Quantum dot microsphere fluorescent probe for trace quaternary ammonium salt detection and preparation method thereof

Abstract

The invention relates to the technical field of analytical chemistry and nano sensing materials, and discloses a quantum dot microsphere fluorescent probe for trace quaternary ammonium salt detection and a preparation method thereof, and the probe is prepared from porous CdSe quantum dot microspheres, fluorescein isothiocyanate and a buffer solution. The FITC is used as an energy donor, the porous microspheres are used as an energy receptor, the FITC and the porous microspheres close the distance through electrostatic adsorption, a fluorescence resonance energy transfer system with an open initial state is constructed, and fluorescence quenching of the FITC and fluorescence enhancement of the quantum dots are shown. When quaternary ammonium salt is detected, the FITC is separated from the surface of the microsphere by utilizing a hydrophobic competitive replacement mechanism, the FRET process is blocked, the fluorescence of the FITC is recovered, and the fluorescence of the quantum dots is reduced, so that the detection is realized through the change of a ratio fluorescence signal. The method has the advantages of high detection sensitivity, good selectivity, high response speed, strong anti-interference capability and the like, and realizes rapid quantitative detection of trace long alkyl quaternary ammonium salt in a complex environment.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and nanosensor materials technology, specifically to a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts and its preparation method. Background Technology

[0002] Quaternary ammonium compounds are widely used in industrial cleaning, medical and health care, and daily chemical products due to their excellent bactericidal, disinfectant, and surfactant properties. However, excessive quaternary ammonium residues not only cause environmental pollution problems such as eutrophication of water bodies, but also pose potential hazards to human health. Therefore, achieving rapid and sensitive detection of trace quaternary ammonium compounds in the environment is of significant practical importance.

[0003] Current quaternary ammonium salt detection technologies mainly rely on high-performance liquid chromatography (HPLC), mass spectrometry (MS / MS), and two-phase titration. While chromatography and mass spectrometry offer high detection accuracy, they are heavily reliant on expensive, large-scale instruments, and their sample pretreatment processes are cumbersome and lengthy, making them unsuitable for rapid, real-time on-site detection. Traditional titration methods are complex to operate, have limited sensitivity for low-concentration samples, and are susceptible to human error.

[0004] In recent years, rapid detection technologies based on fluorescence spectroscopy have attracted widespread attention due to their fast response speed and ease of operation. However, existing quaternary ammonium salt fluorescent probes mostly use changes in fluorescence intensity at a single wavelength as the signal output. In practical applications, they are easily affected by fluctuations in excitation light sources, differences in probe concentration, and background signals from complex sample matrices, resulting in poor reproducibility and accuracy of detection results. Furthermore, when constructing nanofluorescent probes, the commonly used carrier materials are mostly solid microspheres or nanoparticles. This solid structure limits the specific surface area of ​​the material, resulting in a low loading of signal molecules, which in turn restricts the overall luminescence intensity and sensitivity of the probe. More importantly, the solid structure lacks internal mass transfer channels. When long-chain quaternary ammonium salt molecules interact with the probe surface, they often face significant steric hindrance and diffusion resistance, leading to slow displacement reaction kinetics, prolonged detection response time, and difficulty in achieving truly rapid detection. Therefore, developing a self-calibrating ratiometric fluorescent probe with high sensitivity, fast response kinetics, and strong anti-interference capabilities remains a pressing technical challenge in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts and its preparation method, which solves the problems of low sensitivity, strong instrument dependence, cumbersome operation, slow response, and weak anti-interference ability in existing quaternary ammonium salt detection technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts, employing the following technical solution: A quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts, the probe being made from the following raw materials in parts by weight: 80-120 parts porous CdSe quantum dot microspheres; 0.2-1.0 parts fluorescein isothiocyanate; and 20,000-300,000 parts buffer solution.

[0007] By employing the above technical solution, this invention constructs a ratiometric fluorescent probe based on the fluorescence resonance energy transfer (FRET) mechanism. This probe utilizes fluorescein isothiocyanate (FITC) as the energy donor and porous CdSe quantum dot microspheres as the energy acceptor and structural framework. The two bind through electrostatic adsorption. When FITC is excited by light, energy is non-radiatively transferred to the quantum dot microspheres, resulting in a FRET-on state where FITC fluorescence is quenched and the fluorescence of the quantum dot microspheres is enhanced.

[0008] The innovative mechanism and detection process of this invention are as follows: Electrostatic assembly to construct the initial “Turn-on” FRET system: In a buffer environment, the donor molecule is adsorbed onto the acceptor surface by utilizing the electrostatic attraction between positively charged quantum dot microspheres and negatively charged FITC, bringing the two closer to the effective interaction distance of FRET, thereby establishing an efficient energy transfer channel. At this time, the probe exhibits low FITC fluorescence and high quantum dot fluorescence characteristics.

[0009] Hydrophobic interaction recognition: The cadmium acetate-oleylamine complex ligand modified on the surface of the microspheres forms a specific hydrophobic layer. Long-chain quaternary ammonium salts in the test sample can preferentially insert into and anchor to this ligand layer through the hydrophobic interaction of their alkyl chains.

[0010] Competitive displacement response (“Turn-off” FRET): Because the hydrophobic affinity between the long-chain quaternary ammonium salt and the cadmium acetate-oleylamine complex ligand on the microsphere surface is stronger than the electrostatic attraction between FITC and the microsphere, and because the cationic head group of the quaternary ammonium salt repels or neutralizes the positively charged sites on the microsphere surface, FITC molecules detach from the microsphere surface. This detachment increases the donor-acceptor distance, blocks the FRET process, and energy transfer ceases.

[0011] Ratio-rate fluorescence signal output: With FRET blocking, FITC fluorescence significantly recovers (enhances), while the quantum dot microspheres lose their energy source, leading to decreased fluorescence. By detecting the change in the ratio of FITC to the characteristic emission peak intensity of quantum dots from low to high, quantitative detection of trace quaternary ammonium salts is achieved. This ratio-rate measurement mode effectively eliminates light source fluctuations and environmental background interference.

[0012] Preferably, the porous CdSe quantum dot microspheres are prepared by a preparation system comprising the following raw materials: an oil phase component including hydrophobic CdSe quantum dots, cadmium acetate-oleylamine complex ligand, and dichloromethane; and an aqueous phase component including deionized water and polyvinyl alcohol; wherein the concentration of the hydrophobic CdSe quantum dots in dichloromethane is 3-8 mg / mL, and the amount of cadmium acetate-oleylamine complex ligand added is 15-30 μL per milliliter of dichloromethane.

[0013] By adopting the above technical solution, the cadmium acetate-oleylamine complex ligand, as a powerful surface modifier, can not only disperse quantum dots more stably, but its unique metal-organic complex structure can also construct recognition sites with high affinity for long-chain alkyl groups on the surface of microspheres, significantly improving the capture efficiency of probes for long-chain quaternary ammonium salts and accelerating the displacement reaction kinetics.

[0014] Preferably, the porous CdSe quantum dot microspheres have an average particle size of 500 nm-10 μm and a porous structure on the surface and inside; the probe has a Zeta potential of 0 mV to +2 mV.

[0015] By employing the above technical solution, the porous structure of the microspheres increases the specific surface area, provides more reaction sites, and significantly improves the probe loading and detection sensitivity. Simultaneously, the porous channels eliminate steric hindrance to substrate diffusion and reduce mass transfer resistance, allowing long-chain quaternary ammonium salt molecules to rapidly penetrate into the microspheres, thereby shortening the response time and achieving rapid detection. Controlling the zeta potential within the near-neutral range indicates that the positive and negative charge assembly has reached equilibrium, and the probe structure is stable.

[0016] Preferably, the buffer solution is a phosphate buffer with a pH of 7.2-7.6; in the probe, fluorescein isothiocyanate is loaded onto the surface of porous CdSe quantum dot microspheres through electrostatic adsorption, and a fluorescence resonance energy transfer system is constructed.

[0017] By adopting the above technical solution, a near-neutral to weakly alkaline pH environment is maintained to ensure that FITC molecules are fully ionized and negatively charged, while maintaining the positive charge on the surface of quantum dot microspheres. This maintains the stability of electrostatic assembly and prevents structural damage of the probe or non-specific shedding of FITC in non-detection states.

[0018] Secondly, the present invention provides a method for preparing a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts, employing the following technical solution: A method for preparing a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts includes the following steps: preparing porous CdSe quantum dot microspheres with hydrophobic ligands modified on the surface by solvent evaporation-induced self-assembly; dispersing the porous CdSe quantum dot microspheres in a buffer solution to obtain a microsphere dispersion; adding a fluorescein isothiocyanate solution to the microsphere dispersion, stirring under light-protected conditions, and adsorbing the fluorescein isothiocyanate onto the surface of the microspheres by electrostatic interaction; centrifuging to remove unadsorbed fluorescein isothiocyanate, redispersing the precipitate, and obtaining the quantum dot microsphere fluorescent probe.

[0019] By adopting the above technical solution, this invention eliminates the need for complex chemical cross-linking modifications and utilizes a simple physical electrostatic assembly process to construct FRET probes. This method is simple to operate, operates under mild conditions, and avoids the potential damage to the fluorescence performance of quantum dots caused by covalent modification. Centrifugation purification removes free dye molecules, reduces background noise, and ensures a high signal-to-noise ratio for the probe.

[0020] Under the preferred process, the step of preparing porous CdSe quantum dot microspheres with hydrophobic ligands on the surface includes: dispersing CdSe quantum dots in dichloromethane, adding cadmium acetate-oleylamine complex ligands, and allowing the mixture to stand and equilibrate to obtain an oil phase; mixing the oil phase with deionized water at a volume ratio of 4:1-6:1, and shearing at 14,000-20,000 rpm to form a W / O type Pickering emulsion; mixing the W / O type Pickering emulsion with a polyvinyl alcohol aqueous solution at a volume ratio of 1:1.5-1:2.5, and shearing at 8,000-13,000 rpm to form a W / O / W type complex emulsion; heating and stirring to evaporate the dichloromethane, and collecting the product by centrifugation.

[0021] By employing the above-mentioned technical solution, the microstructure of microspheres was cleverly controlled using a W / O / W multi-emulsion combined with solvent evaporation. The pore-forming mechanism is as follows: During primary emulsification, trace amounts of aqueous droplets are dispersed in an oil phase containing quantum dots and polymers, forming a W / O emulsion. After secondary emulsification to form the W / O / W multi-emulsion, the oil phase shrinks and solidifies into spheres as the low-boiling-point organic solvent (dichloromethane) slowly evaporates. The tiny water droplets originally dispersed within the oil phase occupy the space. When the solvent completely evaporates and dries, the spaces occupied by these internal aqueous droplets form interconnected or semi-interconnected pore structures. Simultaneously, by adjusting the shear rate to control the size of the emulsion droplets, the arrangement of amphiphilic molecules at the interface is also promoted, ensuring that the resulting microspheres have uniform particle size and a uniform pore structure distribution.

[0022] By employing the above-mentioned technical solution, the long-term static equilibrium ensures that the cadmium acetate-oleylamine complex ligand is fully modified on the surface of CdSe quantum dots, preventing the quantum dots from agglomerating during subsequent emulsification. At the same time, the quantum dots modified by the cadmium acetate-oleylamine complex ligand endow the microsphere surface with hydrophobicity, ensuring that the final microsphere surface has sufficient hydrophobicity, laying the foundation for subsequent specific recognition.

[0023] Preferably, in the step of heating and stirring to volatilize dichloromethane, the heating and stirring temperature is 38-42 °C and the stirring time is 1.5-3 hours.

[0024] By employing the above technical solution, the solvent evaporation rate is precisely controlled: if the temperature is too high or the evaporation is too fast, the surface of the microspheres is prone to premature skin formation and closure, leading to the collapse of the internal pore structure or the inability to form open pores; if the temperature is too low, the curing time is prolonged, and the stability of the emulsion decreases. This temperature control parameter ensures the integrity of the pore structure.

[0025] Preferably, in the step of adding fluorescein isothiocyanate solution to the microsphere dispersion, the concentration of the microsphere dispersion is 0.4-0.6 mg / mL, and the concentration of fluorescein isothiocyanate solution is 0.8 × 10⁻⁶ mg / mL. -4 -1.2×10 -4 The concentration is mol / L, and the volume ratio of the two is (15-25):1; the stirring time is 20-40 minutes.

[0026] By employing the above technical solution, the molar ratio of acceptor to donor was optimized. This ratio ensures that the coverage of FITC on the microsphere surface is sufficient to generate the FRET effect, while avoiding the aggregation-induced quenching (ACQ) effect caused by excessively dense dye molecule packing, thus placing the probe within its optimal operating sensitivity range.

[0027] This invention provides a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts and its preparation method. It has the following beneficial effects: 1. The quantum dot microspheres prepared by this invention have a rich and interconnected porous structure. This microstructure increases the specific surface area of ​​the microspheres, providing a large number of active sites for the adsorption of fluorescein isothiocyanate, thereby improving the probe loading and detection sensitivity. At the same time, the open pore structure effectively reduces the mass transfer resistance of the substrate inside the microspheres, providing a physical channel for the diffusion of long-chain quaternary ammonium salt molecules into the microspheres. This enables the probe to quickly complete the competitive displacement reaction, shortening the detection response time and overcoming the response lag problem caused by diffusion limitation of traditional solid structure probes.

[0028] 2. The probe constructed in this invention utilizes the hydrophobic interaction between the hydrophobic ligands modified on the surface of microspheres and the long-chain quaternary ammonium salt as a specific recognition mechanism. It can effectively distinguish the target long-chain molecules from hydrophilic short-chain molecules and inorganic ions in the environment, and has excellent anti-interference ability. Combined with the dual-wavelength ratio fluorescence detection mode, the probe performs quantitative analysis by changing the ratio of the intensities of two characteristic emission peaks. It can automatically correct errors caused by light source fluctuations and sample matrix background, ensuring the accuracy of trace quaternary ammonium salt detection in complex real water samples.

[0029] 3. The preparation process adopted in this invention is based on the principle of solvent evaporation-induced self-assembly and electrostatic adsorption, which avoids complex chemical cross-linking reactions and harsh synthesis conditions. The operation is simple and the reaction is mild. It not only effectively prevents the potential damage to the fluorescence performance of quantum dots by chemical modification process, but also effectively preserves the optical activity of the material. Moreover, the preparation cycle is short, the post-processing is simple, and the product can be easily purified by centrifugation, which reduces the production cost and facilitates large-scale preparation. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments and comparative examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Preparation Examples 1-3: Preparation Example 1: This preparation example provides a method for preparing porous CdSe quantum dot microspheres, including the following steps: Preparation of oil phase solution: CdSe quantum dots were dispersed in dichloromethane, and the concentration of CdSe quantum dots in the oil phase was controlled at 5 mg / mL. Cadmium acetate-oleylamine complex ligand was added to the dispersion at a rate of 20 μL of ligand per mL of dichloromethane solution. The mixture was allowed to stand at 25 °C for 12-24 hours to obtain a stable oil phase solution. Primary emulsification: Add the above oil phase solution and deionized water to a centrifuge tube at a volume ratio of 5:1, and use a disperser to shear emulsify at 17020 rpm for 1 minute to form a W / O type Pickering emulsion; Secondary emulsification: The above W / O type Pickering emulsion was mixed with a 1.5% (w / w) polyvinyl alcohol (PVA) aqueous solution at a volume ratio of 1:2, and sheared emulsified for 1 minute at a speed of 10420 rpm using a disperser to form a W / O / W type double emulsion; Pore ​​formation and curing: The above W / O / W type double emulsion was placed on a constant temperature magnetic stirrer and stirred continuously for 2 hours at 40℃ and 150 rpm to allow dichloromethane to fully evaporate. After the solvent was completely evaporated, the product was collected by centrifugation, washed three times with deionized water, and vacuum dried to obtain porous CdSe quantum dot microspheres with an average particle size of about 2 μm.

[0032] Preparation Example 2: This preparation example provides a method for preparing porous CdSe quantum dot microspheres, including the following steps: Preparation of oil phase solution: CdSe quantum dots were dispersed in dichloromethane, and the concentration of CdSe quantum dots was controlled at 5 mg / mL. Cadmium acetate-oleylamine complex ligand was added to the dispersion at a rate of 20 μL of ligand per mL of dichloromethane solution. The mixture was allowed to stand at room temperature of 25℃ for 12-24 hours to obtain a stable oil phase solution. Primary emulsification: Add the above oil phase solution and deionized water to a centrifuge tube at a volume ratio of 5:1, and use a disperser to shear emulsify at 14000 rpm for 1 minute to form a W / O type Pickering emulsion; Secondary emulsification: The above W / O type Pickering emulsion was mixed with a 1.5% (w / w) polyvinyl alcohol (PVA) aqueous solution at a volume ratio of 1:2, and sheared emulsified for 1 minute at 8000 rpm using a disperser to form a W / O / W type double emulsion; Pore ​​formation and curing: The above W / O / W type double emulsion was placed on a constant temperature magnetic stirrer and stirred continuously for 2 hours at 40 ℃ and 150 rpm to allow dichloromethane to fully evaporate. After the solvent was completely evaporated, the product was collected by centrifugation, washed three times with deionized water, and vacuum dried to obtain porous CdSe quantum dot microspheres with an average particle size of about 8 μm.

[0033] Preparation Example 3: This preparation example provides a method for preparing porous CdSe quantum dot microspheres, including the following steps: Preparation of oil phase solution: CdSe quantum dots were dispersed in dichloromethane, and the concentration of CdSe quantum dots was controlled at 5 mg / mL. Cadmium acetate-oleylamine complex ligand was added to the dispersion at a rate of 20 μL of ligand per mL of dichloromethane solution. The mixture was allowed to stand at room temperature of 25℃ for 12-24 hours to obtain a stable oil phase solution. Primary emulsification: Add the above oil phase solution and deionized water to a centrifuge tube at a volume ratio of 5:1, and use a disperser to shear emulsify at 20,000 rpm for 1 minute to form a W / O type Pickering emulsion; Secondary emulsification: The above W / O type Pickering emulsion was mixed with a 1.5% (w / w) polyvinyl alcohol (PVA) aqueous solution at a volume ratio of 1:2, and sheared emulsified for 1 minute at 13000 rpm using a disperser to form a W / O / W type double emulsion; Pore ​​formation and curing: The above W / O / W type double emulsion was placed on a constant temperature magnetic stirrer and stirred continuously for 2 hours at 40℃ and 150rpm to allow dichloromethane to fully evaporate. After the solvent was completely evaporated, the product was collected by centrifugation, washed three times with deionized water, and vacuum dried to obtain porous CdSe quantum dot microspheres with an average particle size of about 600 nm.

[0034] Examples 1-4: Example 1: This example provides a method for preparing a quantum dot microsphere fluorescent probe, using the porous CdSe quantum dot microspheres obtained in Example 1 as a matrix, including the following steps: Dispersion: Accurately weigh the porous CdSe quantum dot microspheres prepared in Example 1 and disperse them in a PBS buffer solution with a pH of 7.4 to prepare a microsphere dispersion with a concentration of 0.5 mg / mL; Mixing: Take the above microsphere dispersion and add a concentration of 1×10⁻⁶ to it. -4 A mol / L fluorescein isothiocyanate (FITC) solution was used, with the volume ratio of the microsphere dispersion to the FITC solution controlled at 20:1. Assembly: The mixture was stirred at room temperature in the dark for 30 minutes, and the donor FITC was adsorbed onto the surface of the acceptor microspheres by the electrostatic attraction between the microspheres and FITC. Purification: Centrifugation was used to remove unadsorbed FITC from the supernatant. The resulting precipitate was the assembled quantum dot microsphere fluorescent probe, which was redispersed in PBS buffer at pH 7.4 for storage. The probe was detected to be in the FRETON state with a zeta potential of approximately 0.5 mV.

[0035] Example 2: This example provides a method for preparing a quantum dot microsphere fluorescent probe, using the porous CdSe quantum dot microspheres with a larger particle size obtained in Example 2 as a matrix, including the following steps: Dispersion: Accurately weigh the porous CdSe quantum dot microspheres (average particle size of about 8 μm) prepared in Example 2, and disperse them in PBS buffer solution at pH=7.4 to prepare a microsphere dispersion with a concentration of 0.5 mg / mL; Mixing: Take the above microsphere dispersion and add a concentration of 1×10⁻⁶ to it. -4 A mol / L fluorescein isothiocyanate (FITC) solution was used, with the volume ratio of the microsphere dispersion to the FITC solution controlled at 20:1. Assembly: Stir the mixture at room temperature in the dark for 30 minutes to allow FITC to be adsorbed onto the surface of the microspheres by utilizing the electrostatic attraction between the microspheres and FITC. Purification: Centrifugation removes unadsorbed FITC from the supernatant. The resulting precipitate is the assembled quantum dot microsphere fluorescent probe. It is then redispersed in PBS buffer at pH 7.4 for storage.

[0036] Example 3: This example provides a method for preparing a quantum dot microsphere fluorescent probe, using the porous CdSe quantum dot microspheres with small particle size obtained in Example 3 as a matrix, including the following steps: Dispersion: Accurately weigh the porous CdSe quantum dot microspheres (average particle size of about 600 nm) prepared in Example 3, and disperse them in PBS buffer solution with pH 7.4 to prepare a microsphere dispersion with a concentration of 0.5 mg / mL; Mixing: Take the above microsphere dispersion and add a concentration of 1×10⁻⁶ to it. -4 A mol / L fluorescein isothiocyanate (FITC) solution was used, with the volume ratio of the microsphere dispersion to the FITC solution controlled at 20:1. Assembly: Stir the mixture at room temperature in the dark for 30 minutes to allow FITC to be adsorbed onto the surface of the microspheres by utilizing the electrostatic attraction between the microspheres and FITC. Purification: Centrifugation removes unadsorbed FITC from the supernatant. The resulting precipitate is the assembled quantum dot microsphere fluorescent probe. It is then redispersed in PBS buffer at pH 7.4 for storage.

[0037] Example 4: This example provides a method for preparing a quantum dot microsphere fluorescent probe, designed to verify the assembly effect under preset concentration fluctuations, including the following steps: Dispersion: Accurately weigh the porous CdSe quantum dot microspheres prepared in Example 1 and disperse them in a PBS buffer solution with a pH of 7.4 to prepare a microsphere dispersion with a concentration of 0.5 mg / mL; Mixing: Take the above microsphere dispersion and add a solution with a concentration of 1.2 × 10⁻⁶. -4 A mol / L fluorescein isothiocyanate (FITC) solution was used, with the volume ratio of the microsphere dispersion to the FITC solution controlled at 20:1. Assembly: Stir the mixture at room temperature in the dark for 30 minutes to allow FITC to be adsorbed onto the surface of the microspheres by utilizing the electrostatic attraction between the microspheres and FITC. Purification: Centrifugation removes unadsorbed FITC from the supernatant. The resulting precipitate is the assembled quantum dot microsphere fluorescent probe. It is then redispersed in PBS buffer at pH 7.4 for storage.

[0038] Comparative Examples 1-2: Comparative Example 1: Compared with Example 1, the difference is that the microsphere matrix used is a solid CdSe quantum dot microsphere; the solid microsphere is prepared by eliminating the first-stage W / O emulsification process, and directly mixing the oil phase solution with the PVA aqueous solution for emulsification, thereby forming a solid structure without internal pores, while the rest are the same.

[0039] Comparative Example 2: Compared with Example 1, the difference is that after mixing the microsphere dispersion with the FITC solution, no stirring assembly was performed for 30 minutes, and no centrifugation purification was performed to remove unadsorbed FITC. Instead, the mixture was used directly as a probe. All other aspects were the same.

[0040] Test Examples 1-5: Test Example 1: Microsphere Morphology Characterization and Particle Size Distribution Testing This test example aims to observe the microstructure and perform particle size statistics on the porous CdSe quantum dot microspheres prepared in Examples 1-3 and the solid CdSe quantum dot microspheres prepared in Comparative Example 1, to verify the ability of the preparation process to control the structure and size of the microspheres.

[0041] Experimental steps: Sample preparation: Take 10 μL of the microsphere dispersions prepared in Preparation Example 1, Preparation Example 2, Preparation Example 3 and Comparative Example 1 respectively, dilute with deionized water to 1 mL, and sonicate for 5 minutes to ensure that the microspheres are monodisperse.

[0042] Scanning electron microscopy (SEM) testing: The diluted sample dispersion was dropped onto a clean silicon wafer surface and allowed to dry naturally at room temperature. The dried silicon wafer carrying the sample was then fixed to the sample stage with conductive adhesive and subjected to surface gold sputtering (sputtering time 30 seconds). The surface morphology and pore distribution of the microspheres were observed using a field emission scanning electron microscope (operating voltage 5.0 kV).

[0043] Transmission electron microscopy (TEM) testing: The diluted sample dispersion was dropped onto a copper grid with a carbon support film and dried under an infrared lamp. The internal structure of the microspheres and the distribution of quantum dots were observed using a transmission electron microscope (accelerating voltage 200 kV).

[0044] Particle size distribution test: The diameter of no less than 200 microspheres in the SEM image was counted using NanoMeasure software, and the average particle size and standard deviation of particle size distribution were calculated; at the same time, the hydrodynamic diameter and polydispersity index (PDI) were determined by dynamic light scattering (DLS).

[0045] Test results: Table 1. Statistical table of structural parameters of microspheres in various preparation examples and comparative examples Results analysis: Based on the data analysis in Table 1, the products obtained from Preparation Examples 1 to 3 all formed the expected spherical structure.

[0046] In terms of morphology and structure, the microspheres prepared in Examples 1, 2, and 3 all exhibited obvious porous features on their surfaces. TEM images showed significant contrast between light and dark areas within the microspheres, with bright areas corresponding to internal cavities or channels and dark areas corresponding to quantum dot and polymer backbone aggregation regions. This confirmed that the W / O / W multi-emulsion method combined with solvent evaporation successfully induced the formation of porous structures. This porous structure originates from the occupancy effect of aqueous droplets within the primary emulsion during preparation. After the organic solvent evaporates, the spaces left by the aqueous phase constitute physical channels. In contrast, Comparative Example 1 used an O / W direct emulsification method, where the oil phase did not contain aqueous droplets. After solvent evaporation, the polymer chains contracted tightly, resulting in a smooth surface under SEM and a uniform, high-electron-density solid structure under TEM.

[0047] Regarding particle size control, the data showed a negative correlation between shear rate and microsphere size. Preparation Example 3 used a higher rotation speed (20,000 rpm for the first stage / 13,000 rpm for the second stage), resulting in high input shear energy and significant droplet fragmentation, ultimately forming submicron-sized microspheres with a diameter of approximately 587 nm. In Preparation Example 2, reducing the rotation speed increased the particle size to approximately 7.89 μm. The PDI values ​​of all samples were below 0.2, indicating that the emulsion self-assembly system exhibited good dispersion stability and size uniformity.

[0048] In summary, the preparation process described in this invention can control the microsphere size within the range of 500 nm to 10 μm by adjusting the emulsification parameters, and stably construct a porous structure with high specific surface area. This structural characteristic provides a physical basis for subsequently increasing the fluorescent probe loading and providing substrate diffusion channels.

[0049] Test Example 2: Probe Assembly Characteristics and FRET System Validation This test case aims to verify the assembly status of the energy donor (quantum dot microspheres) and energy acceptor (FITC) in the quantum dot microsphere fluorescent probe described in Example 1 through potentiometric analysis and fluorescence spectroscopy analysis, and to confirm the effectiveness of electrostatic adsorption and the construction of the fluorescence resonance energy transfer (FRET) system.

[0050] Experimental steps: Sample preparation: Take the following samples for testing: Sample A: Porous CdSe quantum dot microsphere dispersion obtained in Preparation Example 1 (0.5 mg / mL, pH=7.4 PBS solution); Sample B: Pure FITC solution (1×10⁻⁶)-4 (PBS solution with mol / L pH=7.4). Sample C: Quantum dot microsphere fluorescent probe (redispersed solution) prepared in Example 1; Sample D: A simple physical mixture prepared in Comparative Example 4 (without filtration, washing, or equilibration).

[0051] Zeta potential test: Take approximately 1 mL of each of the above groups of samples and inject it into the potential sample cell. Measure the potential using a Zeta potential analyzer at 25℃. Perform the test in triplicate for each sample and record the average Zeta potential.

[0052] Fluorescence spectral characterization: The above samples were spectrally scanned using a fluorescence spectrophotometer. The excitation wavelength was set to 350 nm, and the emission spectrum was scanned from 400 nm to 700 nm. The fluorescence intensity values ​​at 522 nm (FITC characteristic peak) and 581 nm (quantum dot characteristic peak) were recorded.

[0053] Test results: Table 2. Potential and fluorescence intensity parameters before and after probe assembly (excitation wavelength 350 nm) Note: The enhanced fluorescence of quantum dots in sample C is mainly due to the FRET effect.

[0054] Results Analysis: Table 2 reveals the energy transfer process from FITC to quantum dots. Based on the test results at an excitation wavelength of 350 nm, sample A (pure porous quantum dot microspheres, acting as acceptors) has a positively charged surface (Zeta potential +8.6 mV), and its fluorescence emission is predominantly at 581 nm (Ig). 581 (10152), I 522 / I 581 The ratio was only 0.057; sample B (pure FITC solution, used as a donor) had a negatively charged surface (-12.1 mV), and its fluorescence emission was mainly concentrated at 522 nm (I0). 522 (for 13100), I 522 / I 581 The ratio was 2.01; however, the C (FITC probe) potential of the sample obtained after assembly was close to neutral (+0.5mV), and the fluorescence emission characteristics changed significantly—the intensity at 522 nm decreased sharply to 1950, while the intensity at 581 nm increased to 12840. 522 / I 581With a ratio of 0.152, compared to pure donor and acceptor, the probe showed significantly lower fluorescence at 522 nm and significantly enhanced fluorescence at 581 nm, indicating that a fluorescence resonance energy transfer system was successfully constructed between FITC and quantum dots, and the probe was in a low-ratio FRET-on state. In contrast, sample D (comparative example 3 mixture) had a potential of -3.12 mV, and both had high fluorescence intensities at 522 nm and 581 nm (12550 and 13740, respectively). 522 / I 581 The ratio is 0.91, and its signal characteristics are between those of the donor and the acceptor. This indicates that the system simultaneously contains the emission of free FITC that did not participate in FRET (strong signal at 522 nm) and the emission of quantum dots after partial energy transfer (strong signal at 581 nm). This suggests that the FRET system was not completely and exclusively formed under these mixed conditions.

[0055] Test Example 3: Sensitivity and Linearity Range Test This test case aims to evaluate the detection sensitivity and linear response range of the porous CdSe quantum dot microsphere fluorescent probe prepared in Example 1 for the target analyte (DDAC), and to verify the synergistic effect of the porous structure on the sensing performance using Comparative Example 1 (solid microsphere probe) as a control.

[0056] Experimental steps: Preparation of standard solutions: Accurately weigh the diecryldimethylammonium chloride (DDAC) standard, dissolve it in PBS buffer at pH 7.4 and dilute it stepwise to prepare a series of standard solutions with concentrations of 0 μM (blank control), 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM and 15 μM.

[0057] Detection procedure: Take 0.9 mL of each of the fluorescent probe dispersions prepared in Example 1 and Comparative Example 1 and place them in a detection bottle. Add 0.1 mL of the above-mentioned DDAC standard solutions of different concentrations to the bottles respectively, shake gently to mix, and let stand at room temperature for 1 minute to react.

[0058] Spectral Acquisition and Data Processing: Emission spectra were acquired using a fluorescence spectrometer at an excitation wavelength of 350 nm. The emission peak at 522 nm (FITC emission peak, I) was recorded. 522 ) and 581 nm (quantum dot emission peak, I 581 The fluorescence intensity at point ( ) is used to calculate the fluorescence intensity ratio (Ratio = I). 522 / I 581 Linear regression analysis was performed with DDAC concentration as the x-axis and fluorescence intensity ratio as the y-axis.

[0059] Test results: Table 3. Fluorescence intensity ratio response table of Example 1 and Comparative Example 1 at different DDAC concentrations. Results analysis: The porous CdSe quantum dot microsphere fluorescent probe (Example 1) exhibits excellent DDAC detection performance. The sensing mechanism is based on the DDAC disrupting the FRET effect between FITC and the quantum dots, specifically manifested as I 522 Fluorescence intensity increases with increasing DDAC concentration, I 581 The intensity will decrease accordingly, and the ratio of the two (I) 522 / I 581 The ratio monotonically increased from 0.15 (blank) to 3.02 (1500 nM), a change of 20.1 times, demonstrating extremely high signal gain. Within the 0-1500 nM concentration range, this ratio exhibited a good linear relationship with the DDAC concentration (R = 0.001913c + 0.15, r...). 2 >0.999), suitable for quantitative analysis.

[0060] Compared to the solid microsphere probe of Comparative Example 1, the porous structure offers significant advantages. The ratio of Comparative Example 1 only changed from 0.56 to 0.86 (a 1.54-fold change), with a sensitivity far lower than that of Example 1. This difference is mainly attributed to the larger specific surface area provided by the porous structure, which not only increases the number of recognition sites and accelerates the diffusion and binding of DDACs, but also makes the FRET pairs easier to disrupt, thereby achieving higher initial FRET efficiency (lower blank background) and stronger signal response.

[0061] Based on the standard deviation σ = 0.093 (Ratio units) of the blank experiment and the slope of the calibration curve S = 0.001913Ratio / nM, the detection limit (LOD) of this method for DDAC was calculated to be 146 nM. This detection performance indicates that the porous structure design significantly improves the probe's sensitivity, giving it the potential to detect low concentrations of quaternary ammonium disinfectants in environmental water. To further reduce the detection limit, the fluctuation of the blank signal can be reduced by optimizing the probe preparation process, improving labeling efficiency, or improving instrument stability. This probe has a fast response (1 minute) and is easy to operate, providing a feasible technical solution for rapid on-site monitoring.

[0062] Test Example 4: Specificity and Anti-interference Capability Test This test case aims to investigate the recognition specificity of quantum dot microsphere fluorescent probes for long-chain quaternary ammonium salts. By introducing quaternary ammonium salt homologues with different chain lengths and common environmental interfering ions, the core role of hydrophobic interactions in the specific recognition mechanism is verified.

[0063] Experimental steps: Preparation of interfering and target analyte solutions: Prepare aqueous solutions of the following substances as test samples: Long-chain quaternary ammonium salts (target substances): decyl dimethyl ammonium chloride (DDAC), hexadecyl trimethyl ammonium bromide (CTAB); Short-chain quaternary ammonium salts (structural analogs): Tetramethylammonium chloride (TMAC), Tetrabutylammonium bromide (TBAB); Common inorganic interfering ions: sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), magnesium sulfate (MgSO4), and ammonium nitrate (NH4NO3). The concentration of long-chain quaternary ammonium salts was set at 5 μM, and the concentration of other interfering substances was set at 100 μM (i.e., the concentration of interfering substances was 20 times that of the target analyte) to simulate a high background interference environment.

[0064] Cross-test: Take 0.9 mL of each probe dispersion prepared in Example 1, and add 0.1 mL of each of the above-mentioned test solutions. Mix well and let stand at room temperature for 2 minutes.

[0065] Data acquisition: The emission spectra of each mixture system were measured using a fluorescence spectrometer, and I was recorded. 522 with I 581 Numerical values ​​were used to calculate the fluorescence intensity ratio (Ratio) and compared with the blank control group (with an equal amount of PBS buffer added).

[0066] Test results: Table 4. Specific response data for different substances in Example 1 Results analysis: This test case systematically evaluated the specificity and anti-interference ability of a quantum dot microsphere fluorescent probe by introducing quaternary ammonium salt homologues of different chain lengths and common environmental interfering ions. The test results show that the probe has a high specific recognition ability for long-chain quaternary ammonium salts (DDAC, CTAB), while exhibiting excellent anti-interference performance against short-chain quaternary ammonium salts and common inorganic salts, verifying the core role of hydrophobic interactions in the specific recognition mechanism.

[0067] In the test, when 500 nM of long-chain quaternary ammonium salts (DDAC, CTAB) were added, the ratio of the fluorescence intensity of the probes (I0.05) was [not specified]. 522 / I 581The fluorescence intensity (FIN) increased significantly from 0.15 in the blank control to 1.08 and 0.89, respectively, with a 7.2-fold increase in response to DDAC and a 5.9-fold increase in response to CTAB, both exhibiting a "significant response." This result fully demonstrates that the probe can effectively distinguish and specifically bind to quaternary ammonium salt molecules with long alkyl chains. This high selectivity is mainly attributed to the strong hydrophobic interaction between the hydrophobic ligand modified on the probe surface and the long-chain quaternary ammonium salt molecule. This interaction drives the target analyte into the porous structure or binding site of the probe, thereby triggering a change in the fluorescence signal.

[0068] To verify the probe's anti-interference ability, high concentrations (10 μM, 20 times the target concentration) of short-chain quaternary ammonium salts (TMAC, TBAB) and common inorganic salts (NaCl, CaCl2, MgSO4) were introduced into the test. The results showed that for inorganic salts, the probe ratio remained at 0.15, completely consistent with the blank control, exhibiting a "no significant change" state. For short-chain quaternary ammonium salts, although the concentrations of TMAC and TBAB were as high as 10 μM (20 times the DDAC test concentration), the probe only showed a "weak response," with the ratio only slightly increasing to 0.22, a change far less than the response to long-chain quaternary ammonium salts. This phenomenon indicates that short-chain quaternary ammonium salts, lacking sufficiently long hydrophobic chains, cannot form effective hydrophobic binding with the probe; while inorganic salts completely lack hydrophobic structures and cannot interfere with the probe's recognition process.

[0069] Notably, at the same concentration (500 nM), the probe's response to DDAC (1.08) was higher than its response to CTAB (0.89), which may be related to the difference in their alkyl chain structures. DDAC contains two decyl chains, while CTAB contains one hexadecyl chain. This structural difference may lead to different binding abilities and mechanisms with the probe's hydrophobic sites, thus affecting the FRET disruption efficiency and the final signal output. This differential response further demonstrates the close relationship between the probe recognition mechanism and the hydrophobic structure of the target analyte.

[0070] In summary, this quantum dot microsphere fluorescent probe possesses excellent specificity and anti-interference capabilities. It can not only accurately identify long-chain quaternary ammonium salts but also effectively eliminate interference from high-concentration inorganic salts and structural analogs. Furthermore, it exhibits distinguishable response characteristics to long-chain quaternary ammonium salts with different structures, providing a reliable technical basis for the selective detection of specific quaternary ammonium salt pollutants in complex environmental matrices.

Claims

1. A quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts, characterized in that, The probe is made from raw materials comprising the following parts by weight: 80-120 parts of porous CdSe quantum dot microspheres; 0.2-1.0 parts of fluorescein isothiocyanate; 20,000-300,000 portions of buffer solution.

2. The quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts according to claim 1, characterized in that, The porous CdSe quantum dot microspheres are prepared from a preparation system comprising the following raw materials: Oil phase components: hydrophobic CdSe quantum dots, cadmium acetate-oleylamine complex ligand, dichloromethane; Aqueous phase components: deionized water, polyvinyl alcohol; The concentration of the hydrophobic CdSe quantum dots in dichloromethane is 3-8 mg / mL, and the amount of the cadmium acetate-oleylamine complex ligand added is 15-30 μL per milliliter of dichloromethane.

3. The quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts according to claim 2, characterized in that, The method for preparing the porous CdSe quantum dot microspheres includes: The oil phase component is mixed with deionized water for primary emulsification to form a W / O emulsion. The W / O emulsion is then mixed with a polyvinyl alcohol aqueous solution for secondary emulsification to form a W / O / W complex emulsion. Finally, the emulsion is cured by solvent evaporation.

4. The quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts according to claim 1, characterized in that, The porous CdSe quantum dot microspheres have an average particle size of 500 nm-10 μm and a porous structure that extends through the surface and interior. The probe has a zeta potential ranging from 0 mV to +2 mV.

5. The quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts according to claim 1, characterized in that, The buffer solution is a phosphate buffer with a pH of 7.2-7.6; In the probe, fluorescein isothiocyanate serves as the energy donor and is loaded onto the surface of porous CdSe quantum dot microspheres, which act as the energy acceptor, through electrostatic adsorption, thus constructing a fluorescence resonance energy transfer system that is initially in the on state.

6. A method for preparing a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts, characterized in that, The preparation of a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts according to any one of claims 1-5 comprises the following steps: Porous CdSe quantum dot microspheres with hydrophobic ligands of cadmium acetate-oleylamine complex ligands were prepared by solvent evaporation-induced self-assembly method. The porous CdSe quantum dot microspheres were dispersed in a buffer solution to obtain a microsphere dispersion. Add fluorescein isothiocyanate solution to the microsphere dispersion and stir under light-protected conditions. Utilize electrostatic interaction to adsorb fluorescein isothiocyanate onto the surface of the microspheres. Centrifugation was used to remove unadsorbed fluorescein isothiocyanate, and the precipitate was redispersed to obtain the quantum dot microsphere fluorescent probe.

7. The method for preparing a quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts according to claim 6, characterized in that, The step of preparing porous CdSe quantum dot microspheres with surface-modified cadmium acetate-oleylamine complex ligand hydrophobic ligands includes: CdSe quantum dots were dispersed in dichloromethane, and cadmium acetate-oleylamine complex ligands were added. After standing to equilibrate, the oil phase was obtained. The oil phase is mixed with deionized water at a volume ratio of 4:1-6:1 and sheared at a speed of 14,000-20,000 rpm to form a W / O type Pickering emulsion. The W / O type Pickering emulsion was mixed with a polyvinyl alcohol aqueous solution at a volume ratio of 1:1.5-1:2.5 and sheared at a speed of 8000-13000 rpm to form a W / O / W type double emulsion. Heating and stirring cause dichloromethane to evaporate, and centrifugation is used to collect the product.

8. The quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts and its preparation method according to claim 7, characterized in that, In the step of obtaining the oil phase, the cadmium acetate-oleylamine complex ligand is prepared by pre-reaction of cadmium acetate and oleylamine in a molar ratio of 1:(2-4), and the standing equilibration time is 12-24 hours.

9. The quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts and its preparation method according to claim 7, characterized in that, In the step of heating and stirring to volatilize dichloromethane, the heating and stirring temperature is 38-42 °C, and the stirring time is 1.5-3 hours.

10. A quantum dot microsphere fluorescent probe for the detection of trace quaternary ammonium salts according to claim 6, and its preparation method thereof, characterized in that, In the step of adding fluorescein isothiocyanate solution to the microsphere dispersion, the concentration of the microsphere dispersion is 0.4-0.6 mg / mL, and the concentration of fluorescein isothiocyanate solution is 0.8 × 10⁻⁶ mg / mL. -4 -1.2×10 -4 mol / L, and the volume ratio of the two is (15-25):1; The stirring time is 20-40 minutes.

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