Molecularly imprinted polymer ratiometric fluorescent probe based on two-color carbon dots as well as preparation method and application of molecularly imprinted polymer ratiometric fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent probes, and provides a design strategy of a molecularly imprinted polymer ratiometric fluorescent probe based on two-color carbon dots in order to solve the problems that a current molecularly imprinted polymer fluorescent probe based on a single emission carbon dot is poor in detection result precision and is easily influenced by external environment interference and operation errors and the like. The fluorescent probe can be used for detecting catechol. The blue light-emitting carbon dots and the yellow light-emitting carbon dots are synthesized through traditional hydro-thermal synthesis; on the basis of blue luminous carbon dots, a molecularly imprinted polymer based on the blue luminous carbon dots is synthesized through a sol-gel method, and the molecularly imprinted polymer is combined with yellow luminous carbon dots through physical action. The ratiometric fluorescence strategy has the advantages of rapidness, effectiveness, stable performance, strong anti-interference capability, no need of expensive instruments and equipment, simplicity and convenience in operation, low detection cost and the like, solves the problem of low selectivity of single-fluorescence carbon dot detection, increases signal sources of imprinted polymer detection, and more importantly, reduces background interference due to colorimetric imprinting, so that the detection efficiency is improved. And the detection accuracy is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a molecular imprinting polymer ratio fluorescent probe based on dual-color carbon dots, and a preparation method and application thereof. Background Art

[0002] Catechol (CT), also known as catechol, has a unique chemical reactivity due to the positional structure of its benzene ring and hydroxyl group. It is widely used in the production of drugs, cosmetics, and dyes. It has good water solubility, is easy to penetrate and migrate in the environment, and is widely present in soil and water resources. Catechol has high stability and toxicity, has a significant inhibitory effect on the central nervous system of humans and animals, and is a human carcinogen. Therefore, in order to protect the environment and human health, it is of great significance to develop an accurate, sensitive, and fast method to determine catechol in water environments.

[0003] Currently reported methods for detecting CT include spectroscopy, chromatography, and electrochemistry. Spectroscopy requires expensive instruments and complex operating procedures, while the traditional electrodes used in electrochemical methods will produce contamination effects when detecting CT. Therefore, fluorescence spectroscopy, as a fast, simple, and efficient analytical technique, has become a relatively feasible method. Since the fluorescent signal usually has a high contrast with the background noise, this method can be used to track substances at extremely low concentrations, which is particularly valuable in the fields of environmental monitoring, biomarker detection, and drug discovery.

[0004] Carbon dots (CDs), as a fluorescent nanomaterial with excellent photoluminescence properties, are widely used in the design of optical sensing systems for the analysis of trace substances. They have extremely high sensitivity, but poor detection selectivity. Molecular imprinting technology (MIT) is one of the strategies to improve the selectivity of CDs sensing systems. Molecular imprinting polymers (MIPs) not only have the same selectivity and specificity as antibodies, but also make up for the defects of antibodies such as poor stability, difficulty in obtaining, and high prices. The combination of MIPs and CDs can solve the problems of poor stability and poor selectivity of CDs-based fluorescent sensing systems. At present, the detection results of molecular imprinting polymer fluorescent probes based on single-emission carbon dots have poor accuracy and are easily affected by external environmental interference and operational errors. The introduction of dual-color fluorescent carbon dots and the design of ratiometric fluorescent probes can overcome these shortcomings. Summary of the invention

[0005] In order to solve the problems that the current molecular imprinted polymer fluorescent probe based on single-emission carbon dots has poor detection accuracy and is easily affected by external environmental interference and operation errors, the present invention provides a molecular imprinted polymer ratio fluorescent probe based on dual-color carbon dots and a preparation method and application thereof, and the application of the fluorescent probe in the detection of catechol.

[0006] The present invention is realized by the following technical scheme: a molecular imprinting polymer ratio fluorescent probe based on dual-color carbon dots, wherein citric acid is used as a carbon source and ethylenediamine is used as a nitrogen source to hydrothermally synthesize blue luminescent carbon dots; on the basis of the blue luminescent carbon dots, catechol CT is used as a template molecule, 3-aminopropyltriethoxysilane APTES is used as a functional monomer, tetraethyl silicate TEOS is used as a crosslinking agent, and ammonia water is used as a catalyst to synthesize a molecular imprinting polymer based on the blue luminescent carbon dots through a sol-gel method; and o-phenylenediamine is used as a solute and ethanol is used as a solvent to synthesize yellow luminescent carbon dots through high-temperature carbonization.

[0007] The method for preparing the molecularly imprinted polymer ratio fluorescent probe based on dual-color carbon dots comprises the following specific steps: (1) Obtaining the precursor: 2.00 g of citric acid and 1 mL of ethylenediamine were dissolved in 40 mL of water. The solution was transferred to a high-pressure reactor and reacted at 150 °C for 2 h to obtain a brown-yellow solution, which was marked as blue-luminescent carbon dots B-CDs. Take 0.30g of o-phenylenediamine and dissolve it in 50mL of ethanol. Transfer the solution to a high-pressure reactor lined with polytetrafluoroethylene. After reacting at 180℃ for 12h, a bright yellow solution is obtained, which is marked as yellow luminescent carbon dots Y-CDs. (2) Obtaining powdered CDs: The two carbon dot solutions obtained in step (1) were centrifuged at 8000 r / min for 20 min respectively, and the supernatant was purified in a 500 Da dialysis bag for 24 h, wherein the water was changed every 4 h; finally, the purified liquid was freeze-dried for 48 h to obtain a solid powder; (3) Preparation of molecular imprinting polymer B-CDs@MIPs ratiometric fluorescent probe based on blue luminescent carbon dots: Step (1) 30 mg of the obtained B-CDs were added with 3 ml of deionized water to obtain a B-CDs stock solution with a concentration of 10 mg / mL, and ultrasonically dispersed for 30 minutes; The B-CDs stock solution was transferred to a solvent system consisting of 16.67 mL of ethanol and 33.33 mL of water, and 200 μL of NH3•H2O was added, and stirred at a stirring speed of 500 r / min for 15 minutes; then 37.8 mg of the template molecule catechol CT and 484 μL of the functional monomer 3-aminopropyltriethoxysilane (APTES) were added to react for 30 minutes; finally, 0.918 mL of the crosslinker TEOS was dispersed in 9.18 mL of ethanol and added to the reaction system; the reaction was protected from light throughout the process and carried out for 24 hours; After the reaction is completed, centrifuge at 8000 r / min for 10 minutes, discard the supernatant, and retain the precipitate; then, prepare an eluent composed of a mixture of methanol and acetic acid in a volume ratio of 9:1, take 12 mL of the eluent to elute the precipitate, and place it in an ultrasonic instrument for 5 minutes. Finally, centrifuge and retain the supernatant, and elute the precipitate for the second time; retain the supernatant and precipitate obtained from the two elutions respectively, and the precipitate is B-CDs@MIPs, which is placed in a vacuum drying oven at 50°C for 12 hours to obtain solid B-CDs@MIPs.

[0008] The application of the molecular imprinting polymer ratio fluorescence probe based on dual-color carbon dots prepared by the method in CT detection comprises the following steps: (1) Preparation of B-CDs@MIPs stock solution: 10 mg of B-CDs@MIPs was placed in 2 ml of deionized water to prepare a stock solution with a concentration of 5 mg / mL; (2) Preparation of Y-CDs stock solution: 0.5 mg of Y-CDs was placed in 1 ml of deionized water to prepare a stock solution with a concentration of 0.5 mg / mL; (3) Preparation of CT stock solution: Accurately weigh 10 mg of CT powder, add it to 2 mL of secondary water, stir to dissolve, and prepare a stock solution with a concentration of 5 mg / mL; (4) Probe B-CDs@MIPs / Y-CDs was used as a ratiometric fluorescent probe to detect CT: A linear equation was obtained between the CT content and the fluorescence intensity of the B-CDs@MIPs / Y-CDs system: 0.12 mL of B-CDs@MIPs stock solution was added to it with several gradient volumes of CT stock solution and pH=5 BR buffer solution, and the volume was fixed to 1.97 mL. After incubation for 50 minutes, 0.03 mL of Y-CDs stock solution was added; the final concentrations of B-CDs@MIPs, Y-CDs, and CT were controlled to be 0.30 mg / mL, 0.0075 mg / mL, and 0.005−0.5 mg / mL; at an excitation wavelength of 360 nm, the fluorescence intensity ratio F of B-CDs@MIPs / Y-CDs at 440 nm and 555 nm was recorded. b / F y ; Linear fitting of CT concentration and fluorescence intensity ratio F was performed by Origin software b / F y , we get the linear equation: F b / F y =-1.25466 C CT +1.11027, R 2 =0.994, the corresponding linear range is: 0.005-0.5mg / mL, and the minimum detection limit is 0.146 ug / mL; (5) Calculate the imprinting factor IF: IF=-1.25466 / -0.45929 =2.73.

[0009] The dual-color carbon dot molecular imprinting polymer ratio fluorescence probe is used in actual sample detection of CT.

[0010] The actual sample is a tap water sample, a rainwater sample or a lake water sample; the specific steps include: (1) Centrifuge the sample at 8000 r / min for 10 min, filter once with a 0.22 μm microporous membrane, extract the supernatant, and obtain the sample reserve solution for later use; (2) 0.12 mL of B-CDs@MIPs stock solution and different volumes of sample stock solution were diluted to 1.97 mL with BR buffer at pH = 5. After incubation for 50 min, 0.03 mL of Y-CDs stock solution was added to make the final concentration of B-CDs@MIPs in the system 0.30 mg / mL and the final concentration of Y-CDs in the system 0.0075 mg / mL. The fluorescence intensity F of the mixed solution at 440 nm and 555 nm was measured and recorded. b and F y , calculate F b / F y ; Substitute into the linear equation F b / F y =-1.25466 C CT +1.11027, the CT content in the actual sample is calculated.

[0011] The blue luminescent carbon dots and yellow luminescent carbon dots of the present invention are easy to prepare, and can still have bright fluorescence after being coated with molecular imprinting polymers; the probe is based on dual-color fluorescence ratio detection, has good selectivity, high sensitivity, and strong anti-background interference ability. The probe of the present invention can utilize the high fluorescence and high sensitivity of carbon dots, combined with the advantages of ratio detection, to achieve specific detection of CT in water environments. The present invention has the advantages of rapid and effective, stable performance, strong anti-interference ability, no need for expensive instruments and equipment, simple operation, and low detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The fluorescence spectra of B-CDs prepared in Example 1 at different excitation wavelengths; Figure 2 The fluorescence spectra of the Y-CDs prepared in Example 1 at different excitation wavelengths; Figure 3 The UV absorption spectra of B-CDs@MIPs prepared in Example 2 before and after elution; Figure 4 To explore the optimal excitation spectrum of the mixed system of B-CDs@MIPs and Y-CDs prepared in Example 2; Figure 5 IR spectra of B-CDs, B-CDs@MIPs and B-CDs@NIPs prepared in Example 2; Figure 6 X-ray photoelectron spectroscopy (XPS) of B-CDs@MIPs prepared in Example 2; Figure 7 The fluorescence spectrum of the B-CDs@MIPs / Y-CDs system with optimized Y-CDs concentration in Example 2; Figure 8 The fluorescence spectra of B-CDs@MIPs / Y-CDs after adding different concentrations of CT into the B-CDs@MIPs / Y-CDs solution; Fig. 9 This is the linear fitting diagram between CT concentration and the change of B-CDs@MIPs / Y-CDs fluorescence intensity; the corresponding linear range is 0.005−0.500 mg / mL, and the minimum detection limit is 0.146 ug / mL; Fig.10 It is the linear fitting diagram between CT concentration and the change of B-CDs@NIPs / Y-CDs fluorescence intensity; Fig.11 It is the linear fitting diagram between CT concentration and the change of B-CDs@NIPs / Y-CDs fluorescence intensity; the corresponding linear range is 0.005−0.500 mg / mL; Fig.12 For various ions (Mg 2+ , Zn 2+ , K + , Cu 2+ , Br - , SO4 2- , NO3 - , HCO3 - , I - )The interference experiment results of CT detection using ratio fluorescence method; Fig.13 Five substances with similar structures to CT were selected to replace CT and the results of the fluorescence response of B-CDs@MIPs / Y-CDs system or B-CDs@NIPs / Y-CDs were obtained; Fig.14 Schematic diagram of the interaction between the B-CDs@MIPs / Y-CDs system and CT. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0015] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0016] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0017] Example 1: Preparation and characterization of B-CDs, Y-CDs, B-CDs@MIPs and B-CDs@NIPs (1) Take 2.00 g of citric acid and 1 mL of ethylenediamine and dissolve them in 40 mL of water. The solution is transferred to a polytetrafluoroethylene-lined autoclave and reacted at 150 °C for 2 h to obtain a brown-yellow solution, which is labeled as blue-luminescent carbon dots (B-CDs). Take 0.30 g of o-phenylenediamine and dissolve it in 50 mL of ethanol. The solution is transferred to a polytetrafluoroethylene-lined autoclave and reacted at 180 °C for 12 h to obtain a bright yellow solution, which is labeled as blue-luminescent carbon dots (Y-CDs).

[0018] (2) The two carbon dot solutions were centrifuged at 8000 r / min for 20 min, and the supernatant was purified in a 500 Da dialysis bag for 24 h, with water replaced every 4 h. Finally, the purified liquid was freeze-dried for 48 h to obtain solid powders B-CDs and Y-CDs.

[0019] (3) Prepare a 10 mg / mL solution of B-CDs, disperse it by ultrasonic for 10 min, take 3 mL and transfer it to a solvent system consisting of 16.67 mL ethanol and 33.33 mL water, add 200 μL NH3•H2O, and stir at 500 r / min for 15 min; then add 37.8 mg template molecule CT and 484 μL functional monomer APTES (3-aminopropyltriethoxysilane) to react for 30 min; finally, disperse 0.918 mL crosslinker TEOS in 9.18 mL ethanol and put it into the reaction system. The reaction was protected from light and carried out for 24 hours. After the reaction was completed, centrifuge at 8000 r / min for 10 min, discard the supernatant, and retain the precipitate. Then, prepare an eluent composed of a mixture of methanol and acetic acid in a volume ratio of 9:1, take 12 mL to elute the precipitate, and place it in an ultrasonicator for 5 min, finally centrifuge and retain the supernatant, and elute the precipitate for the second time. The supernatant and precipitate obtained from the two elutions were retained respectively, and the precipitate was B-CDs@MIPs. The prepared B-CDs@MIPs were placed in a vacuum drying oven at 50°C for 12 hours and then stored in a drying dish.

[0020] (4) Preparation of non-molecularly imprinted polymers (B-CDs@NIPs) coated with blue luminescent carbon dots: The specific operation is the same as step (3), except that the template molecule CT is not added. The prepared B-CDs@NIPs are placed in a vacuum drying oven at 50°C for 12 hours and then stored in a drying dish.

[0021] The spectral properties of the obtained B-CDs and Y-CDs powders are shown in Figure 1 and Figure 2 .

[0022] Figure 1 Figure 2 is the fluorescence emission spectra of B-CDs under different excitation spectra. It can be seen that B-CDs has a better emission spectrum under 350 nm and 360 nm excitation.

[0023] Figure 2 Figure 2 is the fluorescence emission spectrum of Y-CDs under different excitation spectra. It can be seen that under excitation at 350 nm and 360 nm, it also has a good emission spectrum.

[0024] Figure 3Figure 3 shows the changes in the UV spectrum of B-CDs@MIPs before and after elution. The UV absorption peak of CT appears at 276 nm. CT exists before elution of B-CDs@MIPs, but the absorption peak of CT is greatly weakened or even disappears after elution, proving that the elution of the template molecule CT is successful.

[0025] Figure 4 After B-CDs@MIPs and Y-CDs are mixed, there is a relatively good emission spectrum under 360 nm excitation. Therefore, the optimal excitation wavelength of the ratio system is selected as 360 nm, and the corresponding emission wavelengths are 440 nm and 555 nm.

[0026] Figure 5 The infrared spectra of B-CDs, B-CDs@MIPs and B-CDs@NIPs are shown in Figure 1. As can be seen from the figure, the structure of N-CDs is not changed after the imprinting layer is coated, and the wavelength of 460 cm is increased. -1 Si-O bonds, and Figure 6 In addition, it can be found that the spectrum of B-CDs@NIPs is no different from that of B-CDs@MIPs. This is because the difference between the two during synthesis is whether the template molecule is added or not, and after elution, the element and group composition of the two are almost the same. Example 2: Study of B-CDs@MIPs / Y-CDs as a ratiometric fluorescent probe 1. CT detection based on B-CDs@MIPs / Y-CDs ratio fluorescence (1) Preparation of B-CDs@MIPs stock solution: Accurately weigh 20 mg of B-CDs@MIPs, add 5 mL of deionized water to prepare a B-CDs@MIPs stock solution with a concentration of 5 mg / mL, and ultrasonically disperse for 1 minute.

[0027] (2) Preparation of CT stock solution: Accurately weigh 20 mg of CT, add 4 mL of deionized water to prepare a CT stock solution with a concentration of 5 mg / mL, and ultrasonically disperse it for 1 minute.

[0028] (3) Preparation of Y-CDs stock solution: Accurately weigh 2 mg of CT, add 4 mL of deionized water to prepare a CT stock solution with a concentration of 0.5 mg / mL, and ultrasonically disperse it for 1 minute. Figure 7 To optimize the fluorescence spectrum of Y-CDs concentration, it can be seen from the figure that 0.75 μg / mL was selected as the final Y-CDs concentration.

[0029] (4) CT content and fluorescence signal F of B-CDs@MIPs b / F yObtaining the linear equation between: accurately measure 0.12 mL of B-CDs@MIPs stock solution, add several gradient volumes of CT stock solution and pH=5 BR buffer solution to it, and make it up to 1.97 mL. After incubation for 50 minutes, add 0.03 mL of Y-CDs stock solution; at this time, the final concentration of B-CDs@MIPs is 0.30 mg / mL, and the concentration range of CT is 0.005-0.500 mg / mL; under an excitation wavelength of 360 nm, measure and record the fluorescence intensity values ​​of the mixed system containing different CT concentrations at wavelengths of 440 nm and 555 nm, and mark them as F b / F y .

[0030] (5) Linear fitting of CT concentration and fluorescence intensity ratio F using Origin software b / F y , the results are shown in Figure 8 The results show that with the addition of different concentrations of CT solution, the fluorescence intensity of B-CDs@MIPs at 440 nm gradually decreases, but remains almost unchanged at 555 nm. Therefore, an optical probe for ratiometric detection of CT was constructed. The linear relationship of the detection system for detecting CT is shown in Fig. 9 As shown, the linear range is F b / F y =-1.25466 C CT + 1.11027, detection limit is 0.146 ug / mL.

[0031] 2. Fluorescence detection of CT based on B-CDs@NIPs / Y-CDs ratio Step 1, prepare B-CDs@NIPs stock solution: accurately weigh 20 mg B-CDs@NIPs, add 5 mL deionized water to prepare a B-CDs@NIPs stock solution with a concentration of 5 mg / mL, and ultrasonically disperse for 1 minute.

[0032] Step 2, prepare CT stock solution: accurately weigh 20 mg CT, add 4 mL deionized water to prepare a CT stock solution with a concentration of 5 mg / mL, and ultrasonically disperse for 1 minute.

[0033] Step 3, prepare Y-CDs stock solution: accurately weigh 2 mg of CT, add 4 mL of deionized water to prepare a CT stock solution with a concentration of 0.5 mg / mL, and ultrasonically disperse for 1 minute.

[0034] Step 4: CT content and fluorescence signal F of B-CDs@NIPs b / F yObtaining the linear equation between: Take 0.12 mL of B-CDs@NIPs stock solution, add several gradient volumes of CT stock solution and pH=5 BR buffer solution to it, and make it up to 1.97 mL. After incubation for 50 minutes, add 0.03 mL of Y-CDs stock solution; at this time, the final concentration of B-CDs@NIPs is 0.30 mg / mL, and the concentration range of CT is 0.005-0.500 mg / mL; under the excitation wavelength of 360 nm, measure and record the fluorescence intensity values ​​of the mixed system containing different CT concentrations at wavelengths of 440 nm and 555 nm, and mark them as F b / F y .

[0035] Step 4: Linear fitting of CT concentration and fluorescence intensity ratio F by Origin software b / F y , the results are shown in Fig.10 . It shows that with the addition of different concentrations of CT solution, the fluorescence intensity of B-CDs@NIPs at 440 nm gradually decreases, but remains almost unchanged at 555 nm. Most importantly, compared with B-CDs@MIPs / Y-CDs, the degree of change of B-CDs@NIPs / Y-CDs at 440 nm is very small. This is because when synthesizing B-CDs@MIPs, the template molecule CT is added to form an imprinted cavity, which can specifically recognize CT. However, B-CDs@NIPs has no specificity because no template molecules are added. It can only rely on physical adsorption to adsorb a small amount of CT and quench a small part of its fluorescence. The specific mechanism diagram is shown in the figure. Fig.14 The linear relationship of the detection system to detect CT is as follows Fig.11 As shown, the linear range is F b / F y =-0.45929 C CT +0.97906 R 2 =0.991. Therefore, the imprinting factor is calculated to be 4.15.

[0036] 3. Anti-interference experiment of CT detection based on B-CDs@MIPs / Y-CDs ratio fluorescence Step 1, prepare B-CDs@MIPs stock solution: accurately weigh 20 mg B-CDs, add 5 mL deionized water to prepare a B-CDs@MIPs stock solution with a concentration of 5 mg / mL, and ultrasonically disperse for 1 minute.

[0037] Step 2, prepare CT stock solution: accurately weigh 20 mg CT, add 4 mL deionized water to prepare a CT stock solution with a concentration of 5 mg / mL, and ultrasonically disperse for 1 minute.

[0038] Step 3, prepare Y-CDs stock solution: accurately weigh 2 mg of CT, add 4 mL of deionized water to prepare a CT stock solution with a concentration of 0.5 mg / mL, and ultrasonically disperse for 1 minute.

[0039] Step 4: Weigh a certain mass of ions (Mg 2+ , Zn 2+ , K + , Cu 2+ , Br - , SO4 2- , NO3 - , HCO3 - ,I - ), add 10 mL of secondary water to prepare ion stock solutions with a concentration of 10 mg / mL.

[0040] Step 5: Accurately measure 0.12 mL of B-CDs@MIPs stock solution, add 0.2 mL of CT stock solution and 0.5 mL of ion stock solution, and dilute to 1.97 mL. After incubation for 50 minutes, add 0.03 mL of Y-CDs stock solution. At this time, the final concentration of B-CDs@MIPs is 0.30 mg / mL, the final concentration of Y-CDs is 0.0075 mg / mL, the final concentration of CT is 0.5 mg / mL, and the concentration of various interfering ions is 2.5 mg / mL; at an excitation wavelength of 360 nm, measure and record the fluorescence intensity of the mixed system at wavelengths of 440nm and 555nm, and mark it as F b / F y The experimental results are shown in Fig.12 .

[0041] Fig.12 This is a study on the interference of different ions on the detection of CT by the B-CDs@MIPs / Y-CDs system, indicating that the ions studied will not cause the fluorescence (F b / F y ) changed significantly, indicating that the B-CDs@MIPs / Y-CDs system has good anti-interference ability for CT detection.

[0042] 4. Selective experiments based on ratiometric fluorescence detection of CT by B-CDs@MIPs / Y-CDs or B-CDs@NIPs / Y-CDs Step 1, prepare B-CDs@MIPs stock solution: accurately weigh 20 mg B-CDs@MIPs, add 5 mL deionized water to prepare a B-CDs@MIPs stock solution with a concentration of 5 mg / mL, and ultrasonically disperse for 1 minute.

[0043] Step 2, prepare B-CDs@NIPs stock solution: accurately weigh 20 mg B-CDs@NIPs, add 5 mL deionized water to prepare a B-CDs@NIPs stock solution with a concentration of 5 mg / mL, and ultrasonically disperse for 1 minute.

[0044] Step 2, prepare CT stock solution: accurately weigh 20 mg CT, add 4 mL deionized water to prepare a CT stock solution with a concentration of 5 mg / mL, and ultrasonically disperse for 1 minute.

[0045] Step 3, prepare Y-CDs stock solution: accurately weigh 2 mg of CT, add 4 mL of deionized water to prepare a CT stock solution with a concentration of 0.5 mg / mL, and ultrasonically disperse for 1 minute.

[0046] Step 4: weigh 5 mg of resorcinol, hydroquinone, phenol, m-phenylenediamine and p-phenylenediamine, add 1 mL of secondary water, and prepare structural analog stock solutions with a concentration of 5 mg / mL.

[0047] Step 5: Accurately measure 0.12 mL of B-CDs@MIPs stock solution or B-CDs@NIPs stock solution, add 0.20 mL of CT stock solution and 0.20 mL of CT stock solution or 0.20 mL of structural analog stock solution, and adjust the volume to 1.97 mL. After incubation for 50 min, add 0.03 mL of Y-CDs stock solution. At this time, the final concentration of B-CDs@MIPs or B-CDs@NIPs is 0.30 mg / mL, the final concentration of Y-CDs is 0.0075 mg / mL, the final concentration of structural analogs is 0.50 mg / mL, and the final concentration of CT is 0.50 mg / mL; at an excitation wavelength of 360 nm, measure and record the fluorescence intensity values ​​of the mixed system containing different CT concentrations at wavelengths of 440 nm and 555 nm, and mark them as F b / F y The experimental results are shown in Fig.12 .

[0048] Fig.13 Five substances with similar structures to CT were selected to replace CT in the fluorescence response of the B-CDs@MIPs / Y-CDs system. The results show that the fluorescence change of the B-CDs@MIPs / Y-CDs system will be quenched to the greatest extent only after the addition of CT.

[0049] Example 3: CT content detection in actual samples Step 1: Take 10 mL of tap water from the 515 laboratory of the Institute of Environmental Sciences of Shanxi University, centrifuge it at 8000 r / min for 10 min, filter the supernatant once with a 0.22 μm microporous membrane to obtain a tap water sample stock solution for later use.

[0050] Step 2: Collect 10 mL of rainwater from May 2024, centrifuge at 8000 r / min for 10 min, filter the supernatant once with a 0.22 μm microporous membrane to obtain a rainwater sample reserve solution for later use.

[0051] Step 3: Take 10 mL of water from Lingde Lake of Shanxi University, centrifuge it at 8000 r / min for 10 min, filter the supernatant once with a 0.22 μm microporous membrane to obtain a lake water sample reserve solution for later use.

[0052] Step 4: First add 0.12 mL of B-CDs@MIPs stock solution, then add 1 mL of pH=5 BR buffer, and dilute to 1.97 mL with various sample stock solutions. After incubation for 50 min, add 0.03 mL of Y-CDs stock solution. The final concentration of B-CDs@MIPs in the system is 0.30 mg / mL, and the final concentration of Y-CDs is 0.0075 mg / mL. Measure and record the fluorescence intensity F of the mixed solution at 440 nm and 555 nm respectively. b and F y , calculate F b / F y .

[0053] Step 5: F b / F y Substitute into the linear equation Substitute into the linear equation F b / F y =-1.25466 C CT +1.11027, the CT content in the actual sample was calculated, and the results are shown in Table 1.

[0054] Table 1 shows the results of CT content detection in the reagent samples. The results show that the tap water samples, rain water samples and lake water samples tested all contain a certain amount of CT. This method can be used to detect the content of CT in tap water, rain water samples and lake water samples, and is not affected by other components in the sample solution. It can be seen that the ratio fluorescence method based on B-CDs@MIPs / Y-CDs is suitable for detecting the CT content in actual samples, and the detection results will not be affected by other interferences, and has potential practical application value.

[0055] Table 1: CT content test results in soil samples, rainwater samples and lake water samples ND: Not Detected Example 4: Spiked recovery experiment of CT in actual samples Step 1: Take 1.0 mL of 5 mg / mL CT stock solution for later use; Step 2: First add 0.12 mL of B-CDs@MIPs stock solution, then add 12 μL, 80 μL and 140 μL of CT standard solution to the above system, then add 1 mL of BR buffer solution with pH=5, and finally use various sample stock solutions to make the volume 1.97 mL. After incubation for 50 min, add 0.03 mL of Y-CDs stock solution. The final concentration of B-CDs@MIPs in the system is 0.30 mg / mL, the final concentration of Y-CDs is 0.0075 mg / mL, and the final concentration of CT is 0.03 mg / mL, 0.20 mg / mL and 0.35 mg / mL, respectively. Measure and record the fluorescence intensity F of the mixed solution at 440 nm and 555 nm respectively. b and F y , calculate F b / F y ; Substitute into the linear equation F b / F y =-1.25466 C CT +1.11027 to calculate the content of CT in the system, and further calculate the spiked recovery of CT in the stock solutions of tap water samples, rainwater samples and lake water samples. The results are shown in Table 2.

[0056] Table 2 shows that the spiked recoveries of CT in the three actual samples ranged from 90.3 to 110.5%, and the relative standard deviation was less than 7.7%, indicating that B-CDs@MIPs / Y-CDs had good reproducibility when used for the detection of CT in actual samples.

[0057] Table 2: Results of spike recovery experiments of CT in tap water, lake water and rainwater samples Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A molecularly imprinted polymer ratio fluorescent probe based on dual-color carbon dots, characterized in that: Blue luminescent carbon dots were synthesized by hydrothermal method using citric acid as carbon source and ethylenediamine as nitrogen source. Based on the blue luminescent carbon dots, molecular imprinting polymers based on blue luminescent carbon dots were synthesized by sol-gel method using catechol CT as template molecule, 3-aminopropyltriethoxysilane APTES as functional monomer, tetraethyl silicate TEOS as cross-linking agent and ammonia water as catalyst. Yellow luminescent carbon dots were synthesized by high temperature carbonization using o-phenylenediamine as solute and ethanol as solvent.

2. The method for preparing the molecularly imprinted polymer ratio fluorescent probe based on dual-color carbon dots according to claim 1, characterized in that: The specific steps are as follows: (1) Obtaining the precursor: 2.00 g of citric acid and 1 mL of ethylenediamine were dissolved in 40 mL of water. The solution was transferred to a high-pressure reactor and reacted at 150 °C for 2 h to obtain a brown-yellow solution, which was marked as blue-luminescent carbon dots B-CDs. Take 0.30g of o-phenylenediamine and dissolve it in 50mL of ethanol. Transfer the solution to a high-pressure reactor lined with polytetrafluoroethylene. After reacting at 180℃ for 12h, a bright yellow solution is obtained, which is marked as yellow luminescent carbon dots Y-CDs. (2) Obtaining powdered CDs: The two carbon dot solutions obtained in step (1) were centrifuged at 8000 r / min for 20 min respectively, and the supernatant was purified in a 500 Da dialysis bag for 24 hours, wherein the water was changed every 4 hours; finally, the purified liquid was freeze-dried for 48 hours to obtain a solid powder; (3) Preparation of molecular imprinting polymer B-CDs@MIPs ratiometric fluorescent probe based on blue luminescent carbon dots: Step (1) 30 mg of the obtained B-CDs were added with 3 ml of deionized water to obtain a B-CDs stock solution with a concentration of 10 mg / mL, and ultrasonically dispersed for 30 minutes; The B-CDs stock solution was transferred to a solvent system consisting of 16.67 mL of ethanol and 33.33 mL of water, and 200 μL of NH3•H2O was added, and stirred at a stirring speed of 500 r / min for 15 minutes; then 37.8 mg of the template molecule catechol CT and 484 μL of the functional monomer 3-aminopropyltriethoxysilane (APTES) were added to react for 30 minutes; finally, 0.918 mL of the crosslinker TEOS was dispersed in 9.18 mL of ethanol and added to the reaction system; the reaction was protected from light throughout the process and carried out for 24 hours; After the reaction is completed, centrifuge at 8000 r / min for 10 minutes, discard the supernatant, and retain the precipitate; then, prepare an eluent composed of a mixture of methanol and acetic acid in a volume ratio of 9:1, add 12 mL of the eluent to elute the precipitate, and place it in an ultrasonic instrument for 5 minutes. Finally, centrifuge and retain the supernatant, and elute the precipitate for the second time; retain the supernatant and precipitate obtained from the two elutions respectively, and the precipitate is B-CDs@MIPs, which is placed in a vacuum drying oven at 50°C for 12 hours to obtain solid B-CDs@MIPs.

3. Application of the molecular imprinted polymer ratio fluorescent probe based on dual-color carbon dots prepared by the method of claim 2 in CT detection, characterized in that: The steps include: (1) Preparation of B-CDs@MIPs stock solution: 10 mg of B-CDs@MIPs was placed in 2 ml of deionized water to prepare a stock solution with a concentration of 5 mg / mL; (2) Preparation of Y-CDs stock solution: 0.5 mg of Y-CDs was placed in 1 ml of deionized water to prepare a stock solution with a concentration of 0.5 mg / mL; (3) Preparation of CT stock solution: Accurately weigh 10 mg of CT powder, add it to 2 mL of secondary water, stir to dissolve, and prepare a stock solution with a concentration of 5 mg / mL; (4) Probe B-CDs@MIPs / Y-CDs was used as a ratiometric fluorescent probe to detect CT: A linear equation was obtained between the CT content and the fluorescence intensity of the B-CDs@MIPs / Y-CDs system: 0.12 mL of B-CDs@MIPs stock solution was added to it with several gradient volumes of CT stock solution and pH=5 BR buffer solution, and the volume was fixed to 1.97 mL. After incubation for 50 minutes, 0.030 mL of Y-CDs stock solution was added; the final concentrations of B-CDs@MIPs, Y-CDs, and CT were controlled to be 0.30 mg / mL, 0.0075 mg / mL, and 0.005-0.5 mg / mL; at an excitation wavelength of 360 nm, the fluorescence intensity ratio F of B-CDs@MIPs / Y-CDs at 440 nm and 555 nm was recorded. b / F y ; Linear fitting of CT concentration and fluorescence intensity ratio F was performed by Origin software b / F y , we get the linear equation: F b / F y =-1.25466 C CT +1.11027, R 2 =0.994, the corresponding linear ranges are: 0.005-0.5 mg / mL, and the minimum detection limit is 0.146 ug / mL; (5) Calculate the imprinting factor IF: IF=-1.25466 / -0.45929 =2.

73.

4. The use according to claim 3, characterized in that: The application of the molecular imprinting polymer ratio fluorescence probe of the dual-color carbon dots in detecting CT in actual samples is characterized in that the actual sample is a tap water sample, a rainwater sample or a lake water sample; specifically comprises the following steps: (1) Centrifuge the sample at 8000 r / min for 10 min, filter once with a 0.22 μm microporous membrane, extract the supernatant, and obtain the sample reserve solution for later use; (2) 0.12 mL of B-CDs@MIPs stock solution and different volumes of sample stock solution were added, and finally the volume was adjusted to 1.97 mL using BR buffer with pH = 5. After incubation for 50 minutes, 0.03 mL of Y-CDs stock solution was added; the final concentration of B-CDs@MIPs in the system was 0.30 mg / mL, and the final concentration of Y-CDs was 0.0075 mg / mL; the fluorescence intensity F of the mixed solution at 440 nm and 555 nm was measured and recorded. b and F y , calculate F b / F y ; Substitute into the linear equation F b / F y =-1.25466 C CT +1.11027, the CT content in the actual sample is calculated.

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