at low temperature under vacuum to obtain vinyl-modified R-CdTe@SiO2, recorded as KH-570-R-CdTe@SiO2;

[0041] Step 4: Preparation of sulfamethoxazole derivative solution

[0042] 5 mL of 1 mmol / mL fluorescamine aqueous solution and 5 mL of 1 mmol / mL sulfamethoxazole aqueous solution were mixed and allowed to stand at 20°C for 7 min to allow a derivatization reaction to occur, thereby obtaining a sulfamethoxazole derivative solution with green fluorescence;

[0043] Step 5: Preparation of fluorescence sensor

[0044] Step 5.1, add 3 mL of sulfamethoxazole derivative solution to 25 mL of acetonitrile, then add 0.6 μmol of methacrylic acid, sonicate for 5 min, self-assemble at 4 °C for 4 h, then add 0.02 g of azobisisobutyronitrile, 0.5 mL of ethylene glycol dimethacrylate and 0.5 mg of KH-570-R-CdTe@SiO2 in sequence to obtain a mixed solution A;

[0045] Step 5.2: First, nitrogen is filled into the mixed solution A for 15 minutes to remove oxygen, and then it is transferred to a constant temperature oil bath at 60°C and reacted for 16 hours. After the reaction is completed, the precipitate is separated by centrifugation, and then it is repeatedly washed several times with a 1.5% acetic acid ethanol solution to remove the template molecules and unreacted functional monomers until the template molecules can no longer be detected in the elution solution, thereby obtaining a ratiometric fluorescent sensor.

[0046] Example 2

[0047] Step 1: Preparation of water-soluble red fluorescent CdTe quantum dots

[0048] Step 1.1, weigh 37 mg of tellurium powder, 40 mg of KBH4 and 2 mL of ultrapure water, put them into a round-bottom flask, introduce nitrogen into the round-bottom flask to remove oxygen in the flask, and react at 75°C until a transparent solution is formed, which is the precursor NaHTe solution;

[0049] Step 1.2, first take 45mg CdCl2·2.5H2O and add it to a three-necked flask, then add 70mL ultrapure water, stir well, then add 60μL thioglycolic acid, mix well, use 0.2mol / L NaOH solution to adjust the pH to 10, fill with nitrogen to remove oxygen for 15min, then quickly add 2mL precursor NaHTe solution, stir at 110℃ for 16h, and obtain water-soluble red fluorescent CdTe quantum dots, recorded as R-CdTe;

[0050] Step 2: Preparation of R-CdTe@SiO2

[0051] 1.75 mL of water-soluble red fluorescent CdTe quantum dots, 15 mL of cyclohexane, 3.5 mL of n-hexanol and 3.5 mL of Triton X-100 were added to a 50 mL round-bottom flask and stirred for 30 min. Then, 150 μL of 25% ammonia water and 125 μL of 0.075% diethylene glycol diacrylate solution of phthalate were added and stirred for 25 min. Then, 200 μL of tetraethyl orthosilicate was added and stirred for 20 h. After the reaction was completed, 150 μL of acetone was added to break the emulsion, and the supernatant was removed by centrifugation. The precipitate was washed with anhydrous ethanol and dried at low temperature under vacuum to obtain silica-coated R-CdTe nanospheres, recorded as R-CdTe@SiO2.

[0052] Step 3: Preparation of KH-570-R-CdTe@SiO2

[0053] Weigh 0.75 g of R-CdTe@SiO2 into a round-bottom flask, add 2.0 mL of γ-methacryloxypropyltrimethoxysilane and 50 mL of anhydrous toluene, mix well, react at 80 °C for 10 h, centrifuge, wash the precipitate with anhydrous ethanol, and dry it at low temperature under vacuum to obtain vinyl-modified R-CdTe@SiO2, recorded as KH-570-R-CdTe@SiO2;

[0054] Step 4: Preparation of sulfamethoxazole derivative solution

[0055] 8 mL of 1 mmol / mL fluorescamine aqueous solution and 8 mL of 1 mmol / mL sulfamethoxazole aqueous solution were mixed and allowed to stand at 30°C for 8 min to allow a derivatization reaction to occur, thereby obtaining a sulfamethoxazole derivative solution with green fluorescence;

[0056] Step 5: Preparation of fluorescence sensor

[0057] Step 5.1, add 4 mL of sulfamethoxazole derivative solution to 25 mL of acetonitrile, then add 0.6 μmol of methacrylic acid, sonicate for 7 min, self-assemble at 5 °C for 6 h, then add 0.04 g of azobisisobutyronitrile, 1.0 mL of ethylene glycol dimethacrylate and 1.5 mg of KH-570-R-CdTe@SiO2 in sequence to obtain a mixed solution A;

[0058] Step 5.2: First, nitrogen is filled into the mixed solution A for 25 minutes to remove oxygen, and then it is transferred to a constant temperature oil bath at 60°C and reacted for 20 hours. After the reaction is completed, the precipitate is separated by centrifugation, and then it is repeatedly washed several times with a 1.5% acetic acid ethanol solution to remove the template molecules and unreacted functional monomers until the template molecules can no longer be detected in the elution solution, thereby obtaining a ratiometric fluorescent sensor.

[0059] Example 3

[0060] Step 1: Preparation of water-soluble red fluorescent CdTe quantum dots

[0061] Step 1.1, weigh 40 mg of tellurium powder, 60 mg of KBH4 and 2 mL of ultrapure water, put them into a round-bottom flask, pass nitrogen into the round-bottom flask to remove oxygen in the flask, react at 90°C until a transparent solution is formed, which is the precursor NaHTe solution;

[0062] Step 1.2, first take 60mg CdCl2·2.5H2O and add it into a three-necked flask, then add 80mL ultrapure water, stir well, then add 75μL thioglycolic acid, mix well, use 0.2mol / L NaOH solution to adjust pH to 11, fill with nitrogen to remove oxygen for 15min, then quickly add 2mL precursor NaHTe solution, stir at 120℃ for 16h, and obtain water-soluble red fluorescent CdTe quantum dots, recorded as R-CdTe;

[0063] Step 2: Preparation of R-CdTe@SiO2

[0064] 2 mL of water-soluble red fluorescent CdTe quantum dots, 20 mL of cyclohexane, 4 mL of n-hexanol and 4 mL of Triton X-100 were added to a 450 mL round-bottom flask and stirred for 40 min. Then, 200 μL of 25% ammonia water and 150 μL of 0.075% diethylene glycol diacrylate solution of phthalate were added and stirred for 40 min. Then, 300 μL of tetraethyl orthosilicate was added and stirred for 24 h. After the reaction was completed, 200 μL of acetone was added to break the emulsion, and the supernatant was removed by centrifugation. The precipitate was washed with anhydrous ethanol and dried at low temperature under vacuum to obtain silica-coated R-CdTe nanospheres, recorded as R-CdTe@SiO2.

[0065] Step 3: Preparation of KH-570-R-CdTe@SiO2

[0066] Weigh 1 g of R-CdTe@SiO2 and put it into a round-bottom flask, then add 3.0 mL of γ-methacryloxypropyltrimethoxysilane and 60 mL of anhydrous toluene, mix well, react at 90 °C for 12 h, centrifuge, wash the precipitate with anhydrous ethanol, and dry it at low temperature under vacuum to obtain vinyl-modified R-CdTe@SiO2, recorded as KH-570-R-CdTe@SiO2;

[0067] Step 4: Preparation of sulfamethoxazole derivative solution

[0068] 10 mL of 1 mmol / mL fluorescamine aqueous solution and 10 mL of 1 mmol / mL sulfamethoxazole aqueous solution were mixed and allowed to stand at 35°C for 10 min to allow a derivatization reaction to occur, thereby obtaining a sulfamethoxazole derivative solution with green fluorescence.

[0069] Step 5: Preparation of fluorescence sensor

[0070] Step 5.1, add 6 mL of sulfamethoxazole derivative solution to 25 mL of acetonitrile, then add 0.6 μmol of methacrylic acid, ultrasonicate for 10 min, self-assemble at 6 °C for 8 h, then add 0.06 g of azobisisobutyronitrile, 1.5 mL of ethylene glycol dimethacrylate and 2.5 mg of KH-570-R-CdTe@SiO2 in sequence to obtain a mixed solution A;

[0071] Step 5.2: First, nitrogen is filled into the mixed solution A for 30 minutes to remove oxygen, and then it is transferred to a constant temperature oil bath at 60°C and reacted for 24 hours. After the reaction is completed, the precipitate is separated by centrifugation, and then it is repeatedly washed several times with a 1.5% acetic acid ethanol solution to remove the template molecules and unreacted functional monomers until the template molecules can no longer be detected in the elution solution, thereby obtaining a ratiometric fluorescent sensor.

[0072] Example 4

[0073] Step 1: Preparation of water-soluble red fluorescent CdTe quantum dots

[0074] Step 1.1, weigh 38 mg of tellurium powder, 50 mg of KBH4 and 2 mL of ultrapure water, put them into a round-bottom flask, introduce nitrogen into the round-bottom flask to remove oxygen in the flask, and react at 80°C until a transparent solution is formed, which is the precursor NaHTe solution;

[0075] Step 1.2, first take 50mg CdCl2·2.5H2O and add it to a three-necked flask, then add 75mL ultrapure water, stir well, then add 70μL thioglycolic acid, mix well, use 0.2mol / L NaOH solution to adjust the pH to 11, fill with nitrogen to remove oxygen for 15min, then quickly add 2mL precursor NaHTe solution, stir at 120℃ for 16h, and obtain water-soluble red fluorescent CdTe quantum dots, recorded as R-CdTe;

[0076] Step 2: Preparation of R-CdTe@SiO2

[0077] 1.6 mL of water-soluble red fluorescent CdTe quantum dots, 13 mL of cyclohexane, 3 mL of n-hexanol and 4 mL of Triton X-100 were added to a 50 mL round-bottom flask and stirred for 25 min. Then, 125 μL of 25% ammonia water and 110 μL of 0.075% diethylene glycol diacrylate solution of phthalate were added and stirred for 20 min. Then, 150 μL of tetraethyl orthosilicate was added and stirred for 18 h. After the reaction was completed, 130 μL of acetone was added to break the emulsion, and the supernatant was removed by centrifugation. The precipitate was washed with anhydrous ethanol and dried at low temperature under vacuum to obtain silica-coated R-CdTe nanospheres, recorded as R-CdTe@SiO2.

[0078] Step 3: Preparation of KH-570-R-CdTe@SiO2

[0079] Weigh 0.6 g of R-CdTe@SiO2 into a round-bottom flask, add 1.5 mL of γ-methacryloxypropyltrimethoxysilane and 45 mL of anhydrous toluene, mix well, react at 70 °C for 8 h, centrifuge, wash the precipitate with anhydrous ethanol, and dry it at low temperature under vacuum to obtain vinyl-modified R-CdTe@SiO2, recorded as KH-570-R-CdTe@SiO2;

[0080] Step 4: Preparation of sulfamethoxazole derivative solution

[0081] 7 mL of 1 mmol / mL fluorescamine aqueous solution and 7 mL of 1 mmol / mL sulfamethoxazole aqueous solution were mixed and allowed to stand at 25°C for 9 minutes to allow a derivatization reaction to occur, thereby obtaining a sulfamethoxazole derivative solution with green fluorescence;

[0082] Step 5: Preparation of fluorescence sensor

[0083] Step 5.1, add 5 mL of sulfamethoxazole derivative solution to 25 mL of acetonitrile, then add 0.6 μmol of methacrylic acid, ultrasonicate for 6 min, self-assemble at 4 °C for 5 h, then add 0.03 g of azobisisobutyronitrile, 0.8 mL of ethylene glycol dimethacrylate and 1.0 mg of KH-570-R-CdTe@SiO2 in sequence to obtain a mixed solution A;

[0084] Step 5.2: First, nitrogen is filled into the mixed solution A for 20 minutes to remove oxygen, and then it is transferred to a constant temperature oil bath at 70°C and reacted for 22 hours. After the reaction is completed, the precipitate is separated by centrifugation, and then it is repeatedly washed several times with a 1.5% acetic acid ethanol solution to remove the template molecules and unreacted functional monomers until the template molecules can no longer be detected in the elution solution, thereby obtaining a ratiometric fluorescent sensor.

[0085] Example 5

[0086] Step 1: Preparation of water-soluble red fluorescent CdTe quantum dots

[0087] Step 1.1, weigh 36 mg of tellurium powder, 30 mg of KBH4 and 2 mL of ultrapure water, put them into a round-bottom flask, introduce nitrogen into the round-bottom flask to remove oxygen in the flask, and react at 70°C until a transparent solution is formed, which is the precursor NaHTe solution;

[0088] Step 1.2, first take 40mg CdCl2·2.5H2O and add it to a three-necked flask, then add 65mL ultrapure water, stir well, then add 55μL thioglycolic acid, mix well, use 0.2mol / L NaOH solution to adjust the pH to 9, fill with nitrogen to remove oxygen for 15min, then quickly add 2mL precursor NaHTe solution, stir at 100℃ for 16h, and obtain water-soluble red fluorescent CdTe quantum dots, recorded as R-CdTe;

[0089] Step 2: Preparation of R-CdTe@SiO2

[0090] 1.8 mL of water-soluble red fluorescent CdTe quantum dots, 18 mL of cyclohexane, 3.5 mL of n-hexanol and 3 mL of Triton X-100 were added to a 50 mL round-bottom flask and stirred for 35 min. Then, 180 μL of 25% ammonia water and 140 μL of 0.075% diethylene glycol diacrylate solution of phthalate were added and stirred for 30 min. Then, 250 μL of tetraethyl orthosilicate was added and stirred for 22 h. After the reaction was completed, 170 μL of acetone was added to break the emulsion, and the supernatant was removed by centrifugation. The precipitate was washed with anhydrous ethanol and dried at low temperature under vacuum to obtain silica-coated R-CdTe nanospheres, recorded as R-CdTe@SiO2.

[0091] Step 3: Preparation of KH-570-R-CdTe@SiO2

[0092] Weigh 0.9 g of R-CdTe@SiO2 into a round-bottom flask, add 2.5 mL of γ-methacryloxypropyltrimethoxysilane and 55 mL of anhydrous toluene, mix well, react at 90 °C for 12 h, centrifuge, wash the precipitate with anhydrous ethanol, and dry it at low temperature under vacuum to obtain vinyl-modified R-CdTe@SiO2, recorded as KH-570-R-CdTe@SiO2;

[0093] Step 4: Preparation of sulfamethoxazole derivative solution

[0094] 6 mL of 1 mmol / mL fluorescamine aqueous solution and 6 mL of 1 mmol / mL sulfamethoxazole aqueous solution were mixed and allowed to stand at 35°C for 10 min to allow a derivatization reaction to occur, thereby obtaining a sulfamethoxazole derivative solution with green fluorescence;

[0095] Step 5: Preparation of fluorescence sensor

[0096] Step 5.1, add 6 mL of sulfamethoxazole derivative solution to 25 mL of acetonitrile, then add 0.6 μmol of methacrylic acid, ultrasonicate for 8 min, self-assemble at 6 °C for 7 h, then add 0.05 g of azobisisobutyronitrile, 1.2 mL of ethylene glycol dimethacrylate and 2.0 mg of KH-570-R-CdTe@SiO2 in sequence to obtain a mixed solution A;

[0097] Step 5.2: First, nitrogen is filled into the mixed solution A for 30 minutes to remove oxygen, and then it is transferred to a constant temperature oil bath at 80°C and reacted for 18 hours. After the reaction is completed, the precipitate is separated by centrifugation, and then it is repeatedly washed several times with a 1.5% acetic acid ethanol solution to remove the template molecules and unreacted functional monomers until the template molecules can no longer be detected in the elution solution, thereby obtaining a ratiometric fluorescent sensor.

[0098] The fluorescent sensors prepared in Examples 1 to 5 are uniform spherical nanoparticles, which can form a uniform dispersion when dispersed in ultrapure water and exhibit red fluorescence under a 365nm ultraviolet lamp. When the sensor solution is mixed with a sulfamethoxazole derivative, the sulfamethoxazole derivative is specifically recognized and adsorbed to the imprinted cavity on the surface of the fluorescent sensor. As the concentration of the sulfamethoxazole derivative increases, the content of the derivative adsorbed on the sensor surface gradually increases, and the green fluorescence gradually increases. During the whole process, the red fluorescence of CdTe is not affected by the detection environment, and the fluorescence intensity hardly changes. A correlation is established between the ratio of the green fluorescence (488nm) and the red fluorescence (630nm) intensities and the concentration of the sulfamethoxazole derivative to achieve quantitative detection of sulfamethoxazole.

[0099] Various performance tests were performed on the intermediate product involved in Example 1 and the prepared fluorescent sensor. The test process and results are as follows:

[0100] 1) Test the fluorescence change of sulfamethoxazole during the derivatization process with fluorescent amine

[0101] A 0.2 nmol / mL sulfamethoxazole aqueous solution and a 0.2 nmol / mL fluorescein aqueous solution were prepared respectively, and the sulfamethoxazole aqueous solution and the fluorescein aqueous solution were mixed at a volume ratio of 1:1, shaken well, and the fluorescence spectrum of the mixed solution was measured at regular intervals. The results are as follows: Figure 1 As shown, it can be seen that during the derivatization reaction, as the derivatization time increases, the fluorescence intensity increases rapidly and tends to be stable at 7 minutes. Therefore, when the concentration of the sulfamethoxazole solution is not greater than 0.02 nmol / mL, the derivatization reaction can be completed within 7 minutes.

[0102] Put the sulfamethoxazole derivative solution, sulfamethoxazole aqueous solution and fluorescent amine aqueous solution into a cuvette, observe and take pictures under fluorescent light and ultraviolet light respectively. The results are as follows: Figure 2 As shown, a, b and c in the figure represent sulfamethoxazole aqueous solution, fluorescein aqueous solution and sulfamethoxazole derivative solution, respectively. It can be seen that: under fluorescent light, sulfamethoxazole aqueous solution and fluorescein aqueous solution are both colorless and transparent liquids, and sulfamethoxazole derivative solution exhibits green fluorescence; under 365nm ultraviolet light, sulfamethoxazole aqueous solution and fluorescein aqueous solution have no fluorescence, and sulfamethoxazole derivative solution exhibits strong green fluorescence.

[0103] 2) Test the optimal detection time of the fluorescence sensor

[0104] The fluorescent sensor prepared in Example 1 was dispersed in ultrapure water to prepare a fluorescent sensor solution with a concentration of 1.0 mg / mL. 2 mL of the fluorescent sensor solution was taken into a test tube, and 10 μL of a 2.0 mg / L sulfamethoxazole derivative solution was added dropwise thereto. The fluorescence spectrum of the solution was measured at regular intervals, and the fluorescence intensity within 30 minutes of adding the sulfamethoxazole derivative solution was measured respectively. The optimal detection time was determined based on the ratio of the fluorescence intensity at 488 nm and 630 nm of the fluorescence spectrum. The results are shown in FIG. Figure 3 As shown in Figure 2, with the extension of time, the fluorescence intensity ratio gradually decreases. When the detection time is 10 min, the fluorescence intensity ratio I 630 / I 488 Therefore, the optimal detection time of the fluorescence sensor was determined to be 10 min.

[0105] 3) Test the selectivity of fluorescent sensors for different antibiotics

[0106] The fluorescent sensor prepared in Example 1 was dispersed in ultrapure water to prepare a fluorescent sensor solution with a concentration of 1.0 mg / mL. The fluorescent sensor solution was divided into several portions, each with 2 mL. 10 μL of different antibiotic solutions derived from fluorescent amine with a concentration of 2.0 mg / L were added dropwise to each solution. The mixture was incubated for 10 min. After the fluorescence was stabilized, the fluorescence spectrum of each solution was measured. The selectivity of the sensor was evaluated based on the ratio of the fluorescence intensity at 488 nm and 630 nm. The results are shown in FIG. Figure 4 As shown in the figure, it can be seen that in all test groups, the fluorescent sensor only produced an obvious response to the sulfamethoxazole derivatives. 488 / I 630 The value increased significantly, and the fluorescence sensor did not respond significantly to other antibiotics derived from fluorescent amine, indicating that the fluorescence sensor prepared in Example 1 has a good selective response to sulfamethoxazole derivatives.

[0107] 4) Testing the detection performance of the fluorescent sensor for sulfamethoxazole derivatives

[0108] The fluorescent sensor prepared in Example 1 was dispersed in ultrapure water to prepare a fluorescent sensor solution with a concentration of 1.0 mg / mL. The fluorescent sensor solution was divided into several portions, each with 2 mL. 10 μL of sulfamethoxazole derivative solutions of different concentrations were added dropwise to the solutions respectively. One portion was not added with the sulfamethoxazole derivative solution and was used as a blank sample. Finally, a series of mixed solutions with concentrations of 0 μg / L, 5 μg / L, 10 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, 150 μg / L, 200 μg / L, 300 μg / L, 500 μg / L, 750 μg / L and 1000 μg / L were obtained. After the mixed solution was incubated for 10 min, the fluorescence spectrum of the mixed solution was measured by a fluorescence spectrometer, as shown in FIG. Figure 5 As shown, at the same time, the fluorescence intensity ratio I 488 / I 630 The value is the vertical axis, and the concentration of sulfamethoxazole is the horizontal axis. The fitting curve is drawn. The result is as follows Figure 6 As shown;

[0109] from Figure 5 It can be seen that as the concentration of sulfamethoxazole gradually increases, the fluorescence spectrum at 630 nm hardly changes, while the fluorescence intensity at 488 nm gradually increases with the increase of the concentration of sulfamethoxazole.

[0110] from Figure 6 As you can see, I 488 / I 630 The values ​​of sulfamethoxazole and sulfamethoxazole concentration showed two fitting curves with different slopes. The fitting curve R 2 All of them are greater than 0.989, indicating that the fluorescence sensor has a good linear relationship in response to sulfamethoxazole derivatives and can realize the quantitative detection of sulfamethoxazole. The detection limit is 1.2 ng / mL calculated by 3σ / k, where σ is the standard deviation of the blank sample and k is the slope of the calibration curve.

[0111] 5) Test the detection performance of the fluorescence sensor for sulfamethoxazole derivatives in actual samples

[0112] The fluorescent sensor prepared in Example 1 was dispersed in ultrapure water to prepare a fluorescent sensor solution with a concentration of 1.0 mg / mL for later use;

[0113] Mix 12.9 g C6H8O7·H2O, 10.9 g Na2HPO4·12H2O and 39.2 g EDTA, and dilute to 1000 mL with ultrapure water to obtain Mcllvaine-Na2 EDTA buffer with a pH of 5.2, which is set aside;

[0114] Beef and fish samples were prepared into minced meat by high-speed grinder. Sulfadimethoxazole standard solution was spiked into the minced meat by external standard method. The spiked levels were 10 μg / kg, 30 μg / kg and 50 μg / kg, respectively. 2 g of sample was weighed into a centrifuge tube, 10 mL of Mcllvaine-Na2 EDTA buffer was added, vortexed for 1 min, ultrasonicated for 10 min, vortexed once during the period, centrifuged at 10000 rpm for 5 min at -2 °C, 5 mL of ethyl acetate was added to remove fat, the aqueous phase was collected, and the aqueous phase was directly analyzed by LC-MS after being filtered through a 0.22 μm membrane. At the same time, the solution passed through a 0.22 μm membrane was added with a fluorescent amine solution for derivatization for 7 min, 10 μL of the derivatization solution was added with 2 mL of a 1.0 mg / mL fluorescent sensor solution, incubated for 10 min, and the fluorescence spectrum of the mixed solution was measured by a fluorescence spectrometer. The content of sulfadimethoxazole in the sample was calculated according to the standard curve. The results are shown in Table 1.

[0115] As can be seen from Table 1, no sulfamethoxazole was detected in the unspiked beef and fish samples, and the detection recovery of sulfamethoxazole in the spiked samples was 95.4% to 132%, with an RSD of less than 4.5%, which is consistent with the LC-MS analysis results, indicating that the fluorescent sensor prepared in Example 1 has a good detection ability for sulfamethoxazole in actual samples.

[0116] Table 1 Application of fluorescence sensor in beef and fish sample detection and spike recovery test results

[0117]

[0118] In addition, the reaction formulas for preparing sulfamethoxazole derivatives in Examples 1 to 5 of the present invention are as follows:

[0119]

[0120] Referring to the above reaction formula, it can be seen that sulfamethoxazole and fluorescein undergo a derivatization reaction at a molar ratio of 1:1. Based on this, the content of sulfamethoxazole in the real sample can be inferred according to the detected content of sulfamethoxazole derivatives, thereby indirectly realizing the detection of sulfamethoxazole in environmental and food samples.

Claims

1. A method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs, characterized in that: The steps include: Step 1, take 1.5-2mL water-soluble red fluorescent CdTe quantum dots, 10-20mL cyclohexane, 3-4mL n-hexanol and 3-4mL Triton X-100 and add them into a round-bottom flask, stir, then add 100-200μL of 25% ammonia water and 100-150μL of 0.075% diethylene glycol diacrylate solution by volume, continue stirring, then add 100-300μL tetraethyl orthosilicate, continue stirring for 16-24h, after the reaction is completed, add 100-200μL acetone, demulsify, centrifuge, wash the precipitate, and dry it at low temperature under vacuum conditions to obtain silica-coated R-CdTe nanospheres, recorded as R-CdTe@SiO2; Step 2, weigh 0.5-1 g R-CdTe@SiO2 into a round-bottom flask, add 1.0-3.0 mL γ-methacryloxypropyltrimethoxysilane and 40-60 mL anhydrous toluene, mix well, react at 70-90° C. for 8-12 h, centrifuge, wash the precipitate, and dry it at low temperature under vacuum to obtain vinyl-modified R-CdTe@SiO2, recorded as KH-570-R-CdTe@SiO2; Step 3, taking 5-10 mL of a 1 mmol / mL aqueous solution of fluorescent amine and 5-10 mL of a 1 mmol / mL aqueous solution of sulfamethoxazole, mixing them, standing at 20-35° C. for 7-10 min to allow a derivatization reaction to occur, and obtaining a sulfamethoxazole derivative solution having green fluorescence; Step 4, take 3-6 mL of sulfamethoxazole derivative solution and add it to 25 mL of acetonitrile, then add 0.6 μmol of methacrylic acid, ultrasonicate, self-assemble at 4-6°C for 4-8 hours, then add 0.02-0.06 g of azobisisobutyronitrile, 0.5-1.5 mL of ethylene glycol dimethacrylate and 0.5-2.5 mg of KH-570-R-CdTe@SiO2 in sequence to obtain a mixed solution A, first fill the mixed solution A with nitrogen to deoxygenate, then transfer it to a constant temperature oil bath at 60-80°C, react for 16-24 hours, after the reaction is completed, centrifuge, and wash the precipitate repeatedly several times until the template molecule can no longer be detected in the elution solution to obtain a ratiometric fluorescent sensor.

2. The method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs according to claim 1, characterized in that: The water-soluble red fluorescent CdTe quantum dots in step 1 are prepared by the following method: Step 1.1, weigh 35-40 mg of tellurium powder, 20-60 mg of KBH4 and 2 mL of ultrapure water, put them into a round-bottom flask, introduce nitrogen into the round-bottom flask to remove oxygen in the flask, react at 60-80° C. to form a transparent solution, which is the precursor NaHTe solution; Step 1.2: first take 30-60 mg CdCl2·2.5H2O and add it into a three-necked flask, then add 60-80 mL ultrapure water, stir well, then add 50-75 μL thioglycolic acid, mix well, adjust the pH value to alkaline, fill with nitrogen to remove oxygen for 15 minutes, then quickly add 2 mL of precursor NaHTe solution, stir at 100-120°C for 16 hours to obtain water-soluble red fluorescent CdTe quantum dots.

3. The method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs according to claim 2, characterized in that: In the step 1.2, a NaOH solution with a concentration of 0.2 mol / L is used to adjust the pH to 9-11.

4. The method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs according to claim 1, characterized in that: The stirring time of step 1 is 20 to 40 minutes.

5. The method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs according to claim 1, characterized in that: The stirring time of step 1 is 10 to 40 minutes.

6. The method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs according to claim 1, characterized in that: The washing in step 1 and step 2 is performed by washing with anhydrous ethanol.

7. The method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs according to claim 1, characterized in that: The ultrasonic time in step 4 is 5 to 10 minutes.

8. The method for preparing the fluorescent sensor for detecting sulfamethoxazole drugs according to claim 1, characterized in that: The time for filling with nitrogen to remove oxygen in step 4 is 15 to 30 minutes.

9. The method for preparing a fluorescent sensor for detecting sulfamethoxazole drugs according to claim 1, characterized in that: The washing in step 4 is performed by repeatedly washing several times with a 1.5% by mass acetic acid ethanol solution.

10. A fluorescent sensor for detecting sulfamethoxazole prepared by the method according to any one of claims 1 to 9, characterized in that: It has a fluorescent response to sulfamethoxazole derivatives.

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