Preparation method and application of double-fluorescent ZIF-8 nanoprobe with spatial compartment structure

Abstract

This invention discloses a method for preparing and applying a dual-fluorescent ZIF-8 nanoprobe with a spatially compartmentalized structure. Based on an epitaxial layer-by-layer growth strategy, this invention constructs a multi-layered core-shell structure to obtain a dual-fluorescent ZIF-8 nanoprobe with a spatially compartmentalized structure, ZIF-8@coumarin@ZIF-8@rhodamine B, exhibiting an "inner layer reference - outer layer response" relationship. The nanoprobe prepared by this invention not only suppresses signal crosstalk caused by fluorescence resonance energy transfer but also achieves spatial selectivity of the inner filtration effect. Based on the characteristic absorption of malachite green in the 600-620 nm range, it can selectively quench the fluorescence of the outer rhodamine layer while maintaining a stable signal from the inner coumarin layer, thus achieving ratiometric fluorescence detection. This detection method has advantages such as strong anti-interference ability, high signal stability, and high sensitivity, and can be used for the rapid detection of malachite green in aquatic products.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing and functional nanomaterials technology, specifically relating to the preparation method and application of a dual-fluorescent ZIF-8 nanoprobe with a spatial compartment structure. Background Technology

[0002] Malachite green is a triphenylmethane dye that was once widely used in aquaculture due to its excellent antibacterial and antiparasitic effects. However, this substance has potential carcinogenic and mutagenic properties and has been banned for use in food animals by many countries. Therefore, developing rapid, sensitive, and reliable detection methods for malachite green is of great significance for ensuring the quality and safety of aquatic products. Currently, the main detection methods for malachite green include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). Although these methods have high sensitivity and accuracy, they generally suffer from problems such as expensive instruments, complex operation, and long detection cycles, making it difficult to meet the needs of rapid on-site detection. In contrast, fluorescence-based detection methods have received widespread attention in recent years due to their advantages of simple operation and rapid response.

[0003] In fluorescence detection systems, ratiometric fluorescence, by introducing an internal reference signal, can effectively reduce the impact of environmental factors and probe concentration fluctuations on detection results, thereby improving detection stability. However, in multi-fluorescent probe systems, when multiple fluorescent dyes coexist at the nanoscale, fluorescence resonance energy transfer (FRET) and aggregation quenching effects easily occur, leading to severe signal crosstalk and making it difficult to achieve relatively independent output of dual-channel signals. Furthermore, existing systems typically focus only on enhancing or suppressing fluorescence signals during the design process, lacking regulation of the detection mechanism (such as the internal filtration effect, IFE), making it difficult to achieve efficient and selective response to the target analyte.

[0004] Metal-organic frameworks (MOFs) have been widely used in fluorescence sensing due to their high specific surface area, tunable pore structure, and excellent guest molecule encapsulation capabilities. However, existing "dye@MOF" systems mostly employ monolayer structures, typically co-loading multiple fluorescent dyes within the same channel or crystal. This results in close spatial proximity between different fluorescent units, making it difficult to avoid energy transfer processes such as FRET and hindering the functional specialization of different fluorescence channels. Therefore, there is an urgent need to develop a novel structural design strategy for ratiometric fluorescence detection systems that are stable, sensitive, and highly resistant to interference. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a method for preparing and applying a dual-fluorescent ZIF-8 nanoprobe with a spatially compartmentalized structure. Based on an epitaxial layer-by-layer growth strategy, this invention constructs a functional hierarchical structure of "inner reference - outer response" through the synergistic design of fluorescent dye types and their spatial distribution, thereby achieving fluorescence signal decoupling and regulation of the internal filtering effect pathway at the nanoscale. The provided nanoprobe not only achieves spatial separation of multiple fluorescent dyes at the nanoscale, suppressing FRET at the structural level, but also, by combining the spectral characteristics of the target analyte, regulates the fluorescence signal pathway, thus constructing a stable, sensitive ratiometric fluorescence detection system with high anti-interference capability.

[0006] The first objective of this invention is to provide a dual-fluorescent nanoprobe with a spatial compartment structure. The nanoprobe is a core-shell structure constructed with ZIF-8 as the framework. The core-shell structure includes an inner ZIF-8 layer and an outer ZIF-8 layer, wherein: the inner ZIF-8 layer encapsulates a coumarin-based fluorescent dye as an internal reference channel; and the outer ZIF-8 layer encapsulates a rhodamine-based fluorescent dye as a response channel.

[0007] The provided dual-fluorescent nanoprobe with a spatial compartment structure achieves partitioned loading of different fluorescent dyes at the nanoscale by constructing a multi-layer core-shell structure, thereby effectively suppressing signal crosstalk caused by FRET and realizing relatively independent output of dual fluorescent signals.

[0008] Preferably, the method for preparing the nanoprobe includes the following steps: S1. An aqueous solution containing surfactant and 2-methylimidazole is mixed with an aqueous solution of zinc salt and stirred until homogeneous. A coumarin-based fluorescent dye solution is added, stirred until homogeneous, and allowed to stand for 0.5-24 h. After the reaction is complete, the supernatant is discarded by centrifugation, and the precipitate is washed and dispersed with a polar solvent to obtain a suspension of ZIF-8@coumarin-based fluorescent dye nanoparticles. S2. A suspension of ZIF-8@coumarin fluorescent dye nanoparticles was dispersed in an aqueous solution containing a surfactant and 2-methylimidazole. A zinc salt aqueous solution was added and mixed well. A rhodamine fluorescent dye solution was added and mixed well. The mixture was allowed to stand for 0.5-24 h. After the reaction was completed, the supernatant was discarded by centrifugation. The precipitate was washed with a polar solvent and dried to obtain a dual fluorescent nanoprobe ZIF-8@coumarin fluorescent dye@ZIF-8@rhodamine fluorescent dye with a spatial compartment structure. In the reaction system of step S1 or step S2, the concentration of the surfactant is 0.27~0.69 mM, and the amount added is 0.01%~0.025% of the volume of the reaction system; the concentration of 2-methylimidazole in the reaction system is 60~1200 mM, and the concentration of the zinc salt in the reaction system is 15~150 mM; the molar ratio of 2-methylimidazole to zinc salt is 4:1~80:1. The concentration of the coumarin-based fluorescent dye solution is 1-5 mM, and the amount added is 0.05-0.2 mL; the concentration of the rhodamine-based fluorescent dye is 2-10 mM, and the amount added is 0.05-0.2 mL.

[0009] Preferably, the surfactant is CTAB, TPABr, CTAC, or STAC, the zinc salt is one or more of zinc acetate, zinc nitrate, zinc chloride, or zinc acetate, and the polar solution is one or more of methanol, ethanol, N,N-dimethylformamide, or deionized water.

[0010] Preferably, the standing reaction time in step S1 is 1 to 6 hours, and the standing reaction time in step S1 is 0.5 to 3 hours.

[0011] Preferably, the nanoprobe is ZIF-8@coumarin@ZIF-8@rhodamine B, and its preparation method includes the following steps: S1. An aqueous solution containing CTAB and 2-methylimidazole was mixed with an aqueous solution of Zn(Ac)2·2H2O and stirred until homogeneous. A methanol solution of coumarin was added, and the mixture was stirred until homogeneous. The mixture was then allowed to stand for 3 h. After the reaction was completed, the supernatant was discarded by centrifugation. The precipitate was washed with a polar solvent and dispersed to obtain a suspension of ZIF-8@coumarin nanoparticles. S2. ZIF-8@coumarin nanoparticle suspension was dispersed in an aqueous solution containing CTAB and 2-methylimidazole, zinc salt aqueous solution was added and mixed, rhodamine B solution was added and mixed, and the reaction was allowed to stand for 1 h. After the reaction was completed, the supernatant was discarded by centrifugation, the precipitate was washed with methanol and dispersed to obtain dual fluorescent nanoprobes ZIF-8@coumarin@ZIF-8@rhodamine B with a spatial compartment structure. The aqueous solution containing CTAB and 2-methylimidazole has a CTAB concentration of 0.41~0.47 mM and a 2-methylimidazole concentration of 790 mM; the aqueous solution containing Zn(Ac)2·2H2O has a Zn(Ac)2·2H2O concentration of 97.5 mM; and the volume ratio of the aqueous solution containing CTAB and 2-methylimidazole to the aqueous solution containing Zn(Ac)2·2H2O is 1.75:0.25. The concentration of the coumarin methanol solution is 5 mM, and the amount added is 0.1 mL; the concentration of the rhodamine B aqueous solution is 5 mM, and the amount added is 0.1 mL.

[0012] Preferably, the centrifugation is performed at 8000-9000 rpm for 10 min.

[0013] The above method achieves partitioned loading of coumarin and rhodamine in different spatial regions through the layer-by-layer epitaxial growth and sequential introduction strategy. Coumarin is located in the inner layer as a stable internal reference channel, while rhodamine is located in the outer layer as a response channel. This effectively suppresses signal crosstalk caused by FRET in the structure and achieves relatively independent output of dual fluorescence signals.

[0014] A second object of the present invention is to provide the application of any of the nanoprobes described herein in the detection of malachite green.

[0015] Preferably, the malachite green is derived from environmental water or aquatic products.

[0016] A third objective of this invention is to provide a method for detecting malachite green, characterized by comprising the following steps: S1. Extract malachite green from the sample to be tested and prepare the sample solution; S2. Prepare malachite green standard solutions with different concentration gradients using malachite green samples; S3. Add 100 μL of a suspension of dual fluorescent nanoprobes ZIF-8@coumarin@ZIF-8@rhodamine B with a spatial compartment structure to every 50 μL of the sample solution to be tested or malachite green standard solutions of different concentration gradients, diluted to a concentration of 1 mg / mL. Incubate for 30 s to 2 min, and measure the fluorescence emission spectrum under the condition of excitation wavelength of 350 to 380 nm. Record the fluorescence intensity F1 of the coumarin channel at 450 nm and the fluorescence intensity F2 of the rhodamine channel at 590 nm. S4. Establish standard curves using the fluorescence intensity ratios F2 / F1 or F1 / F2 of malachite green standard solutions with different concentration gradients as response signals; S5. Substitute the fluorescence intensity ratio F2 / F1 or F1 / F2 of the sample solution into the standard curve to calculate the malachite green content in the sample.

[0017] Preferably, when the sample to be tested is ambient water, step S1 involves filtering the sample using a 0.22 μm filter membrane to obtain the sample solution; when the sample to be tested is aquatic product, 4 mL of acetonitrile-water mixed solution is added to every 2.0 g of sample homogenate, vortexed for 2 min, centrifuged, and the supernatant is taken as the sample solution to be tested, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 8:2.

[0018] Preferably, centrifugation is performed at 8000 rpm for 10 min.

[0019] The proposed method for detecting malachite green combines the spectral characteristics of malachite green with the matching relationship between its absorption in the 600-620 nm range and the emission spectrum of rhodamine. This allows the inner filtration effect to preferentially act on the outer responding dye, thereby achieving selective modulation of the fluorescence signal, while the inner coumarin signal remains basically stable. Thus, a detection method based on the ratio of dual-channel fluorescence intensity is constructed to achieve quantitative analysis of malachite green.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a layered ZIF-8 spatial compartment structure through epitaxial layer-by-layer growth, achieving directional and partitioned loading of coumarin and rhodamine at the nanoscale. This not only effectively suppresses signal crosstalk caused by FRET, but also enables stable and relatively independent output of dual fluorescence signals by constructing a functional layered system of "inner reference - outer response". Based on this, combined with the characteristic absorption of malachite green in the 600-620 nm range, the inner filtration effect preferentially acts on the outer rhodamine channel, achieving selective modulation of the fluorescence signal. This constructs a stable, reliable, highly sensitive, and interference-resistant ratiometric fluorescence detection system, improving detection performance from a mechanistic perspective. Comparison with monolayer co-loaded nanoprobes and reverse layered spatial compartment nanoprobes shows that only the forward layered spatial compartment nanoprobe of this invention can simultaneously achieve efficient FRET suppression and inner filtration effects, improving detection sensitivity by approximately one order of magnitude, demonstrating a significant structure-mechanism synergistic advantage. In addition, this method is mild, easy to operate, and has good repeatability and scalability. It can achieve rapid and sensitive detection of malachite green in aquatic products and can be extended to other multi-component fluorescent probe systems, showing good application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the synthetic route and spatial compartment structure of the ZIF-8@coumarin@ZIF-8@rhodamine B bilayer nanoprobe.

[0022] Figure 2 The images show the morphology of the ZIF-8@coumarin@ZIF-8@rhodamine B bilayer structure nanoprobe at different stages of its preparation. Specifically: a is a scanning electron microscope (SEM) image of the ZIF-8@coumarin nanoparticles; b is a SEM image of the ZIF-8@coumarin@ZIF-8@rhodamine B bilayer structure nanoprobe; and c is a magnified SEM image of a single ZIF-8@coumarin@ZIF-8@rhodamine B nanoparticle.

[0023] Figure 3 The images show the optical and physicochemical properties of the ZIF-8@coumarin@ZIF-8@rhodamine B bilayer structure nanoprobes obtained at different stages of the preparation process. Among them: a is the UV-Vis absorption spectrum of ZIF-8@coumarin nanoparticles, b is the UV-Vis absorption spectrum of ZIF-8@coumarin@ZIF-8@rhodamine B nanoprobes, c is the hydration particle size distribution, and d is the zeta potential change.

[0024] Figure 4 The graphs show a comparison of the fluorescence properties of nanoprobes with different structures. In particular, a is a schematic diagram of the structure of the monolayer co-supported nanoprobe (ZIF-8@coumarin / rhodamine B), the reverse hierarchical spatial compartment structure nanoprobe (ZIF-8@rhodamine B@ZIF-8@coumarin), and the forward hierarchical spatial compartment structure nanoprobe (ZIF-8@coumarin@ZIF-8@rhodamine B). b is a comparison of the fluorescence intensity changes of coumarin and rhodamine B in different nanostructures.

[0025] Figure 5 This is a schematic diagram showing the overlap between the absorption spectrum of malachite green and the emission spectrum of the ZIF-8@coumarin@ZIF-8@rhodamine B nanoprobe with a double-layered spatial compartment structure.

[0026] Figure 6 The graph shows a comparison of the analytical performance of different nanoprobe structures for detecting malachite green. In the graph: a is the standard curve of the monolayer co-supported nanoprobe (ZIF-8@coumarin / rhodamine B), b is the standard curve of the reverse hierarchical spatial compartment structure nanoprobe (ZIF-8@rhodamine B@ZIF-8@coumarin), and c is the standard curve of the forward hierarchical spatial compartment structure nanoprobe (ZIF-8@coumarin@ZIF-8@rhodamine B); x is the concentration of malachite green (MG), and y is the ratio of fluorescence intensity at 590 nm to 450 nm. Detailed Implementation

[0027] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0028] Example 1: Fabrication of a dual-fluorescent ZIF-8 nanoprobe with a forward-looking hierarchical spatial compartment structure This embodiment illustrates a method for preparing a dual-fluorescent ZIF-8 nanoprobe with a spatial compartment structure. By employing a sequential control strategy of "inner layer preferential construction - outer layer epitaxial growth," different fluorescent dyes are directionally loaded at the nanoscale, thereby constructing a nanoprobe with a functional hierarchical structure.

[0029] (1) Preparation of inner layer ZIF-8@coumarin nanoparticles First, a mixed solution containing organic ligands and surfactants was prepared. 0.30 mg of cetyltrimethylammonium bromide (CTAB) and 113.5 mg of 2-methylimidazole were weighed and a total volume of 1.75 mL of aqueous solution was prepared, resulting in final concentrations of 0.47 mM and 790 mM, respectively. The solution was magnetically stirred for 5 min at room temperature to ensure thorough mixing. Then, 0.25 mL of a 97.5 mM Zn(Ac)₂·2H₂O aqueous solution was added, and stirring continued for 5 min to promote the initial complexation of zinc ions with the organic ligands. Next, 0.10 mL of a 2 mM coumarin methanol solution was added, and stirring continued for 3 min to allow coumarin to participate in the encapsulation process simultaneously during ZIF-8 nucleation and growth. After stirring, the reaction system was allowed to stand at room temperature for 3 h to form inner-layer ZIF-8@coumarin nanoparticles. After the reaction was complete, the suspension was centrifuged (8000 rpm, 10 min), the supernatant was discarded, and the precipitate was resuspended in methanol and centrifuged again (8000 rpm, 10 min). This process was repeated twice to remove unreacted precursors and free dye. The precipitate was redispersed in 1 mL of methanol to obtain a uniformly dispersed suspension of ZIF-8@coumarin nanoparticles.

[0030] (2) Epitaxial growth of outer ZIF-8 and introduction of rhodamine B Take 0.40 mL of the ZIF-8@coumarin nanoparticle suspension and disperse it in 1.75 mL of a solution containing 2-methylimidazole (790 mM) and CTAB (0.41 mM). Stir at room temperature for 5 min to ensure uniform dispersion of the inner layer particles. Then add 0.25 mL of a 97.5 mM Zn(Ac)2·2H2O aqueous solution, mix rapidly and stir for 30 s to induce epitaxial growth of the outer ZIF-8 layer on the surface of the inner layer particles. Next, add 0.10 mL of a 5 mM rhodamine B aqueous solution and continue stirring for 3 min to ensure simultaneous encapsulation of rhodamine B during the formation of the outer ZIF-8 layer. Allow the system to stand at room temperature for 1 h to complete the growth of the outer ZIF-8 layer. The precipitate was then centrifuged (8000 rpm, 10 min), the supernatant was discarded, and the precipitate was resuspended in methanol and centrifuged again (8000 rpm, 10 min). This process was repeated twice to obtain the final product, ZIF-8@coumarin@ZIF-8@rhodamine B nanoprobe. The precipitate was dried overnight in a vacuum drying oven. When using, a suspension of a certain concentration was prepared with water or an organic solvent such as methanol.

[0031] like Figure 1 As shown, this embodiment demonstrates the process of achieving spatial partitioning of fluorescent dyes through layer-by-layer epitaxial growth, clearly illustrating the construction path of a forward-layered spatial compartment structure.

[0032] like Figure 2 As shown, SEM characterization was performed on the materials obtained at different stages in this embodiment. Figure 2 Figure 'a' shows that the inner ZIF-8@coumarin nanoparticles have a regular cubic morphology with a particle size of about 100~150 nm, indicating that the inner layer structure is uniformly formed. Figure 2 The b in the figure shows that the particle size increased to about 200~300 nm after epitaxial growth, indicating that the outer ZIF-8 layer was successfully grown; Figure 2 The fact that the single particles in c maintain a regular morphology indicates that the epitaxial process did not destroy the crystal structure, verifying the successful construction of the core-shell structure.

[0033] like Figure 3 As shown, the optical and physicochemical properties of the stepwise synthesis process of the material are characterized. Figure 3 Figure a shows that ZIF-8@coumarin nanoparticles have an absorption peak at about 350 nm, indicating that coumarin was successfully encapsulated in the inner layer; Figure 3 b in the figure shows that ZIF-8@coumarin@ZIF-8@rhodamine B has an absorption peak at about 555 nm, indicating that rhodamine was successfully introduced into the outer structure; Figure 3 c shows that the particle size (d) of ZIF-8@coumarin particles is 123.6 nm, and the particle size of ZIF-8@coumarin@ZIF-8@rhodamine B particles increases to 211.3 nm, verifying the outer layer growth; Figure 3 The d-value shows that the zeta potential of ZIF-8@coumarin particles is 23.5 mV, while the zeta potential of ZIF-8@coumarin@ZIF-8@rhodamine B particles rises to 29.3 mV, indicating that the surface properties have changed, which is consistent with the formation of the outer layer structure.

[0034] The above results demonstrate that this embodiment successfully constructed a bilayer ZIF-8 nanoprobe with a spatially compartmentalized structure using a sequential epitaxial growth strategy, achieving directional and partitioned loading of coumarin and rhodamine in different spatial regions. This structure not only effectively increases the spatial distance between the two fluorescent dyes, thereby suppressing FRET, but also, by positioning coumarin in the inner layer as a stable internal reference channel and rhodamine B in the outer layer as a response channel, makes it easier for rhodamine B to interact spectrally with the target analyte, thus providing a structural basis for the selective action of the subsequent internal filtration effect. Therefore, this positively oriented hierarchical spatially compartmentalized structure provides a key material basis for achieving fluorescence signal decoupling and internal filtration effect pathway regulation, laying an important prerequisite for constructing a high-sensitivity ratio fluorescence detection system.

[0035] The preparation method of bilayer nanoprobes with spatial compartment structure can be adjusted as follows: CTAB can be replaced with other surfactants, such as tetrapropylammonium bromide (TPABr), hexadecyltrimethylammonium chloride (CTAC), and stearyltrimethylammonium chloride (STAC).

[0036] Zn(Ac)2·2H2O can be replaced with other zinc salts, such as one or more of zinc nitrate, zinc chloride, or zinc acetate.

[0037] Methanol used for washing and dispersion can be replaced with one or more other polar solutions, such as ethanol, N,N-dimethylformamide, or deionized water.

[0038] In the reaction system of inner ZIF-8 construction and outer ZIF-8 epitaxial growth, the concentration of the surfactant is 0.27~0.69 mM, and the addition amount is 0.01%~0.025% of the reaction system volume; the concentration of 2-methylimidazole in the reaction system is 60~1200 mM, the concentration of the zinc salt in the reaction system is 15~150 mM, and the molar ratio of 2-methylimidazole to zinc salt is 4:1~80:1. Coumarin can be replaced by other types of coumarin fluorescent dyes, added to the reaction system in a 1~5 mM solution, and the addition amount is 0.05~0.2 mL; Rhodamine B can be replaced by other rhodamine fluorescent dyes, added to the reaction system in a 2~10 mM solution, and the addition amount is 0.05~0.2 mL.

[0039] The static reaction time for preparing the inner ZIF-8@coumarin fluorescent dye nanoparticles is 0.5~24 h, preferably 1~6 h; the static reaction time required for the epitaxial growth of the outer ZIF-8 and the introduction of the rhodamine fluorescent dye is 0.5~24 h, preferably 0.5~3 h.

[0040] Example 2: Construction and fluorescence performance comparison of ZIF-8 nanoprobes with different spatial distribution structures This embodiment constructs three ZIF-8 nanoprobes with different spatial distribution structures of fluorescent dyes, including a monolayer co-supported structure, a reverse layered spatial compartment structure, and a forward layered spatial compartment structure (the preparation method of the ZIF-8 nanoprobe with the forward layered spatial compartment structure is described in Example 1). The fluorescence performance of these nanoprobes is systematically compared to clarify the influence of the spatial distribution of fluorescent dyes on fluorescence interaction and detection performance, thereby verifying the necessity and superiority of the fluorescent probe structure design.

[0041] (1) Preparation of ZIF-8@coumarin / rhodamine B nanoprobes with monolayer co-supported structure Monolayer nanoparticles were prepared using a one-step co-loading method. First, 2 mL of an aqueous solution containing 2-methylimidazole (987 mM) and CTAB (0.59 mM) was prepared and stirred at room temperature for 3 min. Then, 0.25 mL of a 120 mM Zn(Ac)₂·2H₂O aqueous solution was added, and the mixture was rapidly stirred for 1 min. A mixed solution of coumarin and rhodamine B (specifically 0.10 mL of 2 mM coumarin solution and 0.15 mL of 2 mM rhodamine B solution) was added to the system, and stirring was continued for 1 min to ensure that the two fluorescent dyes were simultaneously encapsulated in the same structure during ZIF-8 nucleation and growth. After standing at room temperature for 2 h, the mixture was centrifuged (9000 rpm, 10 min), resuspended in methanol, and centrifuged again. This process was repeated twice to obtain the precipitate, which was the monolayer co-loaded ZIF-8@coumarin / rhodamine B nanoprobe. The precipitate was dried overnight in a vacuum drying oven. Before use, a suspension of a specific concentration was prepared using water or an organic solvent such as methanol.

[0042] (2) Preparation of reverse hierarchical spatial compartment structured nanoprobe ZIF-8@Rhodamine B@ZIF-8@Coumarin (i) Preparation of inner layer ZIF-8@Rhodamine B nanoparticles Add 0.25 mL of a 97.5 mM Zn(Ac)₂·2H₂O aqueous solution to 1.75 mL of an aqueous solution containing 2-methylimidazole (approximately 790 mM) and CTAB (0.41 mM). After stirring for several minutes, add 0.1 mL of a 5 mM Rhodamine B solution and continue stirring for 2–3 minutes. After allowing the reaction to stand at room temperature for 1 hour, centrifuge (9000 rpm, 10 min), resuspend in methanol, and centrifuge again (9000 rpm, 10 min). Repeat this process twice to obtain ZIF-8@Rhodamine B nanoparticles as the precipitate. Redisperse the precipitate in 1 mL of methanol to obtain a uniformly dispersed suspension of ZIF-8@Rhodamine B nanoparticles.

[0043] (ii) Outer layer growth and introduction of coumarin 0.4 mL of the above suspension was dispersed in 1.75 mL of an aqueous solution containing 2-methylimidazole (790 mM) and CTAB (0.47 mM). 0.25 mL of a 97.5 mM Zn(Ac)₂·2H₂O aqueous solution was added to induce epitaxial growth, and 0.1 mL of a 2 mM coumarin solution was added for simultaneous encapsulation. After standing at room temperature for 3 h, the mixture was centrifuged (9000 rpm, 10 min), resuspended in methanol, and centrifuged again (9000 rpm, 10 min). This process was repeated twice to obtain the precipitate, which was a reverse-layered spatial compartment structured nanoprobe ZIF-8@Rhodamine B@ZIF-8@Coumarin. The precipitate was dried overnight in a vacuum drying oven. Before use, a suspension of a specific concentration was prepared using water or an organic solvent such as methanol.

[0044] (3) Fluorescence performance test The two control probes and the positively layered spatial compartment structure nanoprobe prepared by the method in Example 1 were subjected to fluorescence tests under the same conditions. The emission spectra were recorded at a fixed excitation wavelength, and the fluorescence intensity changes of the coumarin (about 450 nm) and rhodamine B (about 590 nm) channels were compared.

[0045] like Figure 4 Figure a shows a schematic diagram of a monolayer co-loaded nanoprobe (ZIF-8@coumarin / rhodamine B), a reverse hierarchical spatial compartment structure nanoprobe (ZIF-8@rhodamine B@ZIF-8@coumarin), and a forward hierarchical spatial compartment structure nanoprobe (ZIF-8@coumarin@ZIF-8@rhodamine B).

[0046] Figure 4 b in the figure represents the comparison results of the fluorescence performance of the three structures.

[0047] In a monolayer system, the two dyes coexist in the same spatial region at close proximity, resulting in a significant FRET effect. This manifests as a decrease in coumarin fluorescence intensity of approximately 40% and an increase in rhodamine fluorescence of approximately 10%, exhibiting typical donor-acceptor energy transfer characteristics. The fluorescence signal in this system exhibits significant crosstalk, which is unfavorable for use as a stable dual-channel detection signal.

[0048] In the reverse hierarchical spatial compartment structure nanoprobe (ZIF-8@Rhodamine B@ZIF-8@Coumarin), although spatial separation reduces FRET, the fluorescence of Rhodamine B is reduced by about 35% and the fluorescence of coumarin is enhanced by about 20% because Rhodamine B is located in the inner layer. This indicates that there is a "spatial shielding effect" of the inner dye, which weakens the response signal and is not conducive to subsequent detection response based on the inner filtration effect. In contrast, in the forward hierarchical spatial compartment structure nanoprobe of Example 1 (ZIF-8@Coumarin@ZIF-8@Rhodamine B), coumarin is located in the inner spatial region and Rhodamine B is located in the outer spatial region. Their interaction is effectively suppressed, the fluorescence emission peaks remain relatively independent, and no obvious FRET phenomenon was observed, thus achieving stable output of dual-channel fluorescence signals.

[0049] Further analysis of subsequent detection applications reveals that the monolayer co-supported nanoprobe structure exhibits a significant FRET effect, leading to instability in the internal reference signal (coumarin) and thus reducing the accuracy of ratiometric detection. In the reverse layered spatial compartment structure nanoprobe, the interaction between the responding dye Rhodamine B and the target analyte is limited due to the inner layer, hindering the effective occurrence of the internal filtration effect and resulting in reduced detection sensitivity. In contrast, the forward layered spatial compartment structure nanoprobe, with Rhodamine B located in the outer layer, preferentially interacts with malachite green, effectively modulating the internal filtration effect, while maintaining a stable coumarin signal in the inner layer. This results in a ratiometric fluorescence detection system with high stability and reliability.

[0050] The above results indicate that the spatial distribution of fluorescent dyes has a decisive influence on fluorescence behavior and detection performance. This invention, by constructing an "inner reference-outer response" spatial compartment structure, not only structurally suppresses signal crosstalk caused by FRET, but also achieves spatially directional modulation of the inner filtering effect, thus providing a key structural foundation for constructing a highly sensitive and stable ratiometric fluorescence detection system.

[0051] Example 3: Ratio fluorescence detection method and performance comparison of malachite green in aquatic products using ZIF-8 nanoprobes with different structures This embodiment is used to evaluate the performance of the positively layered spatial compartment dual-fluorescent ZIF-8 nanoprobe prepared in Example 1 in the detection of malachite green, and compare it with the monolayer co-loaded and reverse layered spatial compartment structure nanoprobe constructed in Example 2, thereby verifying the influence of the spatial distribution mode of fluorescent dye on the detection performance.

[0052] (1) Detection principle This embodiment constructs a ratiometric fluorescence detection system based on a dual-fluorescent nanoprobe. In the forward-oriented hierarchical spatial compartment structure nanoprobe, coumarin and rhodamine are located in the inner and outer spatial regions, respectively, enabling effective decoupling of the fluorescence signals. Malachite green exhibits characteristic absorption in the 600–620 nm range, and its absorption spectrum overlaps with the emission spectrum of rhodamine (e.g., ...). Figure 5 As shown in the figure, the fluorescence of the rhodamine channel is selectively quenched through the internal filtration effect, while the coumarin channel remains essentially stable. Therefore, the concentration of malachite green can be quantitatively analyzed by measuring the intensity ratio of the two fluorescence channels (F1 / F2 or F2 / F1).

[0053] (2) Standard curve Preparation of malachite green standard solution: Weigh an appropriate amount of malachite green standard, dissolve it in deionized water or a methanol-water mixture, and prepare a 10 mM malachite green stock solution (mother liquor). Store it in the dark for later use. Subsequently, based on the stock solution, prepare malachite green standard working solutions of different concentrations using a stepwise gradient dilution method. The working solution concentrations are 0, 0.01, 0.025, 0.1, 0.2, 0.5, 1, 2, and 5 μM, respectively, for subsequent standard curve establishment and detection performance evaluation.

[0054] 100 μL of 1 mg / mL suspensions of forward-layered spatial compartment nanoprobes, monolayer co-supported nanoprobes, and reverse-layered spatial compartment nanoprobes were added to 50 μL of the sample solution to be tested. After mixing, the mixtures were incubated at room temperature for 30 s to 2 min. Fluorescence emission spectra were measured at excitation wavelengths of 350–380 nm. The fluorescence intensity F1 of the coumarin channel (approximately 450 nm) and the fluorescence intensity F2 of the rhodamine channel (approximately 590 nm) were recorded, and a standard curve was established using F2 / F1 as the response signal.

[0055] like Figure 6 As shown in Figure a, the ZIF-8@coumarin / rhodamine monolayer co-supported nanoprobe exhibits a linear response in the range of 0.1–5 μM, with the linear equation being y = -0.89lgx + 1.15 (R0). 2 =0.976), and the limit of detection (LOD) is 0.083 μM; Figure 6 As shown in b, the ZIF-8@Rhodamine B@ZIF-8@Coumarin reverse-layered spatial compartment nanoprobe exhibits a linear response in the range of 0.1~5 μM, with the linear equation being y=-0.17lgx+0.30(R 2 =0.986), LOD is 0.079 μM; such as Figure 6As shown in c, the ZIF-8@coumarin@ZIF-8@rhodamine B positive hierarchical spatial compartment nanoprobe exhibits good linearity in the range of 0.01~5 μM, with the linear equation being y=-0.41lgx+0.36(R 2 =0.982), LOD is 0.0076 μM.

[0056] The above results indicate that the spatial distribution of different fluorescent dyes has a decisive influence on the detection performance of malachite green. In the monolayer co-supported structure, the coexistence of coumarin and rhodamine in the same space easily leads to FRET, causing coupling between the internal reference signal and the response signal, thereby reducing the stability of the ratio signal and the detection sensitivity. In the reverse layered spatial compartment structure nanoprobe, although FRET is suppressed to some extent by spatial separation, the optical interaction between rhodamine and malachite green is shielded by the outer ZIF-8 structure due to the inner layer, limiting the effective occurrence of the internal filtering effect and making it difficult to achieve high-sensitivity detection. In contrast, in the forward layered spatial compartment structure, coumarin is located in the inner layer as a stable internal reference signal, and rhodamine is located in the outer layer as a response signal. It can preferentially overlap with malachite green and produce an internal filtering effect, while avoiding FRET interference. This achieves a synergistic regulation mechanism of "signal decoupling-selective response", significantly improving the detection sensitivity and achieving a performance improvement of about one order of magnitude.

[0057] (3) Preparation of the sample solution to be tested For aquatic product samples, the sample tissue was first homogenized. 2.0 g of the homogenized sample was weighed and placed in a 50 mL centrifuge tube. 4 mL of an acetonitrile-water mixture (8:2 volume ratio) was added, and the sample was vortexed for 2 min to extract malachite green. The sample was then centrifuged at 8000 rpm for 10 min, and the supernatant was collected as the test solution. For environmental water samples, the samples were filtered through a 0.22 μm pore size membrane and used directly as the test solution.

[0058] (4) Detection of Malachite Green in Aquatic Products To evaluate the accuracy of this method, spiked recovery experiments were conducted on environmental water, fish meat, and shrimp meat samples. Specifically, fish meat, shrimp meat, and shellfish tissue were selected as representative aquatic product samples. First, homogenization was performed, and 2.0 g of the homogenized sample was weighed into centrifuge tubes. Malachite green standard solution was added to achieve final spike concentrations of 0.05 μM, 0.20 μM, and 1.00 μM, respectively. After spiking, the samples were allowed to stand at room temperature for 10 min. Then, the sample solutions were prepared according to the method described in step (3). During the detection, 50 μL of the sample solution to be tested was taken, and 100 μL of a 1 mg / mL forward-stratified spatial compartment nanoprobe (ZIF-8@coumarin@ZIF-8@rhodamine B) suspension was added, i.e., the volume ratio of the probe suspension to the sample solution to be tested was 2:1. After mixing evenly, the mixture was incubated at room temperature for 1 min. The fluorescence emission spectrum was measured under the condition of excitation wavelength 350~380 nm. The fluorescence intensity F1 of the coumarin channel (about 450 nm) and the fluorescence intensity F2 of the rhodamine channel (about 590 nm) were recorded. The F2 / F1 response signal was used as the standard curve to calculate the malachite green content in the sample solution to be tested.

[0059] Recovery rate (%) = (measured concentration / added concentration) × 100%; Coefficient of variation (%) = (standard deviation of measured concentration / average of measured concentration) × 100%.

[0060] Table 1. Results of Detection of Malachite Green in Aquatic Products by ZIF-8@Coumarin@ZIF-8@Rhodamine B Nanoprobe The results are shown in Table 1. The ZIF-8@coumarin@ZIF-8@rhodamine B nanoprobe demonstrated good recovery and repeatability in detecting malachite green in different matrix samples. Specifically, the recovery rate in environmental water samples was 94.13%–104.87%, with a coefficient of variation not exceeding 11.97%; the recovery rate in fish samples was 85.67%–99.37%, with a coefficient of variation not exceeding 4.28%; and the recovery rate in shrimp samples was 85.53%–111.03%, with a coefficient of variation not exceeding 5.00%. These results indicate that the ZIF-8@coumarin@ZIF-8@rhodamine B nanoprobe can effectively detect malachite green in different matrix samples, exhibiting good accuracy and precision.

Claims

1. A bifluorescent nanoprobes having a spatial compartmentalized structure, characterized in that, The nanoprobe is a core-shell structure constructed with ZIF-8 as the framework. The core-shell structure includes an inner ZIF-8 layer and an outer ZIF-8 layer, wherein: the inner ZIF-8 layer encapsulates a coumarin-based fluorescent dye as an internal reference channel; and the outer ZIF-8 layer encapsulates a rhodamine-based fluorescent dye as a response channel.

2. The nanoprobe of claim 1, wherein, Its preparation method includes the following steps: S1. An aqueous solution containing surfactant and 2-methylimidazole is mixed with an aqueous solution of zinc salt and stirred until homogeneous. A coumarin-based fluorescent dye solution is added, stirred until homogeneous, and allowed to stand for 0.5-24 h. After the reaction is complete, the supernatant is discarded by centrifugation, and the precipitate is washed and dispersed with a polar solvent to obtain a suspension of ZIF-8@coumarin-based fluorescent dye nanoparticles. S2. A suspension of ZIF-8@coumarin fluorescent dye nanoparticles was dispersed in an aqueous solution containing a surfactant and 2-methylimidazole. A zinc salt aqueous solution was added and mixed well. A rhodamine fluorescent dye solution was added and mixed well. The mixture was allowed to stand for 0.5-24 h. After the reaction was completed, the supernatant was discarded by centrifugation. The precipitate was washed with a polar solvent and dried to obtain a dual fluorescent nanoprobe ZIF-8@coumarin fluorescent dye@ZIF-8@rhodamine fluorescent dye with a spatial compartment structure. In the reaction system of step S1 or step S2, the concentration of the surfactant is 0.27~0.69 mM, and the amount added is 0.01%~0.025% of the volume of the reaction system; the concentration of 2-methylimidazole in the reaction system is 60~1200 mM, and the concentration of the zinc salt in the reaction system is 15~150 mM; the molar ratio of 2-methylimidazole to zinc salt is 4:1~80:

1. The concentration of the coumarin-based fluorescent dye solution is 1-5 mM, and the amount added is 0.05-0.2 mL; the concentration of the rhodamine-based fluorescent dye is 2-10 mM, and the amount added is 0.05-0.2 mL.

3. The nanoprobe of claim 2, wherein, The surfactant is CTAB, TPABr, CTAC, or STAC; the zinc salt is one or more of zinc acetate, zinc nitrate, zinc chloride, or zinc acetate; and the polar solution is one or more of methanol, ethanol, N,N-dimethylformamide, or deionized water.

4. The nanoprobe of claim 3, wherein, The static reaction time in step S1 is 1~6 h, and the static reaction time in step S1 is 0.5~3 h.

5. The nanoprobe of claim 4, wherein, It is ZIF-8@coumarin@ZIF-8@rhodamine B, and its preparation method includes the following steps: S1. An aqueous solution containing CTAB and 2-methylimidazole was mixed with an aqueous solution of Zn(Ac)2·2H2O and stirred until homogeneous. A methanol solution of coumarin was added, and the mixture was stirred until homogeneous. The mixture was then allowed to stand for 3 h. After the reaction was completed, the supernatant was discarded by centrifugation. The precipitate was washed with a polar solvent and dispersed to obtain a suspension of ZIF-8@coumarin nanoparticles. S2. ZIF-8@coumarin nanoparticle suspension was dispersed in an aqueous solution containing CTAB and 2-methylimidazole, zinc salt aqueous solution was added and mixed, rhodamine B aqueous solution was added and mixed, and the reaction was allowed to stand for 1 h. After the reaction was completed, the supernatant was discarded by centrifugation, the precipitate was washed with methanol and dispersed to obtain dual fluorescent nanoprobes ZIF-8@coumarin@ZIF-8@rhodamine B with a spatial compartment structure. The aqueous solution containing CTAB and 2-methylimidazole has a CTAB concentration of 0.41~0.47 mM and a 2-methylimidazole concentration of 790 mM; the aqueous solution containing Zn(Ac)2·2H2O has a Zn(Ac)2·2H2O concentration of 97.5 mM; and the volume ratio of the aqueous solution containing CTAB and 2-methylimidazole to the aqueous solution containing Zn(Ac)2·2H2O is 1.75:0.

25. The concentration of the coumarin methanol solution is 5 mM, and the amount added is 0.1 mL; the concentration of the rhodamine B aqueous solution is 5 mM, and the amount added is 0.1 mL.

6. The application of the nanoprobe according to any one of claims 1 to 5 in the detection of malachite green.

7. Use according to claim 6, characterized in that, The malachite green mentioned is derived from environmental water or aquatic products.

8. A method of detecting malachite green, characterized by, Includes the following steps: S1. Extract malachite green from the sample to be tested and prepare the sample solution; S2. Prepare malachite green standard solutions with different concentration gradients using malachite green samples; S3. Add 100 μL of the dual fluorescent nanoprobe ZIF-8@coumarin@ZIF-8@rhodamine B with a spatial compartment structure to every 50 μL of the sample solution to be tested or malachite green standard solutions of different concentration gradients, dilute to a concentration of 1 mg / mL, and incubate for 30 s to 2 min. Measure the fluorescence emission spectrum under the condition of excitation wavelength of 350 to 380 nm, and record the fluorescence intensity F1 of the coumarin channel at 450 nm and the fluorescence intensity F2 of the rhodamine channel at 590 nm. S4. Establish standard curves using the fluorescence intensity ratios F2 / F1 or F1 / F2 of malachite green standard solutions with different concentration gradients as response signals; S5. Substitute the fluorescence intensity ratio F2 / F1 or F1 / F2 of the sample solution into the standard curve to calculate the malachite green content in the sample.

9. The method of claim 8, wherein, When the sample to be tested is ambient water, step S1 involves filtering the sample through a 0.22 μm filter membrane to obtain the sample solution; when the sample to be tested is aquatic product, 4 mL of acetonitrile-water mixed solution is added to every 2.0 g of sample homogenate, vortexed for 2 min, centrifuged, and the supernatant is taken as the sample solution; the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 8:2.

Images