Preparation of CQD@Zn-MOF Ratio Fluorescent Sensor and Its Application in Dopamine Detection

Abstract

This invention provides a method for preparing a CQD@Zn-MOF ratiometric fluorescence sensor and its application in dopamine detection. The invention uses urea and ethylenediamine as carbon sources to prepare carbon quantum dots (CQDs), and then uses a solvothermal method to composite CQDs with Zn-MOF to obtain a CQD@Zn-MOF composite material. Characterization by X-ray powder diffraction (PXRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) demonstrates that CQDs are uniformly loaded on the surface and channels of Zn-MOF without damaging the MOF crystal framework. The composite material possesses good crystal phase purity, thermal stability, and structural integrity. As a ratiometric fluorescence sensor, this material exhibits a specific fluorescence response only to dopamine (DA): fluorescence is significantly enhanced at 325 nm and significantly quenched at 556 nm, with a fluorescence intensity ratio of I0.05. 325 / I 556 It exhibits a good linear relationship with dopamine concentration, with a detection limit as low as 9 nM, and possesses high selectivity, high sensitivity, rapid response, wide pH tolerance, and excellent anti-interference properties. This invention enables precise quantification and visual detection of dopamine in complex biological samples such as blood, urine, and tears, providing novel sensing materials and technical solutions for rapid neurotransmitter detection and early screening of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation technology and fluorescence sensing detection, specifically involving a method for constructing a CQD@Zn-MOF ratio fluorescence sensor, and the application of this sensor in the highly selective and sensitive detection of dopamine (DA) in biological samples. Background Technology

[0002] Dopamine (DA) is a key catecholamine neurotransmitter, widely involved in various physiological processes such as central nervous system conduction, motor regulation, mood regulation, and hormone secretion. Abnormal dopamine levels are closely related to a variety of neurological diseases, including Parkinson's disease, Alzheimer's disease, schizophrenia, and depression. Therefore, achieving rapid and accurate dopamine detection is of significant scientific importance and practical application value for the early diagnosis, pathological mechanism research, and clinical monitoring of related diseases.

[0003] Existing methods for dopamine detection mainly include high performance liquid chromatography (HPLC), electrochemical methods, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Although these methods have high accuracy and sensitivity, they generally suffer from drawbacks such as expensive instruments, cumbersome operation, complex sample pretreatment, and long detection cycles, making it difficult to achieve rapid on-site detection and large-scale screening, which greatly limits their application in point-of-care testing (POCT) and visual screening.

[0004] Fluorescence sensing technology, with its unique advantages such as ease of operation, rapid response, high sensitivity, and the ability to achieve visual detection, has become a research hotspot in the field of small molecule detection. Traditional single-emission fluorescence sensors are easily affected by factors such as the concentration of the detection system, ambient temperature, and instrument drift, resulting in large measurement errors and poor repeatability. In contrast, ratiometric fluorescence sensors use the intensity ratio of two characteristic emission peaks as the detection signal, which can effectively eliminate background interference, matrix effects, and systematic errors, significantly improving the accuracy and reliability of detection. This has become the mainstream development direction in the field of fluorescence sensing.

[0005] Metal-organic frameworks (MOFs) are novel porous crystalline materials formed by inorganic metal nodes and organic bridging ligands linked by coordination bonds. They possess high specific surface area, regular pore structure, tunable coordination environment, and excellent fluorescence performance, making them promising candidates for fluorescence sensing. Among them, Zn-MOFs are preferred materials for biosensing due to their good biocompatibility and excellent fluorescence stability. However, single Zn-MOFs suffer from problems such as a single emission peak, strong environmental sensitivity, and limited selectivity for target molecules, which restricts their application in the detection of complex biological samples. Carbon quantum dots (CQDs), as an emerging class of carbon-based luminescent nanomaterials, possess characteristics such as good photostability, excellent water solubility, high biocompatibility, abundant surface functional groups, and ease of modification. Combining CQDs with Zn-MOFs can not only introduce new fluorescence emission channels but also regulate energy transfer and electron transfer processes through interfacial interactions, thereby constructing a highly stable, highly selective, and highly sensitive ratiometric fluorescence sensing system.

[0006] Currently, there is a lack of simple and efficient preparation methods for uniformly doping carbon quantum dots into Zn-MOFs and constructing sensors for highly selective ratiometric fluorescence detection of dopamine in complex biological samples, and the sensing mechanism has not yet been systematically and clearly elucidated. Therefore, this invention provides a construction strategy for a CQD@Zn-MOF ratiometric fluorescence sensor, achieving specific recognition, accurate quantification, and visual detection of dopamine, thus overcoming the shortcomings of existing detection methods. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a CQD@Zn-MOF ratio fluorescence sensor. This method is simple, has mild conditions, good repeatability, and can achieve efficient composite of CQD and Zn-MOF.

[0008] Another objective of this invention is to provide the CQD@Zn-MOF composite material as a ratiometric fluorescence sensor for dopamine detection, achieving high selectivity, high sensitivity, and rapid response detection of dopamine, and applicable to complex biological samples such as blood, urine, and tears.

[0009] I. Fabrication of CQD@Zn-MOF Ratiofluorescence Sensor

[0010] 1. Preparation of CQD@Zn-MOF composite material: Accurately weigh zinc chloride (ZnCl2), 4,4',4''-triaminotriphenyltricarboxylic acid (H3TATB), and 1,4-bis(imidazolyl)benzene (bimb), wherein the molar ratio of ZnCl2, H3TATB, and bimb is 1:2.5:2.5~4:1:1. Dissolve in 10 mL of a mixed solvent of N,N-dimethylformamide (DMF) and deionized water (DMF:H2O = 2:3~3:2, volume ratio), and sonicate for 15 min until completely dissolved; then add 2 μL~20 μL of the prepared CQD solution, and continue sonicating for 5 min to ensure uniform dispersion of CQD in the mixture; transfer the mixed solution to a 25.0 mL Teflon-lined autoclave, place it in a constant temperature drying oven at 80 ℃~100 ℃ for 68~76 h, and then cool down to room temperature. The product was repeatedly washed with DMF and deionized water and dried at 60 °C for 24 h to obtain CQD@Zn-MOF composites with different CQD doping amounts. Among them, the composite material with the best fluorescence response performance was obtained when the CQD addition amount was 10 μL, which was determined to be the optimal doping amount for subsequent experiments.

[0011] 2. Preparation of Zn-MOF: The preparation method of Zn-MOF is the same as that of CQD@Zn-MOF, except that carbon quantum dot solution is not added. The product is colorless bulk Zn-MOF crystals.

[0012] 3. Preparation of carbon quantum dots (CQDs): Dissolve 1 mmol of urea and 3 mmol of ethylenediamine in 10 mL of deionized water, and sonicate for 4-6 min until both reagents are completely dissolved to obtain a homogeneous and transparent mixed solution. Place the mixed solution in a sealed 25.0 mL Teflon-lined autoclave and keep it in a constant temperature drying oven at 150-170 ℃ for 5-7 h. After the reaction is completed, allow it to cool naturally to room temperature to obtain a light yellow carbon quantum dot (CQD) solution. No further purification is required, and it can be directly used for subsequent composite reactions.

[0013] II. Characterization of the structure and properties of CQD@Zn-MOF materials

[0014] 1. Phase structure and thermal stability (PXRD, TGA)

[0015] The phase structures of Zn-MOF and CQD@Zn-MOF were characterized using X-ray powder diffraction (PXRD). Figure 1a) The results show that the PXRD spectrum of the original Zn-MOF has sharp and high-intensity diffraction peaks, which are completely consistent with the diffraction peak positions obtained by simulation based on the single crystal structure of Zn-MOF, indicating that the synthesized Zn-MOF has high crystallinity and high crystal phase purity. After CQD doping, the PXRD spectrum of CQD@Zn-MOF is basically consistent with Zn-MOF in terms of diffraction peak positions, relative intensities and peak shapes, and no new impurity peaks or obvious peak position shifts appear, proving that the introduction of CQD does not destroy the crystal framework structure of Zn-MOF, and the crystal phase of the composite material remains intact and has good purity.

[0016] The thermal stability of the two materials was tested using a thermogravimetric analyzer (TGA) under a nitrogen atmosphere. Figure 1 b、 Figure 1 c) The results showed that the thermal decomposition process of Zn-MOF can be divided into three stages: slight weight loss (approximately 7.293%) from room temperature to 268.58 °C, attributed to the removal of adsorbed water on the material surface and residual solvent in the pores; significant weight loss (approximately 17.787%) from 268.58 °C to 430.36 °C, corresponding to the initial decomposition of organic ligands; and severe weight loss after 430.36 °C, corresponding to the collapse of the Zn-MOF framework. Finally, the residual mass at 800 °C is approximately 37.24%, indicating that Zn-MOF has good thermal stability below 268.58 °C. The thermal decomposition process of CQD@Zn-MOF is similar to that of Zn-MOF, exhibiting a three-stage decomposition characteristic. However, the initial weight loss is slightly increased (approximately 7.718%), and the decomposition initiation temperature of the framework is slightly reduced to 261 °C. This may be related to the functional groups on the CQD surface and their regulatory effect on the local chemical environment of Zn-MOF. Nevertheless, CQD@Zn-MOF can still maintain good structural stability below 261 °C, which can meet the temperature requirements in actual detection processes.

[0017] 2. Microstructure and elemental distribution (SEM, TEM, EDS)

[0018] The microstructure of Zn-MOF and CQD@Zn-MOF was observed using scanning electron microscopy (SEM). Figure 2 a, Figure 2 (b) The results show that Zn-MOF has a regular rhombic blocky crystal structure with relatively uniform crystal size distribution, clear boundaries, and a relatively flat surface, but some layered texture is visible. After being composited with CQD, the overall rhombic blocky morphology of CQD@Zn-MOF is still well maintained, indicating that the composite process did not destroy the main framework structure of MOF. At the same time, the crystal surface is significantly smoother and flatter, and the surface texture is weakened. It is speculated that CQD forms a uniform covering or interface modification layer on the crystal surface, thereby improving the surface morphology of the material.

[0019] The microstructure of CQD and CQD@Zn-MOF was observed using transmission electron microscopy (TEM). Figure 2 (c ~ 2f) The results showed that CQD consisted of uniformly sized, well-dispersed near-spherical nanoparticles with particle sizes within the nanoscale range, and no obvious agglomeration was observed; low-magnification TEM images ( Figure 2 In image e), the rhomboid blocky outline of CQD@Zn-MOF can be clearly identified, further proving that the introduction of CQD did not damage the overall crystal structure; high-magnification TEM image ( Figure 2 d、 Figure 2 In f), several nanoparticles can be observed on the crystal surface and edge regions. Their size is consistent with that of the independent CQD, indicating that the CQD is successfully loaded onto the Zn-MOF surface and is relatively uniformly dispersed.

[0020] The elemental composition and distribution of CQD@Zn-MOF were analyzed using energy-dispersive spectroscopy (EDS). Figure 2 (g ~ 2k), the results showed that the four elements C, N, O, and Zn were uniformly distributed and highly overlapped throughout the entire crystal region, with no obvious elemental segregation or local enrichment; EDS mass distribution map ( Figure 2 In k), characteristic signals of C, O, N and Zn elements can be clearly detected. The elemental composition is consistent with the material structure. No obvious impurity peaks were found, further proving that CQD has been uniformly doped in Zn-MOF.

[0021] 3. Surface chemical state analysis (XPS)

[0022] The surface elemental composition and chemical state of Zn-MOF and CQD@Zn-MOF were characterized using X-ray photoelectron spectroscopy (XPS). Figure 3 a ~ 3e), XPS full spectrum ( Figure 3 a) shows that both materials contain four elements: Zn, O, C, and N, consistent with the composition of the materials; compared with Zn-MOF, the relative intensity of the C 1s peak in CQD@Zn-MOF is significantly enhanced, providing direct evidence for the successful introduction of CQD.

[0023] Zn 2p high-resolution spectrum ( Figure 3 In b), Zn 2p of Zn-MOF 3 / 2 and Zn 2p 1 / 2 The binding energies are approximately 1021.8 eV and 1044.9 eV, respectively, and the spin-orbit splitting energy is approximately 23.1 eV, consistent with Zn. 2+ Characteristic binding energy; Zn2p in CQD@Zn-MOF 3 / 2 and Zn 2p 1 / 2 The peak position did not shift significantly, indicating that the load of CQD Zn did not change. 2+The valence state and its basic coordination environment with organic ligands. O 1s high-resolution spectrum ( Figure 3 In c), the main peak of Zn-MOF at 531.6 eV is attributed to -COO. - The oxygen atom involved in coordination, with a peak at approximately 533.0 eV, is related to adsorbed water or hydroxyl groups (-OH) on the material surface. The O1s main peak in CQD@Zn-MOF remains stable, but a weak shoulder peak appears in the high binding energy region, possibly originating from oxygen-containing functional groups (such as -OH, -COOH) introduced onto the CQD surface, further confirming the successful recombination of CQD. (C 1s high-resolution spectrum) Figure 3 In d), Zn-MOF can be decomposed into three characteristic peaks: CC / C=C (approximately 284.8 eV), CN / CO (approximately 285.6 eV), and O=CO (approximately 288.8 eV). The C 1s signal of CQD@Zn-MOF is significantly enhanced overall, and sp 1s signals appear in the low binding energy region, similar to those in CQD. 2 The characteristic contributions related to the carbon structure, accompanied by the enhancement of oxygen-containing functional group peaks, indicate that CQD has been successfully loaded onto the MOF surface. (N 1s high-resolution spectrum) Figure 3 In e), both materials exhibit characteristic peaks of pyridine nitrogen or imidazole nitrogen in the ligands at approximately 399.5 eV, and the peak positions remain essentially unchanged before and after CQD composite, indicating that the introduction of CQD did not significantly affect the chemical environment of nitrogen in the MOF ligands, and the interfacial bonding of the composite material is stable.

[0024] 4. Optimization of optical performance and doping concentration

[0025] The optical properties of CQD@Zn-MOF composites with different CQD doping concentrations (5 μL, 10 μL, 15 μL) were tested using a fluorescence spectrophotometer. Figure 4 With the excitation wavelength set to 276 nm, the results showed that all three doped materials exhibited obvious dual emission peaks at 325 nm and 556 nm, indicating that the introduction of CQD successfully endowed the composite system with dual emission response characteristics, laying the foundation for the construction of a ratiometric fluorescence sensing system.

[0026] Meanwhile, the fluorescence intensity of the two emission peaks changed significantly with the CQD doping concentration: as the CQD doping concentration increased, the fluorescence intensity at 325 nm (the characteristic emission peak of CQD) gradually increased, while the fluorescence intensity at 556 nm (the characteristic emission peak of Zn-MOF) gradually decreased. When the CQD doping concentration was 10 μL, the initial fluorescence intensities at 325 nm and 556 nm were at a relatively moderate level. This avoided both excessively high emission peak intensities that would compress the subsequent fluorescence enhancement response space and excessively weak signals that would adversely affect detection sensitivity and signal-to-noise ratio. It retained a reasonable response control margin, which is more conducive to the generation of significant fluorescence enhancement or quenching changes under the action of the target analyte (dopamine), thereby obtaining a wider detection range and a more stable ratiometric fluorescence output signal. Based on the above analysis, CQD@Zn-MOF prepared with a CQD doping concentration of 10 μL was selected as the fluorescence sensing material for all subsequent experiments.

[0027] III. Fluorescence Sensing Performance of CQD@Zn-MOF for Dopamine

[0028] 1. Specific identification

[0029] CQD@Zn-MOF (2.0 mg) with the optimal doping amount (10 μL CQD) was dispersed in 5.0 mL of deionized water and sonicated for 20 min to obtain a homogeneous and stable suspension, which served as the fluorescence sensing system. 500 μL of common biological matrix solutions of equal concentration (40 μM), including CaCl2, H2O2, NaCl, KCl, glucose, uric acid, glutathione (GSH), phenylalanine (Phe), and dopamine (DA), were added to this sensing system, and the fluorescence spectra of each system were measured at an excitation wavelength of 276 nm. Figure 5 a, Figure 5 b).

[0030] The results showed that, only in the presence of dopamine, the CQD@Zn-MOF sensing system exhibited a significant fluorescence on-off (enhancement) phenomenon at 325 nm and a significant fluorescence quenching phenomenon at 556 nm, forming a ratiometric fluorescence response with a self-calibrated background. However, after adding other biological matrices, the fluorescence spectrum of the sensing system did not change significantly, and the above ratiometric response characteristics were not observed. This demonstrates that CQD@Zn-MOF has excellent specificity and selectivity for dopamine and can effectively distinguish dopamine from other common biological matrices.

[0031] 2. Quantitative detection and sensitivity

[0032] To investigate the quantitative detection capability and sensitivity of CQD@Zn-MOF for dopamine, different concentrations of dopamine solution (1.0 μM ~ 218.8 μM) were gradually added to the CQD@Zn-MOF suspension, and fluorescence titration experiments were performed. The fluorescence emission spectra at each concentration were collected, and the fluorescence intensity ratio was used as the quantification factor. 325 / I 556 Construct a quantitative relationship curve with dopamine concentration as the x-axis and dopamine concentration as the y-axis. Figure 5 c. Figure 5 d、 Figure 5 f). Quantitative analysis results showed that within the dopamine concentration range of 37.7 μM ~ 87.7 μM, the fluorescence intensity ratio I... 325 / I 556 It exhibits a good linear relationship with dopamine concentration, and the linear equation is: y = -2.888 +0.09072x (where x is the dopamine concentration in μM; y is the fluorescence intensity ratio I). 325 / I 556 ), correlation coefficient R 2 = 0.9919 (R) 2 A value close to 1 indicates a good linear relationship. (Based on 3σ / K) sv Formula (σ is the standard deviation of fluorescence intensity of 10 blank samples, K) sv The sensor, calculated from the slope of the linear equation, has a limit of detection (LOD) of 9 nM and a limit of quantitation (LOQ) of 30 nM for dopamine. Its detection sensitivity is far superior to traditional dopamine detection methods, and it can meet the requirements for accurate detection of low concentrations of dopamine.

[0033] 3. Anti-interference properties, response speed, and pH stability

[0034] Anti-interference experiment: In the CQD@Zn-MOF sensing system, dopamine (40 μM) and other biological interferences (CaCl2, H2O2, NaCl, KCl, glucose, uric acid, GSH, Phe, etc.) of equal concentration were added simultaneously. The fluorescence intensity ratio I of the system was tested. 325 / I 556 The fluorescence ratio was compared with that when only dopamine was added. Figure 5 e). The results showed that under the condition of coexistence of multiple biological interfering substances, the fluorescence response of CQD@Zn-MOF to dopamine was not significantly affected, and the fluorescence intensity ratio did not decrease significantly, indicating that the sensor has excellent anti-interference performance and can be used for the detection of dopamine in complex biological matrices.

[0035] Response speed test: After adding dopamine solution to the CQD@Zn-MOF sensing system, the fluorescence intensity ratio I was monitored in real time. 325 / I 556Changes ( Figure 6 a) The results showed that after the addition of dopamine, the fluorescence signal reached a stable equilibrium within about 40 s, indicating that the sensing process responded quickly, which met the time efficiency requirements of actual analysis and was conducive to the dynamic or real-time monitoring of dopamine.

[0036] pH stability test: The pH value of the CQD@Zn-MOF sensing system was adjusted (2~10), and a fixed concentration of dopamine (40 μM) was added. The fluorescence intensity ratio I of the system under different pH conditions was tested. 325 / I 556 ( Figure 6 (b) The results showed that the fluorescence intensity ratio remained relatively stable within the pH range of 2 to 10, without significant fluctuations. This indicates that the sensor can work effectively under acidic to weakly alkaline conditions and has a wide pH tolerance, providing an important guarantee for its application in biological samples with different pH environments (such as blood pH ≈ 7.4 and urine pH ≈ 4.5~8.0).

[0037] 4. Cyclic stability

[0038] The stability and reusability of the CQD@Zn-MOF sensor were evaluated through cyclic experiments: After each detection, the CQD@Zn-MOF was centrifuged (8000 rpm, 5 min), washed three times with deionized water, dried at 60 ℃, and redispersed in deionized water for the next dopamine detection. This process was repeated 5 times. Figure 6 c). The results showed that after multiple cycles of use, the fluorescence response intensity of CQD@Zn-MOF to dopamine remained at more than 90% of the initial response intensity, indicating that the material has good structural stability and recycling potential, which can effectively reduce the actual detection cost and enhance its sustainable application value.

[0039] IV. Sensing Mechanism

[0040] Combining material structure characterization, fluorescence spectroscopy analysis, fluorescence lifetime testing, and theoretical calculations, this study systematically elucidates the ratiometric fluorescence recognition mechanism of dopamine by CQD@Zn-MOF. Figure 7 a~ Figure 7 f):

[0041] 1. Dopamine forms hydrogen bonds or coordination interactions with oxygen-containing functional groups (-OH, -COOH, etc.) on the surface of CQD. This interaction stabilizes the surface excited state of CQD and suppresses its nonradiative transition process, thereby significantly enhancing the characteristic emission (fluorescence intensity at 325 nm) of CQD, and extending the fluorescence lifetime from 10.0 μs to 12.0 μs. Figure 7 d).

[0042] 2. Dopamine, as an electron donor, has a high highest occupied molecular orbital (HOMO) energy level, while the Zn-MOF framework (constructed from H3TATB and bimb ligands) has a low lowest unoccupied molecular orbital (LUMO) energy level. The energy level distributions of these two components satisfy the energy matching conditions required for photoinduced electron transfer (PET). Dopamine can transfer photogenerated electrons to the Zn-MOF framework, leading to the quenching of the characteristic emission of Zn-MOF (at 556 nm), and a slight decrease in fluorescence lifetime from 24.89 μs to 24.51 μs. Figure 7 e).

[0043] 3. The UV-Vis absorption spectrum of dopamine significantly overlaps with the excitation spectrum of CQD@Zn-MOF. Figure 7 (b) indicates that dopamine can absorb the excitation light energy of CQD@Zn-MOF, leading to a decrease in the excited state energy of CQD@Zn-MOF, which further synergistically promotes fluorescence quenching at 556 nm and enhances the ratiometric fluorescence response.

[0044] In addition, the PXRD spectrum shows ( Figure 7 a) Before and after dopamine adsorption, the position and intensity of the diffraction peaks of CQD@Zn-MOF remained basically consistent, indicating that the MOF framework structure was not destroyed during the recognition process. The interaction between dopamine and CQD@Zn-MOF mainly occurred on the material surface or near the surface of the pores, further proving the rationality of the above sensing mechanism.

[0045] V. Practical Sample Application

[0046] 1. Visualized inspection

[0047] Based on the fluorescence properties of CQD@Zn-MOF, fluorescent test strips were prepared for the visual qualitative detection of dopamine. Figure 8): Cut filter paper into strips of 1 cm × 5 cm, immerse them in CQD@Zn-MOF suspension (2.0 mg / 5.0 mL) for 10 min, then remove and air dry to obtain CQD@Zn-MOF fluorescent test strips. Immerse the test strips in different biological matrix solutions (CaCl2, H2O2, NaCl, KCl, Phe, GSH, DA, etc.) for 5 min, then remove and observe the color changes of the test strips under natural light and 365 nm ultraviolet light. The results showed that under natural light, all test strips soaked in different biological matrices displayed the same color, making them indistinguishable to the naked eye. However, under 360 nm UV light, the test strips exhibited significant fluorescence differences: in the presence of most biomolecules, the test strips displayed uniform and bright fluorescence, indicating that CQD@Zn-MOF had little or no response to these substances; while in the presence of dopamine, the test strips showed a distinct bright cyan fluorescence, and the fluorescence intensity increased with increasing dopamine concentration. This phenomenon directly demonstrates that CQD@Zn-MOF has a strong fluorescent selective recognition ability for dopamine. This fluorescent test strip transforms the excellent fluorescence sensing properties of the material into a simple, portable, and instrument-free on-site detection tool. Qualitative detection of dopamine can be achieved simply by observing the fluorescence color under UV light with the naked eye, providing important technical support and theoretical basis for the development of portable biosensors.

[0048] 2. Real biological sample testing

[0049] To verify the practical application performance of the CQD@Zn-MOF sensor, fresh poultry blood, healthy dog ​​urine, and volunteer tears were selected as real biological samples. The samples were processed according to the optimized pretreatment method (specific pretreatment steps: ① Blood samples: centrifuge at 10,000 rpm for 10 min, collect the upper plasma layer, add a mixed solvent of acetonitrile and methanol (v:v = 1:3) to precipitate proteins, centrifuge again at 10,000 rpm for 10 min, and collect the supernatant for later use; ② Tear samples: after non-contact collection, centrifuge at 10,000 rpm for 5 min to remove impurities, add a mixed solvent of acetonitrile and methanol (v:v = 1:3), centrifuge at 10,000 rpm for 10 min to remove proteins, and adjust to neutral with PBS buffer solution at pH = 7.4 for later use; ③ Urine samples: centrifuge at 10,000 rpm for 10 min to collect the supernatant, filter through a 0.22 μm ultrafiltration membrane to remove suspended particles, and adjust to neutral with PBS buffer solution at pH = 7.4 for later use).

[0050] Using the standard spiking method, 0 μM, 40 μM, 60 μM, and 80 μM dopamine standard solutions were added to the treated real samples, respectively, for spiked recovery testing. The recovery rate and relative standard deviation (RSD) were calculated using fluorescence spectroscopy. The results are shown below. Figure 9 ( Figure 9 a: Urine sample Figure 9 b: Blood sample Figure 9 c: tear sample) As shown, the recovery results are summarized in Table 1 (see Specific Example 3 for details).

[0051] The results showed that dopamine was not detected in the real samples without added dopamine (ND); after spiked detection, the recovery rate of dopamine was 98% ~ 102.1%, and the RSD was <1.82%, indicating that the sensor has good accuracy and repeatability and can be used for the accurate quantitative detection of dopamine in complex biological samples such as blood, urine, and tears. Attached Figure Description

[0053] Figure 1 PXRD spectra of Zn-MOF and CQD@Zn-MOF (a), TGA curve of Zn-MOF (b), and TGA curve of CQD@Zn-MOF (c).

[0054] Figure 2 SEM image of Zn-MOF (a), SEM image of CQD@Zn-MOF (b), TEM image of CQD (c), TEM images of CQD@Zn-MOF at different magnifications (df), EDS energy spectrum of CQD@Zn-MOF (g) and elemental distribution map (hk).

[0055] Figure 3 XPS full spectrum of Zn-MOF and CQD@Zn-MOF (a), Zn 2p high-resolution spectrum (b), O 1s high-resolution spectrum (c), C 1s high-resolution spectrum (d), N 1s high-resolution spectrum (e).

[0056] Figure 4 Fluorescence spectra of CQD@Zn-MOF with different CQD doping amounts (5 μL, 10 μL, 15 μL), with an excitation wavelength of 276 nm.

[0057] Figure 5 Fluorescence response of CQD@Zn-MOF to common biological matrices (a), fluorescence selectivity for dopamine (b), fluorescence intensity ratio I 325 / I 556Linear relationship with dopamine concentration and detection limit (c), CIE chromatogram of dopamine recognition by CQD@Zn-MOF (d), anti-interference ability (e), fluorescence titration curves of dopamine at different concentrations (f).

[0058] Figure 6 Time response curve (a), pH stability curve (b), and recycling performance curve (c) of CQD@Zn-MOF to dopamine.

[0059] Figure 7 PXRD spectra of CQD@Zn-MOF before and after dopamine detection (a), overlay of dopamine absorption spectrum with excitation and emission spectra of CQD@Zn-MOF (b), FT-IR spectra of CQD@Zn-MOF before and after dopamine detection (c), fluorescence lifetime curve of CQD@Zn-MOF at 325 nm (d), fluorescence lifetime curve at 556 nm (e), electrostatic potential of H3TATB, bimb and dopamine and HOMO-LUMO analysis (f).

[0060] Figure 8 Photographs showing the CQD@Zn-MOF fluorescent test strips identifying different biological matrices under natural light and 365 nm UV light.

[0061] Figure 9 Fluorescence spectra of CQD@Zn-MOF for detecting different concentrations of dopamine spiked in urine (a), blood (b), and tears (c). Detailed Implementation

[0062] The following detailed embodiments illustrate the preparation and dopamine detection application of the CQD@Zn-MOF ratio fluorescence sensor of the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0063] All chemical reagents used in this invention are of analytical grade and were used directly without further purification or separation after purchase. The instruments used include: Hitachi F-7000 fluorescence spectrophotometer, Shanghai Jinghong DHG-9036A electric thermostatic drying oven, Shenzhen Jie Cleaning Equipment AK-060SD ultrasonic cleaner, Japanese Hitachi UV-3900 ultraviolet spectrophotometer, X-ray powder diffractometer, thermogravimetric analyzer, scanning electron microscope, transmission electron microscope, and X-ray photoelectron spectrometer.

[0064] Example 1: Preparation of CQD@Zn-MOF

[0065] 1. Preparation of Zn-MOF: Zinc chloride (ZnCl2, 2.0 mg, 0.01 mmol), 4,4',4''-triaminotriphenyltricarboxylic acid (H3TATB, 4.4 mg, 0.01 mmol), and 1,4-bis(imidazolyl)benzene (bimb, 2.4 mg, 0.01 mmol) were accurately weighed and dissolved in 10 mL of a mixed solvent of N,N-dimethylformamide (DMF) and deionized water (DMF:H2O = 4:1, volume ratio). The mixture was ultrasonically stirred for 15 min until all three reagents were completely dissolved. The mixed solution was transferred to a 25 mL Teflon-lined high-pressure reactor and placed in a constant temperature drying oven at 90 °C for 72 h. The temperature was then programmed to decrease to room temperature at a rate of 10 °C / h. The reaction product was washed three times with DMF and deionized water and dried in a constant temperature oven at 60 °C for 24 h to obtain colorless blocky Zn-MOF crystals.

[0066] 2. Preparation of CQD: Dissolve 1 mmol of urea and 3 mmol of ethylenediamine in 10 mL of deionized water, sonicate for 5 min until completely dissolved, transfer to a 25 mL Teflon-lined autoclave, place in a constant temperature drying oven at 160 ℃ for 6 h, and allow to cool naturally to room temperature to obtain a light yellow carbon quantum dot (CQD) solution.

[0067] 3. Preparation of CQD@Zn-MOF: Following the Zn-MOF preparation steps, accurately weigh ZnCl2, H3TATB, and bimb, dissolve them in 10 mL of a mixed solvent of DMF:H2O = 4:1 (volume ratio), and sonicate for 15 min until completely dissolved; add 5 μL, 10 μL, and 15 μL of the prepared CQD solution respectively, and continue sonicating for 5 min to ensure uniform dispersion; the subsequent autoclave reaction, washing, and drying conditions are the same as those for Zn-MOF preparation, to obtain CQD@Zn-MOF composite materials with different CQD doping amounts, denoted as CQD@Zn-MOF ... 5μL @Zn-MOF、CQD 10μL @Zn-MOF、CQD 15μL @Zn-MOF.

[0068] Example 2: Specific recognition of dopamine by CQD@Zn-MOF

[0069] Take the CQD prepared in Example 1 10μL@Zn-MOF (2.0 mg) was dispersed in 5 mL of deionized water and sonicated for 20 min to obtain a homogeneous and stable suspension, which served as the sensing system. 500 μL of a common biological matrix solution with a concentration of 40 μM, including CaCl2, H2O2, NaCl, KCl, glucose, uric acid, glutathione (GSH), phenylalanine (Phe), and dopamine (DA), were added to this sensing system. After mixing thoroughly and allowing to stand for 5 min, the fluorescence spectra of each system were measured using a fluorescence spectrophotometer under the conditions of an excitation wavelength of 276 nm and a test voltage of 450 V.

[0070] Test results showed that the system with only dopamine added exhibited a ratiometric response with fluorescence enhancement at 325 nm and fluorescence quenching at 556 nm. The fluorescence spectra of the other biological matrix systems were not significantly different from the blank system (without any biological matrix added), proving that CQD... 10μL @Zn-MOF exhibits excellent specific recognition ability for dopamine.

[0071] Example 3: Detection of dopamine in real samples

[0072] Fresh poultry blood, healthy dog ​​urine, and volunteer tears were selected as real samples and pretreated according to the following method:

[0073] (1) Blood sample pretreatment: Take 5 mL of fresh poultry blood, centrifuge at 10000 rpm for 10 min, and collect the upper plasma layer; add 2 mL of acetonitrile and methanol (v:v = 1:3) mixed solvent to the plasma, vortex for 5 min, centrifuge at 10000 rpm for 10 min, collect the supernatant, and make up to 5 mL with PBS buffer solution at pH = 7.4 for later use.

[0074] (2) Urine sample pretreatment: Take 5 mL of fresh urine from healthy dogs, centrifuge at 10,000 rpm for 10 min, and collect the supernatant; filter the supernatant through a 0.22 μm ultrafiltration membrane to remove suspended particles, and make up to 5 mL with PBS buffer solution at pH = 7.4 for later use.

[0075] (3) Pretreatment of tear samples: 0.5 mL of volunteer tears were collected by capillary non-contact method, centrifuged at 10000 rpm for 5 min to remove impurities; 1 mL of acetonitrile and methanol (v:v = 1:3) mixed solvent was added to the centrifuged tears, vortexed for 5 min, centrifuged at 10000 rpm for 10 min, the supernatant was collected, and the volume was adjusted to 5 mL with PBS buffer solution at pH = 7.4 for later use.

[0076] Using the standard spiking method, 0 μM, 40 μM, 60 μM, and 80 μM dopamine standard solutions were added to the three pretreated real samples, respectively, with three replicates for each concentration. 2 mL of the treated spiked sample was then taken and 2.0 mg of CQD was added. 10μL @Zn-MOF, sonicated for 20 min, fluorescence spectrum measured at excitation wavelength 276 nm, fluorescence intensity ratio I calculated. 325 / I 556 The detection limit of dopamine was calculated based on the linear equation, and then the recovery rate and relative standard deviation (RSD) were calculated. The results are shown in the table below:

[0077] Table 1. Detection results of DA in real samples by CQD@Zn-MOF

Claims

1. A method for preparing a CQD@Zn-MOF ratiometric fluorescent sensor, characterized in that, The specific steps are as follows: Zinc chloride (ZnCl2), 4,4',4''-triaminotriphenyltricarboxylic acid (H3TATB), and 1,4-bis(imidazolyl)benzene (bimb) are dissolved in a mixed solvent of N,N-dimethylformamide (DMF) and water. Carbon quantum dot (CQD) solution is added, and the mixture is ultrasonically dispersed until the system is homogeneous. The mixture is then placed in an autoclave and kept at a constant temperature of 80-100 ℃ for 68-76 h. The temperature is then programmed to decrease to room temperature at a rate of 8-12 ℃ / h. After washing and drying, CQD@Zn-MOF composite material is obtained.

2. The production method according to claim 1, characterized by, The carbon quantum dot solution is prepared as follows: 1 mmol of urea and 3 mmol of ethylenediamine are dissolved in 10 mL of deionized water, sonicated for 4-6 min until completely dissolved, placed in a 25.0 mL Teflon-lined autoclave, and kept at a constant temperature of 150-170 ℃ for 5-7 h. After natural cooling to room temperature, a light yellow CQD solution is obtained.

3. The preparation method according to claim 1, characterized in that, The volume ratio of DMF to H2O in the mixed solvent is 2:3 to 3:2; the molar ratio of ZnCl2, H3TATB, and bimb is 1:2.5:2.5 to 4:1:

1.

4. The method of claim 1, wherein, The volume of the carbon quantum dot solution added is 2 μL to 20 μL, with the optimal addition being 10 μL.

5. The CQD@Zn-MOF composite material prepared by the method according to claim 1, used as a ratiometric fluorescence sensor in dopamine detection.

6. Use according to claim 5, characterized in that, With an excitation wavelength of 276 nm, the CQD@Zn-MOF exhibits a specific ratiometric fluorescence response only in the presence of dopamine: the fluorescence intensity is significantly enhanced at 325 nm and significantly quenched at 556 nm. It shows no obvious fluorescence response to common biological matrices such as CaCl2, H2O2, NaCl, KCl, glucose, uric acid, glutathione, and phenylalanine.

7. Use according to claim 5, characterized in that, When dopamine concentration is in the range of 37.7 ~ 87.7 μM, the fluorescence intensity ratio I 325 / I 556 It showed a good linear relationship with dopamine concentration, and the linear equation was y = -2.888 + 0.09072x (where x is the dopamine concentration in μM; y is the fluorescence intensity ratio I). 325 / I 556 ), correlation coefficient R 2 = 0.9919, Limit of Detection (LOD) = 9 nM, Limit of Quantification (LOQ) = 30 nM.

8. Use according to claim 5, characterized in that, The sensor exhibits stable sensing performance within a pH range of 2 to 10, and the fluorescence response signal reaches equilibrium within 40 seconds. Even after repeated washing, centrifugation, drying, and recycling, it still maintains good sensing performance.

9. Use according to claim 5, characterized in that, The sensor can be used for spiked recovery detection of dopamine in real biological samples such as blood, urine, and tears, with a spiked recovery rate of 98% to 102.1% and a relative standard deviation (RSD) of <1.82%.

10. Use according to claim 5, characterized in that, The sensor can be fabricated as a fluorescent test strip, which allows for the visual qualitative detection of dopamine by observing the change in fluorescence color (bright cyan fluorescence is only observed when dopamine is present) under a 365nm ultraviolet lamp.

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