Ratio-type molecular imprinting fluorescence sensing detection method based on metal organic framework and nitrogen-doped carbon dots

Abstract

The invention provides a ratio type molecular imprinting fluorescence sensing detection method based on a metal organic framework and nitrogen-doped carbon dots, the advantages of N-CDs, Eu-MOF and a molecular imprinting layer are fused, in the presence of target molecules, the blue fluorescence (430nm) of the N-CDs is effectively enhanced due to non-radiative recombination caused by the surface defect state of the CDs, and the red fluorescence (660nm) of the Eu-MOF remains unchanged. According to the ratio type molecularly imprinted fluorescent sensing detection method, while a fluorescent response signal is displayed, the color of the imprinted polymer changes along with different concentrations of the target molecules, and sensitive visual detection of the target molecules can be realized, so that specific sensitive detection of the target molecules in a complex sample is realized; the method has the advantages of high sensitivity, simplicity and convenience in operation, good reproducibility, strong anti-interference performance and the like. According to the method, rapid and sensitive detection of organophosphorus pesticides, toxic agents and other imprinted molecules can be realized by preparing imprinted polymers of different target molecules without fluorescence interference, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical sensing detection and analysis, and specifically relates to a ratiometric molecular imprinting fluorescence sensing detection method based on a metal organic framework and nitrogen-doped carbon dots, including preparation of a europium-based metal organic framework and nitrogen-doped carbon dots, construction of a molecular imprinting fluorescence sensing polymer, ratiometric fluorescence sensitive detection, and visualized rapid detection. Background Art

[0002] Molecularly imprinted polymers (MIPs) are three-dimensional networked recognition materials that are polymerized by functional monomers, crosslinkers and initiators in the presence of target analytes called "template molecules". MIPs provide specific three-dimensional cavity binding sites with complementary shapes, sizes and functions for target analytes, thus having high selectivity. MIPs have the advantages of high sensitivity, strong specificity and simple synthesis, and have been used in sensing, environmental science, food safety, biomedicine and other fields. Fluorescent sensors can convert the recognition between molecular recognition units and targets into fluorescent response signals, and detect the concentration of targets by monitoring the fluorescence intensity. By introducing fluorescent materials into MIPs, a molecularly imprinted fluorescence sensor (MIFs) system can be constructed. MIFs are an organic combination of molecular imprinting technology and fluorescent nanomaterials. Therefore, this sensor has the dual advantages of highly sensitive detection of fluorescent probes and specific recognition of adsorbed MIPs using changes in fluorescent signals, which makes up for the disadvantage that MIPs can only recognize but not transmit signals. Compared with traditional sensors, MIFs have high selectivity, high sensitivity, high specificity and high stability, and have been widely used in the detection of a variety of pollutants. Recently, ratiometric fluorescence sensors have attracted attention due to their excellent sensitivity, self-calibration characteristics, and sufficient color change. Compared with single-channel fluorescence response, which is easily interfered by instruments, environment, and probe concentration, dual-channel ratiometric fluorescence sensors provide built-in signal self-calibration functions, thus obtaining more sensitive and reliable analysis results in practical applications.

[0003] The development of luminescent materials has promoted the development of ratiometric fluorescence imprinted sensors. As a new type of carbon nanofluorescent material, carbon dots (CDs) have the advantages of wide excitation spectrum, good dispersibility, strong hydrophilicity, good biocompatibility and low toxicity, so they are widely used in photocatalysis, nanosensors and bioimaging. However, CDs often suffer from severe quenching due to fluorescence aggregation, which limits their further application.

[0004] Metal-organic frameworks (MOFs) are self-assembled by organic ligands and inorganic metal ions or clusters through coordination bonds. MOFs have been used in the construction of fluorescent sensors due to their extremely high specific surface area, uniform pore size, strong chemical tailoring ability and good thermal stability. Lanthanide metal-organic frameworks (Ln-MOFs) have excellent luminescence properties. Ratiometric fluorescence sensors prepared based on Ln-MOFs can combine the luminescence properties of nanoparticles and Ln-MOFs, making the performance of the sensor better than that of a single component. In addition, CDs encapsulated in luminescent metal-organic frameworks (LMOFs) such as Ln-MOFs have excellent optical properties, physicochemical stability, biocompatibility and low toxicity, giving the sensor a more prominent advantage. However, sensors based on MIPs / LMOFs still have disadvantages in practical applications, such as poor portability, which is not conducive to real-time and on-site detection. Therefore, the development of portable detection methods and equipment based on MIPs / LMOFs is of great significance for rapid and real-time detection of targets on site.

[0005] In summary, based on the demand for developing highly sensitive molecular imprinting sensing detection methods, it is extremely necessary to organically combine luminescent metal organic frameworks and luminescent materials such as carbon dots to construct efficient molecular imprinting fluorescence sensors and establish a specific and sensitive new portable ratiometric imprinting fluorescence sensing detection method. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] The present invention proposes a ratiometric molecular imprinting fluorescence sensing detection method based on metal organic frameworks and nitrogen-doped carbon dots. The red light-emitting source europium-based metal organic framework (Eu-MOF) and blue nitrogen-doped carbon dots (N-CDs) are used as reference signals and response signals, respectively, to construct a new ratiometric fluorescence imprinting sensor to solve the technical problem of how to effectively achieve specific and sensitive detection of detection samples and establish a molecular imprinting fluorescence sensing detection method with high sensitivity, strong selectivity and simple operation.

[0008] (II) Technical solution

[0009] In order to solve the above technical problems, the present invention proposes a ratio-type molecular imprinting fluorescence sensing detection method based on metal organic frameworks and nitrogen-doped carbon dots. The ratio-type molecular imprinting fluorescence sensing detection method comprises the following steps: preparing blue nitrogen-doped carbon dots N-CDs as a response signal and a red light-emitting source europium-based metal organic framework Eu-MOF as a reference signal; using a target molecule as a template, 3-aminopropyltriethoxysilane APTES as a functional monomer, tetraethyl orthosilicate as a crosslinker, N-CDs as a fluorophore, and Eu-MOF as a carrier to prepare a molecular imprinting fluorescent polymer N-CDs@Eu-MOF@MIP, and eluting the target molecule template; after combining the target molecule to be detected with the molecular imprinting polymer, measuring the enhanced blue fluorescence of N-CDs, and the red fluorescence of Eu-MOF remains unchanged, while displaying the response signal, the color of the imprinting polymer changes with the concentration of the target molecule, thereby realizing the visual detection of the imprintable target molecule.

[0010] Furthermore, the preparation of blue nitrogen-doped carbon dots N-CDs includes the following steps: dissolving citric acid in ultrapure water, gradually adding ethylenediamine under magnetic stirring to form a uniform solution; adding the above solution to a stainless steel autoclave lined with polytetrafluoroethylene, heating, and cooling to room temperature after the reaction is completed; purifying the above solution using dialysis to obtain an N-CDs solution.

[0011] Furthermore, the preparation of blue nitrogen-doped carbon dots N-CDs includes the following steps: dissolving 10 g of citric acid in 40 mL of ultrapure water, gradually adding 5 mL of ethylenediamine under magnetic stirring to form a uniform solution; adding the above solution into a 100 mL polytetrafluoroethylene-lined stainless steel autoclave, heating to 200 ° C for 3 hours, and cooling to room temperature after the reaction is completed; purifying the above solution using dialysis to obtain an N-CDs solution.

[0012] Furthermore, the preparation of the red light emitting source europium-based metal organic framework Eu-MOF comprises the following steps: 3 ) 3 and 3,5-dicarboxyphenylboronic acid were dissolved in water and N,N-dimethylformamide and stirred vigorously at room temperature. The mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated. After the reaction was completed, it was cooled to room temperature, washed with N,N-dimethylformamide and ethanol, and centrifuged; it was dried in a vacuum drying oven overnight to obtain white Eu-MOF powder.

[0013] Furthermore, the preparation of the red light emitting source europium-based metal organic framework Eu-MOF includes the following steps: 0.4 mmol of Eu(NO 3 ) 30.4 mmol of 3,5-dicarboxyphenylboronic acid was dissolved in 12 mL of water and 28 mL of N,N-dimethylformamide, and stirred vigorously at room temperature for 2 h. The mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 130 ° C for 12 h. After the reaction was completed, it was cooled to room temperature, washed with N,N-dimethylformamide and ethanol three times each, and centrifuged; dried in a vacuum oven at 60 ° C overnight to obtain white Eu-MOF powder.

[0014] Furthermore, the preparation of molecularly imprinted fluorescent polymer N-CDs@Eu-MOF@MIP includes the following steps: dispersing the synthesized Eu-MOF and N-CDs in a phosphate buffer solution, and dissolving the target molecule in a phosphate buffer solution; mixing the two solutions, adding APTES, stirring, and then adding TEOS and NH 3 ·H 2 O; the mixture was reacted at 25°C for 12h; the crude product was centrifuged and washed with methanol solution for multiple times until the template could not be detected by ultraviolet visible light at a wavelength of 222nm in the supernatant, and the molecularly imprinted fluorescent polymer N-CDs@Eu-MOF@MIP was obtained after freeze-drying.

[0015] Furthermore, the preparation of molecularly imprinted fluorescent polymer N-CDs@Eu-MOF@MIP includes the following steps: 20 mg of synthesized Eu-MOF and 50 μL of N-CDs are dispersed in 5 mL of pH = 7, 10 mM phosphate buffer solution, and 10 mg of target molecule is dissolved in 1 mL of phosphate buffer solution; the two solutions are mixed, and 100 μL of APTES is added, and after stirring for 1 h, 120 μL of TEOS and 120 μL of NH 3 ·H 2 O; the mixture was reacted at 25°C for 12h; the crude product was centrifuged and washed with methanol solution for multiple times until the template could not be detected by ultraviolet visible light at a wavelength of 222nm in the supernatant, and the molecularly imprinted fluorescent polymer N-CDs@Eu-MOF@MIP was obtained after freeze-drying.

[0016] (III) Beneficial effects

[0017] The present invention proposes a ratio-type molecular imprinting fluorescence sensing detection method based on metal organic framework and nitrogen-doped carbon dots, which integrates the advantages of N-CDs, Eu-MOF and molecular imprinting layer. In the presence of target molecules, the blue fluorescence (430nm) of N-CDs is effectively enhanced due to the non-radiative recombination caused by the surface defect state of CDs, while the red fluorescence (660nm) of Eu-MOF remains unchanged. While displaying the fluorescence response signal, the color of the imprinted polymer also changes with the concentration of the target molecule, which can realize sensitive visual detection of the target molecule, thereby realizing specific sensitive detection of the target molecule in complex samples, and has the advantages of high sensitivity, simple operation, good reproducibility, strong anti-interference, etc. This method can realize rapid and sensitive detection of organophosphorus pesticides, toxic agents and other imprintable molecules by preparing imprinted polymers of different target molecules without fluorescence interference, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 TEM photos of the products obtained in the examples of the present invention;

[0019] In the figure: A is N-CDs; B is Eu-MOF; C is BR@MIP; D is BR@NIP;

[0020] Figure 2 Element mapping diagram of the product BR@MIP obtained in the embodiment of the present invention;

[0021] Figure 3 Elemental energy spectrum of the product BR@MIP obtained in the embodiment of the present invention;

[0022] Figure 4 is the XRD spectrum of the product obtained in the embodiment of the present invention;

[0023] In the figure: a is Eu-MOF; b is BR@MIP; c is BR@NIP;

[0024] Figure 5 FT-IR spectrum of the product obtained in the embodiment of the present invention;

[0025] In the figure: a is Eu-MOF; b is BR@MIP; c is BR@NIP;

[0026] Figure 6 The XPS spectrum of the product N-CDs obtained in the embodiment of the present invention;

[0027] In the figure: A is the full XPS spectrum of N-CDs; B is the high-resolution spectrum of C1s; C is the high-resolution spectrum of N1s; D is the high-resolution spectrum of O1s;

[0028] Figure 7The fluorescence spectra of Eu-MOF, Eu-MOF@MIP, N-CDs, BR@MIP, BR@NIP and combined with Mal in the embodiments of the present invention;

[0029] In the figure: A is the fluorescence spectra of Eu-MOF and Eu-MOF combined with 5μM Mal; B is the fluorescence spectra of Eu-MOF@MIP and Eu-MOF@MIP combined with 5μM Mal; C is the fluorescence spectra of N-CDs and BR@MIP; D is the fluorescence spectra of BR@MIP, BR@NIP, and BR@MIP and BR@NIP combined with 5μM Mal;

[0030] Figure 8 Schematic diagram of the principle of ratiometric molecular imprinting fluorescence sensing for detecting Mal based on metal organic frameworks and carbon dots;

[0031] Fig. 9 The fluorescence spectra and Stern-Volmer plots of BR@MIP and BR@NIP at different Mal concentrations in the embodiments of the present invention;

[0032] In the figure: A is the fluorescence spectrum of BR@MIP at different concentrations of Mal (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM); B is the fluorescence spectrum of BR@NIP at different concentrations of Mal (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM); C is the Stern-Volmer plot of BR@MIP; D is the Stern-Volmer plot of BR@NIP;

[0033] Fig.10 Fluorescence spectra and Stern-Volmer plots of N-CDs@MIP at different Mal concentrations;

[0034] In the figure: A is the fluorescence spectra of N-CDs@MIP at different Mal concentrations (1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5 μM); B is the Stern-Volmer plot of N-CDs@MIP;

[0035] Fig.11 is the fluorescence response of BR@MIP and BR@NIP to Mal and its analogs (concentration of 5 μM);

[0036] Fig.12 The fluorescence responses of BR@MIP and BR@NIP to Mal and other interfering ions (concentration of 5 μM);

[0037] Fig.13 is the stability of BR@MIP;

[0038] Fig.14is the reproducibility of BR@MIP after adding 5 μM Mal;

[0039] Fig.15 The reusability of BR@MIP for detecting 5 μM Mal;

[0040] Fig.16 Schematic diagram of the combined smartphone detection of Mal, the fluorescence images of BR@MIP after adding different concentrations of Mal, and the corresponding linear relationship between B / R and Mal concentration (Mal concentration: 2, 3, 4, 5, 6, 7 μM). DETAILED DESCRIPTION

[0041] In order to make the purpose, content and advantages of the present invention more clear, the specific implementation methods of the present invention are further described in detail below in conjunction with the drawings and examples.

[0042] This embodiment proposes a ratiometric molecular imprinting fluorescence sensing detection method based on metal organic frameworks and nitrogen-doped carbon dots, comprising the following steps:

[0043] 1. Preparation of N-CDs

[0044] Dissolve 10 g of citric acid in 40 mL of ultrapure water, and gradually add 5 mL of ethylenediamine under magnetic stirring to form a uniform solution; add the above solution into a 100 mL polytetrafluoroethylene-lined stainless steel autoclave, heat to 200 ° C for 3 hours, and cool to room temperature after the reaction; finally, purify the brown solution using dialysis to obtain N-CDs solution.

[0045] 2. Preparation of Eu-MOF

[0046] 0.4 mmol of Eu(NO 3 ) 3 0.4 mmol of 3,5-dicarboxyphenylboronic acid (BBDC) was dissolved in 12 mL of water and 28 mL of DMF, and stirred vigorously at room temperature for 2 h. The mixture was then transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 130 ° C for 12 h. After the reaction was completed, it was cooled to room temperature, washed three times with DMF and ethanol, and centrifuged; finally, it was dried in a vacuum oven at 60 ° C overnight to obtain white Eu-MOF powder.

[0047] 3. Preparation of BR@MIP Molecularly Imprinted Fluorescent Polymer

[0048] In this example, malathion (Mal) was used as the detection target molecule to prepare molecular imprinting polymers. 20 mg of synthesized Eu-MOF and 50 μL of N-CDs were dispersed in 5 mL of phosphate buffer solution (PBS, pH = 7, 10 mM), and 10 mg of Mal target molecule was dissolved in 1 mL of PBS. The two solutions were mixed, and 100 μL of APTES was added. After stirring for 1 h, 120 μL of TEOS and 120 μL of NH 3 ·H 2 O; the mixture was reacted at 25 ° C for 12 hours; the crude product was centrifuged and washed with methanol solution for several times until the template could not be detected by ultraviolet visible light at a wavelength of 222 nm in the supernatant, and the molecularly imprinted polymer N-CDs@Eu-MOF@MIP (BR@MIP) was obtained after freeze-drying. In addition, as a comparison, the non-imprinted polymer BR@NIP was prepared by the same procedure except that the template molecule was not added. Similarly, N-CDs@MIP was also prepared by the same method except that Eu-MOF was not added.

[0049] 4. Characterization of N-CDs, Eu-MOF and Molecularly Imprinted Polymers

[0050] The products obtained in the above steps were characterized.

[0051] (1) TEM characterization

[0052] The morphology and structure of N-CDs, Eu-MOF, BR@MIP and BR@MIP were characterized by TEM. Figure 1 As shown in Figure A, the diameter of N-CDs is about 4 nm, with good dispersion and uniform size; Figure 1 As shown in Figure B, Eu-MOF microcrystals are spherical with an average size of 500 nm. Figure 1 As shown in Figures C and D, the average size of BR@MIP and BR@NIP is about 600 nm, and the size of the imprinted polymer is larger than that of Eu-MOF, indicating that the imprinted polymer was successfully prepared.

[0053] (2) Element mapping and element spectrum characterization

[0054] Elemental Spectrum ( Figure 2 ) and EDS spectra ( Figure 3 ) showed that the imprinted polymer contained characteristic elements (N, C, Eu, B, Si), which also proved the successful preparation of BR@MIP.

[0055] (3) XRD characterization

[0056] The crystal structures of Eu-MOF, BR@MIP and BR@NIP were characterized by XRD. Figure 4The X-ray diffraction pattern of Eu-MOF shown is consistent with the literature. In addition, the crystallinity of BR@MIP also changes significantly. All results indicate that the imprinted polymer was successfully prepared.

[0057] (4) FT-IR characterization

[0058] The surface functional groups of Eu-MOF, BR@MIP and BR@NIP were characterized by FT-IR. Figure 5 As shown, Eu-MOF has a peak at 1389 cm -1 The peak at 3320cm corresponds to the vibration of BO. -1 and 1656cm -1 The peaks at 2966cm are related to the stretching vibration of OH / NH and the bending vibration of NH, respectively, indicating that N-CDs are embedded in Eu-MOF, indicating that Eu-MOF was successfully prepared. -1 The characteristic peak at 1056cm belongs to the asymmetric stretching vibration of CH. -1 The peak at 781cm belongs to the asymmetric stretching of Si-O-Si. -1 and 448cm -1 The peaks at are related to Si-O vibration and Si-O-Si bending vibration, respectively, indicating the successful formation of the imprinted silica layer.

[0059] (5) XPS characterization

[0060] Figure 6 Showing the XPS spectra of N-CDs, Figure 6 A in the figure shows the full XPS spectrum of N-CDs. The XPS spectrum of C1s has three peaks at 284.1eV, 285.2eV and 287.6eV, which belong to CC / C=C, CO and C=O respectively. Figure 6 B). The N1s XPS spectrum shows peaks at 399.7eV, 400.7eV and 401.5eV ( Figure 6 C), which are related to NC, NH and NO respectively. The O1s XPS spectrum shows C=O peak (530.4eV) and CO peak (532.3eV) ( Figure 6 D). The above results show that N element is successfully doped to form N-CDs.

[0061] 5. Verification and interpretation of detection mechanism

[0062] from Figure 7 As can be seen in Figure A, the fluorescence intensity did not change after adding Mal to Eu-MOF. In addition, the fluorescence intensity of the Eu-MOF imprinted material (Eu-MOF@MIP) hardly changed after adding 5 μM Mal ( Figure 7(B). Therefore, Mal has no reaction to Eu-MOF and thus cannot enhance the luminescence. Instead, CDs play a key role in the luminescence behavior of BR@MIP.

[0063] The photoluminescence mechanisms proposed so far include surface states, quantum confinement effects, molecular states, synergistic effects, conjugation effects, etc. The surface states of CDs play a more important role in their luminescence behavior. For example, some traps and non-radiative relaxation processes on the surface of CDs will reduce the fluorescence signal due to the presence of defect states, which is attributed to the presence of certain groups on the surface of CDs, such as alkylamines and carbonyls. These groups will induce non-radiative recombination, thereby reducing the luminescence efficiency of CDs. Therefore, passivating or reducing these groups can restore the fluorescence signal of CDs. Figure 7 As shown in Figure C, the fluorescence signal of BR@MIP is significantly weaker than that of N-CDs. This is because the imprinted silicon layer on the N-CDs is thicker, which masks the fluorescence of the internal N-CDs, resulting in a weaker fluorescence signal of BR@MIP. In addition, the presence of alkylamine groups in APTES induces the non-radiative nature of excited electrons in N-CDs, which also causes the fluorescence signal of BR@MIP to decay. However, in the presence of Mal, the fluorescence signal of BR@MIP gradually recovers ( Figure 7 This is because the alkylamine groups of APTES can be passivated by interacting with Mal, reducing non-radiative recombination, thereby gradually recovering the fluorescence signal of N-CDs in MIPs. In contrast, since there is no molecular imprinting cavity on BR@NIP, only a small amount of Mal binds nonspecifically to APTES, resulting in only a slight recovery of the fluorescence signal of BR@NIP.

[0064] 6. Detection of Malathion by Ratio-based Molecular Imprinting Fluorescence Sensing Based on Metal-Organic Frameworks and Nitrogen-Doped Carbon Dots

[0065] The principle of ratiometric molecular imprinting fluorescence sensing for malathion based on metal organic frameworks and nitrogen-doped carbon dots is as follows Figure 8As shown. Based on the preparation of reference signal Eu-MOF response signal N-CDs, the molecular imprinting polymer was synthesized by sol-gel method with Mal as template, APTES as functional monomer, TEOS as cross-linker, N-CDs as fluorophore and Eu-MOF as carrier. After template elution, BR@MIP obtains a specific recognition cavity matching the size and shape of Mal. In the presence of Mal, the blue fluorescence (430nm) of N-CDs is effectively enhanced due to the non-radiative recombination caused by the surface defect state of CDs, while the red fluorescence (660nm) of Eu-MOF remains unchanged. Therefore, this ratiometric molecular imprinting fluorescence sensing detection method not only displays the fluorescence response signal, but also the color of the imprinted polymer changes with the concentration of the target molecule, and can also realize sensitive visual detection of the target molecule, thereby realizing specific and sensitive detection of the target molecule in complex samples.

[0066] Under the best optimized conditions, the fluorescence emission curves of BR@MIP and BR@NIP for Mal were measured by fluorescence spectrometer after Mal was combined with the imprinted polymer. Fig. 9 As shown in A and B, as the concentration of Mal increases from 0 μM to 10 μM, the fluorescence intensity at 430 nm gradually increases, while the fluorescence intensity at 616 nm remains unchanged. However, the fluorescence enhancement effect of BR@MIP is stronger than that of BR@NIP, because there are specific imprinting sites on the surface of BR@MIP that match the functional groups, structure and size of the template molecule, while these imprinting sites do not exist on the surface of BR@NIP. Fig. 9 C and D show the SternVolmer diagram. In the concentration range of 1-10 μM, BR@MIP has a good linear fluorescence enhancement effect on Mal. 2 is 0.995. The linear fluorescence enhancement equation of BR@MIP for Mal is y=0.4927x+0.9991, while the linear equation of BR@NIP is y=0.1186x+0.4973. The imprinting factor (IF) was used to evaluate the imprinting effect of BR@MIP, and the calculated IF was 4.15, proving that BR@MIP has a high recognition ability for Mal. At the same time, according to LOD=3σ / k, the detection limit of BR@MIP for Mal is 0.05μM. In addition, Fig. 9 As shown in the upper part of A, under 365nm ultraviolet light, benefiting from the intensity change of two clearly resolved emission peaks, a clear color change from red to blue can be easily observed, which provides the feasibility of detecting Mal with the naked eye. Fig. 9 It can be seen that the fluorescence color change of BR@MIP is more obvious than that of BR@NIP, which is due to the presence of Mal imprinting sites on BR@MIP.

[0067] For comparison, N-CDs were used as fluorescent probes to detect the single signal of Mal. Fig.10 As shown in A, with the increase of Mal concentration, the fluorescence of N-CDs gradually increased, and the relationship between Mal concentration and fluorescence enhancement efficiency (F 430 / F 616 ) / (F 430 / F 616 ) 0 ) can be expressed as y = 0.0850x + 1.0759 (R 2 =0.991), with a good linear relationship and an LOD of 0.5 μM. Compared with the single-signal sensing method based on N-CDs, the ratiometric fluorescence method based on N-CDs / Eu-MOF has a wider linear range and higher sensitivity, which may be attributed to the self-correction characteristics of ratiometric fluorescence. More importantly, the ratiometric fluorescence method based on N-CDs / Eu-MOF shows obvious color changes for different concentrations of Mal, while the single-signal sensing method based on N-CDs only shows brightness changes for Mal ( Fig.10 The above results show that the ratiometric fluorescence method based on N-CDs / Eu-MOF has more advantages than the single fluorescence method.

[0068] 7. Selectivity and anti-interference performance of ratiometric molecular imprinting fluorescence sensing based on metal organic frameworks and nitrogen-doped carbon dots

[0069] The selectivity of BR@MIP to malathion, thiamethoxam, imidacloprid, diazinon, phoxim, glyphosate, dimethoate, chlorpyrifos, etc. was investigated by recording the fluorescence response values. Fig.11 As shown in the figure, the fluorescence response of BR@MIP to Mal is significantly higher than that of other substances. This is because the specific recognition site of BR@MIP is complementary to Mal in size, shape and functional group. The results show that BR@MIP has good selectivity.

[0070] In order to evaluate the anti-interference ability of BR@MIP, some potential interferents, such as K + , Ca 2+ 、Na + , Cl - , Cu 2+ 、SO 4 2- , Pb 2+ Mg 2+ 、Zn 2+ , Mn 2+ 、NO 3 - 、Ni + 、Co 2+ wait( Fig.12 ). Fluorescence response of BR@MIP to Mal (F430 / F 616 ) / (F 430 / F 616 ) 0 ) was significantly higher than other interferents. The results showed that these interferents would not affect the recognition and detection of Mal by BR@MIP.

[0071] 8. Stability, reproducibility and repeatability of sensor detection

[0072] Stability is an important indicator of sensor detection. Fig.13 As shown in the figure, within 20 days, the stability of BR@MIP did not change significantly, indicating that BR@MIP has good fluorescence stability. In order to investigate the reproducibility of BR@MIP, 6 groups of samples were measured to monitor the changes in the fluorescence response of BR@MIP. The results are shown in the figure. Fig.14 As shown, BR@MIP has good reproducibility. Five removal-binding cycle experiments were performed to evaluate the reusability of BR@MIP. Fig.15 As shown, it can be seen that as the number of repetitions increases, ((F 430 / F 616 ) / (F 430 / F 616 ) 0 ) value decreased slightly and could be maintained at 96.78% of the original value, indicating that BR@MIP has good reuse performance.

[0073] 9. Simulation sample testing

[0074] Lettuce, tap water and soil were used as simulated samples to verify the practicality of the BR@MIP ratio fluorescence sensing detection method. Different concentrations of Mal (1μM, 5μM, 10μM) were added to the samples for recovery test. The results are shown in the table below, and the recovery rate is between 93.0% and 99.3%. The results show that the BR@MIP ratio fluorescence sensing detection method can effectively detect Mal in actual samples.

[0075]

[0076] 10. Detection of Mal in samples using a smartphone-assisted fluorescence sensing platform

[0077] To achieve on-site detection of Mal, a smartphone-assisted fluorescence sensing platform was prepared. Different concentrations of Mal and BR@MIP were added to the cuvette, and a series of fluorescence images were taken with a smartphone and further converted into RGB values ​​using software (Colorpicker). In the range of 2-7 μM, B / R showed a good linear relationship with Mal concentration, i.e., y=0.1882x+0.3166(R 2=0.984), with a detection limit of 1.45 μM. In addition, the color change from pink to blue of BR@MIP was more significant compared with BR@NIP ( Fig.16 ). The above results indicate that the combination of this detection method with the prepared smartphone-assisted fluorescence sensor platform can provide a reliable strategy for on-site analysis and detection of Mal.

[0078] Lettuce, tap water and soil were also used to investigate the practicality of the smartphone-based sensing platform. As shown in the table below, the recovery rates were 91.6%-98.0%, 94.4%-98.2% and 92.0%-95.6%, respectively, with RSD < 3.1%. The results show that the sensing platform is satisfactory in the field rapid detection of Mal and has great application potential.

[0079]

[0080] According to the above embodiments, the present invention uses red light-emitting sources europium-based metal organic framework (Eu-MOF) and blue nitrogen-doped carbon dots (N-CDs) as reference signals and response signals, respectively, to establish a new ratio fluorescence imprinting sensing detection method, which can effectively realize rapid and sensitive detection of target molecule malathion, with a detection limit of 0.05 μM, and has the advantages of high sensitivity, simple operation, good reproducibility, and strong anti-interference. On this basis, a smartphone-assisted fluorescence sensing platform is constructed to realize portable on-site rapid detection of malathion in samples. This method can realize rapid and sensitive detection of organophosphorus pesticides, toxic agents and other imprintable molecules by preparing imprinted polymers of different target molecules without fluorescence interference, and has broad application prospects.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ratiometric molecular imprinting fluorescence sensing detection method based on metal organic frameworks and nitrogen-doped carbon dots, characterized in that: The ratio-type molecular imprinting fluorescence sensing detection method comprises the following steps: preparing blue nitrogen-doped carbon dots N-CDs as a response signal and a europium-based metal organic framework Eu-MOF as a red light-emitting source as a reference signal; using a target molecule as a template, 3-aminopropyltriethoxysilane APTES as a functional monomer, tetraethyl orthosilicate as a crosslinker, N-CDs as a fluorophore, and Eu-MOF as a carrier to prepare a molecular imprinting fluorescent polymer N-CDs@Eu-MOF@MIP, and eluting the target molecule template; after combining the target molecule to be detected with the molecular imprinting polymer, measuring the enhanced blue fluorescence of N-CDs, while the red fluorescence of Eu-MOF remains unchanged, and while displaying the response signal, the color of the imprinting polymer changes with the concentration of the target molecule, thereby realizing the visual detection of the imprintable target molecule.

2. The ratiometric molecular imprinting fluorescence sensing detection method based on metal organic framework and nitrogen doped carbon dots according to claim 1, characterized in that: The preparation of the blue nitrogen-doped carbon dots N-CDs includes the following steps: dissolving citric acid in ultrapure water, gradually adding ethylenediamine under magnetic stirring to form a uniform solution; adding the above solution into a stainless steel autoclave lined with polytetrafluoroethylene, heating, and cooling to room temperature after the reaction is completed; purifying the above solution using a dialysis method to obtain an N-CDs solution.

3. The ratiometric molecular imprinting fluorescence sensing detection method based on metal organic framework and nitrogen doped carbon dots according to claim 2, characterized in that: The preparation of the blue nitrogen-doped carbon dots N-CDs includes the following steps: dissolving 10 g of citric acid in 40 mL of ultrapure water, gradually adding 5 mL of ethylenediamine under magnetic stirring to form a uniform solution; adding the above solution into a 100 mL polytetrafluoroethylene-lined stainless steel autoclave, heating to 200° C. for 3 hours, and cooling to room temperature after the reaction is completed; purifying the above solution using dialysis to obtain an N-CDs solution.

4. The ratiometric molecular imprinting fluorescence sensing detection method based on metal organic framework and nitrogen doped carbon dots according to claim 1, characterized in that: The preparation of the red light-emitting source europium-based metal organic framework Eu-MOF includes the following steps: dissolving Eu(NO3)3 and 3,5-dicarboxyphenylboric acid in water and N,N-dimethylformamide, vigorously stirring at room temperature, transferring the mixture to a stainless steel autoclave lined with polytetrafluoroethylene for heating, cooling to room temperature after the reaction is completed, washing with N,N-dimethylformamide and ethanol, and centrifuging; drying in a vacuum drying oven overnight to obtain white Eu-MOF powder.

5. The ratiometric molecular imprinting fluorescence sensing detection method based on metal organic framework and nitrogen doped carbon dots according to claim 4, characterized in that: The preparation of the red light-emitting europium-based metal organic framework Eu-MOF includes the following steps: dissolving 0.4 mmol of Eu(NO3)3 and 0.4 mmol of 3,5-dicarboxyphenylboronic acid in 12 mL of water and 28 mL of N,N-dimethylformamide, stirring vigorously at room temperature for 2 hours, transferring the mixture to a stainless steel autoclave lined with polytetrafluoroethylene, and heating at 130°C for 12 hours. After the reaction is completed, cool to room temperature, wash with N,N-dimethylformamide and ethanol three times each, centrifuge; dry overnight in a vacuum drying oven at 60°C to obtain white Eu-MOF powder.

6. The ratiometric molecular imprinting fluorescence sensing detection method based on metal organic framework and nitrogen doped carbon dots according to claim 1, characterized in that: The preparation of the molecular imprinted fluorescent polymer N-CDs@Eu-MOF@MIP includes the following steps: dispersing the synthesized Eu-MOF and N-CDs in a phosphate buffer solution, and at the same time, dissolving the target molecule in the phosphate buffer solution; mixing the two parts of the solution, adding APTES, stirring, and then adding TEOS and NH3·H2O in sequence; reacting the mixture at 25°C for 12 hours; centrifuging the crude product and washing it with a methanol solution for multiple times until the template cannot be detected by ultraviolet visible light in the supernatant at a wavelength of 222nm, and freeze-drying to obtain the molecular imprinted fluorescent polymer N-CDs@Eu-MOF@MIP.

7. The ratiometric molecular imprinting fluorescence sensing detection method based on metal organic framework and nitrogen doped carbon dots according to claim 6, characterized in that: The preparation of the molecular imprinted fluorescent polymer N-CDs@Eu-MOF@MIP includes the following steps: dispersing 20 mg of synthesized Eu-MOF and 50 μL of N-CDs in 5 mL of pH=7, 10 mM phosphate buffer solution, and dissolving 10 mg of the target molecule in 1 mL of phosphate buffer solution; mixing the two parts of the solution, adding 100 μL of APTES, stirring for 1 hour, and then adding 120 μL of TEOS and 120 μL of NH3·H2O in sequence; reacting the mixture at 25°C for 12 hours; centrifuging the crude product and washing it with a methanol solution for multiple times until the template cannot be detected by ultraviolet visible light at a wavelength of 222 nm in the supernatant, and freeze-drying to obtain the molecular imprinted fluorescent polymer N-CDs@Eu-MOF@MIP.

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