Preparation method and application of fluorescence closing probe for detecting CTC

Abstract

The invention belongs to the technical field of fluorescence analysis, and discloses a preparation method and application of a fluorescence closing probe for detecting CTC. H2SDC, anhydrous AlCl3 and glacial acetic acid react at a certain temperature, and the fluorescence closed probe CYCU-3 is obtained through the steps of centrifugal separation, washing, drying and the like and is used for detecting the concentration of chlortetracycline (CTC). According to the present invention, 4, 4 '-stilbene dicarboxylic acid (H2SDC) is adopted as the raw material to synthesize the aluminum-based fluorescence closing probe, such that CTC detection is achieved, and the detection limit is extremely low (45.551 nM);

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence analysis, and more specifically to the preparation of an aluminum-based fluorescent probe material for detecting chlortetracycline hydrochloride. Background Art

[0002] CTC is a tetracycline antibiotic that was initially isolated from Streptomyces aureus in the soil matrix in the mid-1940s. This antibacterial agent is widely used in three fields: promoting livestock veterinary growth, aquaculture disease control, and human clinical treatment. The oral bioavailability of this compound is low, and most of it is excreted from the body through the kidneys and intestines without metabolism, forming a persistent environmental reservoir. Current wastewater management infrastructure shows serious deficiencies in its ability to degrade CTC. The gradual bioaccumulation of CTC residues in multiple environmental compartments poses a significant ecological hazard, especially chronic ecosystem disruption. CTC has significant trophic transfer potential in agricultural and animal products, facilitating its entry into the human biological system through dietary exposure vectors. Chronic low-dose bioaccumulation ultimately manifests as a risk of multi-system toxicity. Chronic exposure to CTC residues through dietary intake can induce multi-organ toxicity, manifested as liver damage, neurodevelopmental defects, and osteogenesis inhibition.

[0003] CTC is a broad-spectrum tetracycline antibiotic that is widely used in the livestock industry to inhibit bacterial proliferation. Long-term exposure to food contaminated with CTC may cause skin manifestations including erythema and rashes, while the pediatric population is at risk of enamel hypoplasia and osteogenesis inhibition.

[0004] Nowadays, fluorescent probes have become a transformative analytical tool, characterized by high detection efficiency, simple operation, and fast reaction speed. This versatile platform has demonstrated unparalleled versatility in multiple fields, including bioimaging mode, photocatalytic water splitting system, electrocatalytic carbon dioxide reduction architecture, next-generation battery system, and targeted drug delivery carrier - these capabilities have been achieved through the latest breakthroughs in Nature Nanotechnology and Advanced Functional Materials.

[0005] Therefore, developing reliable analytical methods to detect CTC in the real world is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a preparation method and application of a fluorescence-off probe for detecting CTC, specifically an aluminum-based fluorescent probe material for detecting chlortetracycline hydrochloride. The present invention synthesized an aluminum-based fluorescence-off probe using 4,4'-stilbenedicarboxylic acid (H2SDC) as a raw material, achieving CTC detection with an extremely low detection limit (45.551 nM).

[0007] One of the objectives of the present invention is to provide a method for preparing a fluorescence-off probe for detecting CTC, comprising the following steps:

[0008] S11. Dissolve H2SDC and anhydrous AlCl3 in DMF, and ultrasonically dissolve for 10 - 30 min to obtain a homogeneous solution;

[0009] S12. Transfer the homogeneous solution to the inner lining of a high-temperature reaction kettle, add glacial acetic acid CH3COOH, mix well, seal the reaction vessel, and react at a certain temperature;

[0010] S13. After the reaction is completed, cool to room temperature, centrifuge the obtained mixture, and separate the product to obtain a solid product;

[0011] S14. Wash the solid product with DMF 3 - 5 times, ultrasonicate, centrifuge, and dry to obtain the fluorescence-off probe CYCU-3.

[0012] Further, in step S11, the molar ratio of H2SDC to anhydrous AlCl3 is 0.5 - 1.5:0.5 - 1.5, and the concentration of H2SDC in DMF is 20 - 30 mM.

[0013] The beneficial effect of adopting the above dosage relationship is: to improve the coordination rate between the two materials and increase the fluorescence intensity value of the material.

[0014] Further, in step S12, the molar dosage relationship between CH3COOH and H2SDC is 5:1 - 2.

[0015] The beneficial effect of adopting the above dosage relationship is: to increase the pore structure of the material, which is beneficial for CYCU-3 to adsorb CTC.

[0016] Further, in step S12, the reaction temperature is maintained at 150 - 200 °C for 24 - 72 h.

[0017] The beneficial effect of adopting the above temperature conditions is: to accelerate the reaction rate of the material and is beneficial for improving the stability of the material.

[0018] Further, in step S14, the ultrasonication time is 10 - 20 min, the centrifugation is at 8000 - 10000 rpm for 5 - 20 min; the drying is vacuum drying at 50 - 100 °C for 2 - 24 h.

[0019] Another objective of the present invention is to provide an application of a fluorescence-off probe for detecting CTC, including:

[0020] S21. Disperse the fluorescence-off probe CYCU-3 in HEPES buffer solution (pH = 7.4) to obtain a CYCU-3 colloidal suspension;

[0021] S22. Adjust the pH value of the CYCU-3 colloidal suspension to neutral;

[0022] S23. Take the sample to be tested, add it to the CYCU-3 colloidal suspension, mix well, and detect the concentration of CTC at a fixed excitation wavelength.

[0023] Further, in step S21, the concentration of the CYCU-3 colloidal suspension is 0.1-1 mg / mL.

[0024] Further, in step S23, the volume ratio of the sample to be tested to the CYCU-3 solution is 1:99.

[0025] Further, in step S23, the fixed excitation wavelength is 254-365 nm.

[0026] Further, in step S23, the detection time is 2-10 min.

[0027] Further, during the detection process, the fluorescence intensity values at 400-500 nm in the emission spectrum are analyzed.

[0028] The technical concept of the present invention is that the fluorescent material based on MILs has simple raw material acquisition, convenient preparation, and is environmentally friendly. Compared with the traditional method for detecting CTC, the defective structure of CYCU-3 increases the adsorption amount of the material and the analyte. Based on the detection mechanism of coordination and inner filter effect, the material has high sensitivity, good selectivity, and strong anti-interference ability, and is suitable for general water environments.

[0029] Through the above technical solutions, compared with the prior art, the beneficial effects obtained by the present invention are as follows: The present invention is a fluorescence turn-off probe based on the coordination and inner filter effect of CYCU-3 and CTC. During the detection process, after adding CTC, the fluorescence intensity values at 400-500 nm decrease. The probe can detect CTC through the fluorescence mode, which is more convenient in practical applications, and the results of the detection mode can be verified, with higher accuracy. Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0031] Figure 1 It is the synthesis process and detection olive map of CYCU-3;

[0032] Figure 2 It is the selective fluorescence spectrum diagram of CYCU-3;

[0033] Figure 3 is the bar chart of the selectivity and anti-interference of CYCU-3 at the fluorescence ratio F0 429 / F 429 Selectivity and anti-interference bar chart;

[0034] Figure 4 is the bar chart of the selectivity and anti-interference of CYCU-3 at the fluorescence ratio F0 456 / F 456 Selectivity and anti-interference bar chart;

[0035] Figure 5 is the fluorescence waterfall chart of different concentrations (0 - 20 μM) of CTC added to the optimized CYCU-3 solution under 365 nm excitation light;

[0036] Figure 6 are the daylight photos of different concentrations (0 - 20 μM) of CTC added to the optimized CYCU-3 solution and the fluorescence photos irradiated with 365 nm ultraviolet light;

[0037] Figure 7 is the relationship diagram of F 429 、F 456 、F0 429 / F 429 and F0 456 / F 456 when the probe is irradiated with 365 nm excitation light and different concentrations (0 - 20 μM) of CTC are added to the optimized CYCU-3 solution. Detailed implementation manners

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] Example 1

[0040] Preparation of fluorescence-off probe:

[0041] Based on the traditional preparation method of metal-organic frameworks (MOFs), the CYCU-3 framework was prepared by the solvothermal method. 0.1073 g of H2SDC and 0.0533 g of anhydrous AlCl3 were dissolved in 20 mL of DMF and sonicated for 10 minutes. Subsequently, the homogeneous solution was transferred to a 25 mL inner liner of a high-temperature autoclave, and 0.114 mL of glacial acetic acid was added and mixed thoroughly. The reaction vessel was sealed, and the oven temperature was controlled to be maintained at 180 °C for 72 hours. After the reaction was completed and subsequently cooled to room temperature, the resulting mixture was centrifuged for product separation. The obtained solid was washed three times with DMF, sonicated for 10 minutes, centrifuged at 10000 rpm for 5 minutes. Finally, the purified product was vacuum dried at 90 °C for 12 hours to obtain the desired CYCU-3 solid.

[0042] To study the fluorescence response of CYCU-3 to CTC detection, 8 mg of CYCU-3 was dispersed in 8 mL of HEPES buffer and sonicated for 10 minutes to prepare a stable CYCU-3 colloidal suspension (1 mg / mL). When measuring fluorescence, 200 μL of the stock suspension was taken and diluted to a final volume of 2 mL with buffer to obtain a CYCU-3 concentration of 0.1 mg / mL. Different concentrations of CTC standard solutions were successively added to the diluted CYCU-3 suspension for CTC detection. After each addition, the mixture was mixed well and equilibrated for 2 minutes at ambient temperature (25 ± 1 °C). To ensure reproducibility, all measurements were performed three times under the same experimental conditions, and the average value of the fluorescence intensity ratio (F0 / F) was quantitatively analyzed with the CTC concentration.

[0043] Example 2

[0044] 0.0536 g of H2SDC and 0.0799 of anhydrous AlCl3 were dissolved in 10 mL of DMF and sonicated for 30 minutes. Subsequently, the homogeneous solution was transferred to a 25 mL inner liner of a high-temperature autoclave without adding glacial acetic acid. The reaction vessel was sealed, and the oven temperature was controlled to be maintained at 150 °C for 24 hours. After the reaction was completed and subsequently cooled to room temperature, the resulting mixture was centrifuged for product separation. The obtained solid was washed three times with DMF, sonicated for 20 minutes, centrifuged at 8000 rpm for 10 minutes. Finally, the purified product was vacuum dried at 100 °C for 2 hours to obtain the desired CYCU-3 solid.

[0045] To study the fluorescence response of CYCU-3 to CTC detection, 8 mg of CYCU-3 was dispersed in 8 mL of HEPES buffer. After sonication for 30 minutes, a stable CYCU-3 colloidal suspension (1 mg / mL) was prepared. For fluorescence measurement, 200 μL of the stock suspension was taken and diluted to a final volume of 2 mL with the buffer to obtain a CYCU-3 concentration of 0.1 mg / mL. Different concentrations of CTC standard solutions were successively added to the diluted CYCU-3 suspension for CTC detection. After each addition, the mixture was mixed well and equilibrated for 2 minutes at ambient temperature (25 ± 1 °C). To ensure reproducibility, all measurements were carried out three times under the same experimental conditions, and the average value of the fluorescence intensity ratio (F0 / F) was quantitatively analyzed with the CTC concentration.

[0046] Example 3

[0047] 0.1610 g of H2SDC and 0.0266 g of anhydrous AlCl3 were dissolved in 20 mL of DMF and sonicated for 10 minutes. Subsequently, the homogeneous solution was transferred to a 25 mL inner liner of a high-temperature reaction kettle, and 0.114 mL of glacial acetic acid was added and mixed well. The reaction vessel was sealed, and the oven temperature was controlled to be maintained at 200 °C for 72 hours. After the reaction was completed and subsequently cooled to room temperature, the resulting mixture was centrifuged for product separation. The obtained solid was washed three times with DMF, sonicated for 20 minutes, and centrifuged at 90000 rpm for 20 minutes. Finally, the purified product was dried in vacuo at 50 °C for 12 hours to obtain the desired CYCU-3 solid.

[0048] To study the fluorescence response of CYCU-3 to CTC detection, 8 mg of CYCU-3 was dispersed in 8 mL of HEPES buffer. After sonication for 20 minutes, a stable CYCU-3 colloidal suspension (1 mg / mL) was prepared. For fluorescence measurement, 200 μL of the stock suspension was taken and diluted to a final volume of 2 mL with the buffer to obtain a CYCU-3 concentration of 0.1 mg / mL. Different concentrations of CTC standard solutions were successively added to the diluted CYCU-3 suspension for CTC detection. After each addition, the mixture was mixed well and equilibrated for 2 minutes at ambient temperature (25 ± 1 °C). To ensure reproducibility, all measurements were carried out three times under the same experimental conditions, and the average value of the fluorescence intensity ratio (F0 / F) was quantitatively analyzed with the CTC concentration.

[0049] Example 4

[0050] Dissolve 0.1073 g of H2SDC and 0.0533 g of anhydrous AlCl3 in 20 mL of DMF and sonicate for 10 minutes. Subsequently, transfer the homogeneous solution to a 25 mL high-temperature reactor liner, add 0.057 mL of glacial acetic acid and mix well. Seal the reaction vessel and maintain the oven temperature at 160 °C for 48 hours. After the reaction is completed and subsequently cooled to room temperature, the resulting mixture is centrifuged for product separation. The obtained solid is washed three times with DMF, sonicated for 10 minutes, centrifuged at 10000 rpm for 5 minutes. Finally, the purified product is dried in vacuo at 80 °C for 18 hours to obtain the desired CYCU-3 solid.

[0051] To study the fluorescence response of CYCU-3 to CTC detection, disperse 8 mg of CYCU-3 in 8 mL of HEPES buffer and sonicate for 15 minutes to prepare a stable CYCU-3 colloidal suspension (1 mg / mL). For fluorescence measurement, take 200 μL of the stock suspension and dilute it to a final volume of 2 mL with buffer to obtain a CYCU-3 concentration of 0.1 mg / mL. Add different concentrations of CTC standard solutions to the diluted CYCU-3 suspension in sequence for CTC detection. After each addition, mix the mixture well and equilibrate for 2 minutes at ambient temperature (25 ± 1 °C). To ensure reproducibility, all measurements were performed three times under the same experimental conditions, and the average value of the fluorescence intensity ratio (F0 / F) was quantitatively analyzed with the CTC concentration.

[0052] Experiment 1 Selectivity and anti-interference detection:

[0053] Selectivity and anti-interference are important indicators of probe performance. Prepare stock solutions of antibiotics, cationic chlorides, and anionic sodium salts at a concentration of 1 mM in HEPES buffer (pH = 7.4). To evaluate selectivity, mix 200 μL of CYCU-3 stock solution (1 mg / mL) with 20 μL of interfering ions (1 mM) and 1780 μL of HEPES buffer to a final volume of 2 mL. To evaluate the interference effect, prepare a mixture containing 200 μL of CYCU-3 stock solution (1 mg / mL), 20 μL of interfering substances (1 mM), and 20 μL of CTC stock solution (1 mM) in 1760 μL of HEPES buffer. All prepared samples are gently stirred at room temperature (25 ± 1 °C) for 2 minutes. After thorough mixing and equilibration, obtain the fluorescence emission spectrum in the range of 400 - 550 nm using the following instrument parameters: excitation wavelength = 365 nm, excitation slit width = 5 nm, emission slit width = 5 nm, scan speed = 1200 nm / min.

[0054] Utilize the cationic chlorides (K + 、Na + 、Al3+ 、Ba 2+ 、Mg 2+ 、Mn 2+ 、Zn 2+ 、Ca 2+ 、Ni 2 + 、Cr 3+ 、Fe 3+ )、 anionic sodium salts (SO4 2- 、HSO3 - 、HPO4 2- 、H2PO4 - 、CO3 2- 、HCO3 - 、S2 - 、Br - 、HS - 、CH3COO−, NO2 - ) and antibiotics (CTC, CLA, SM, AMP, SDZ, KAN, NFX, CL), and evaluate the recognition ability of CYCU-3 for CTC. As Figure 2 shown, the fluorescence intensity of CYCU-3 showed significant stability after introducing various selective substances at the same concentration. In sharp contrast, the addition of CTC induced obvious fluorescence quenching of CYCU-3, and the quenching efficiency was significantly higher than that of other analytes. Figure 3 and Figure 4 shown fluorescence intensity ratios (F0 429 / F 429 and F0 456 / F 456 ) quantitative analysis showed that the fluorescence ratio changes of non-detected substances were very small, while the quenching ratio induced by CTC was approximately three times that of other compounds, confirming the excellent selectivity of CYCU-3 for CTC detection. In addition, to evaluate the anti-interference ability of CYCU-3, the same concentration of potential interfering ions was introduced into the CYCU-3-CTC complex solution. It is not difficult to find that when CTC and interfering substances coexist, CYCU-3 does not significantly interfere with the detection of CTC.

[0055] Experiment 2 Sensitivity test:

[0056] As Figure 5 shown, after adding different concentrations of CTC (0 - 20 μM) to the CYCU-3 solution, the fluorescence intensity gradually decreased and the fluorescence curve tended to be flat. When the CTC concentration reached 20 μM, the fluorescence intensity dropped below 100. Under natural light, Figure 6 showed that the CYCU-3 solutions with different CTC concentrations were clear and transparent. Under 365 nm ultraviolet lamp irradiation, as the CTC concentration increased, the fluorescence of these solutions gradually darkened.

[0057] In view of the significant luminescence response of CYCU-3 to CTC, its potential as a ultrasensitive fluorescence-off probe for CTC detection was investigated. The fluorescence of the probe solution decreased with the increase of CTC concentration, and a strong linear relationship was shown in the fluorescence intensities at 429 nm and 456 nm. The results are as Figure 7 shown as follows: F 429 = -35.8048X + 814.4242 and F 456 = -40.9416X + 925.4918. CYCU-3 can detect CTC as low as 45.551 nM (Em = 429 nm) and 54.834 nM (Em = 456 nm). Meanwhile, F0 429 / F 429 and F0 456 / F 456 showed good exponential relationships, with R 2 values of 0.9991 and 0.9967 respectively. The fitting equations are Y = 0.2880×EXP(0.1422X) + 0.6937 and

[0058] Y = 0.3009×EXP(0.1410X) + 0.7101.

[0059] Experiment 3 Detection of actual samples:

[0060] To evaluate the practical application potential of CYCU-3 in CTC detection, tap water and commercially available low-fat calcium milk were used for analysis and detection. The low-fat calcium milk was purchased from a local supermarket, and 10 mL aliquots were used for experimental analysis. A series of pretreatment steps were carried out on the milk samples: when preparing the samples, the milk was diluted with 20 μL of ultrapure water and vortexed evenly. Then, it was sonicated for 15 minutes to ensure complete dispersion, centrifuged at 10000 rpm for 10 minutes at 4 °C to separate the supernatant, and filtered through a 0.22 μm microporous filter membrane to remove particulate matter. Similarly, the tap water sample was filtered through a 0.22 μm microporous filter membrane to eliminate insoluble impurities. After these pretreatment procedures, fluorescence measurements were performed using the same instrument parameters as described above to evaluate the CTC detection performance in these complex matrices.

[0061] The spiking recoveries and relative deviations were obtained at 429 nm and 456 nm respectively.

[0062] Table 1 Content, recovery rate (n = 3) and standard deviation of CTC in actual samples:

[0063]

[0064] As shown in Table 1, the recovery rate of CTC in tap water was 97.55% - 98.79%, and the relative standard deviation was 1.33% - 4.92%. The recovery rate of CTC spiked samples was 95.03% - 97.56%, and the relative standard deviation was 3.25% - 4.31%. The sensitivity and reliability of the fluorescence sensor for detecting CTC in actual samples were verified.

[0065] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a fluorescence-off probe for detecting CTCs, characterized in that, It includes the following steps: S11. Dissolve H2SDC and anhydrous AlCl3 in DMF and ultrasonically dissolve to obtain a homogeneous solution; S12. Transfer the homogeneous solution to the inner lining of a high-temperature reaction kettle, add CH3COOH, mix well, seal the reaction vessel, and react at a certain temperature; S13. After the reaction is completed, cool to room temperature, centrifuge to obtain a solid product; S14. Wash the solid product with DMF multiple times, ultrasonicate, centrifuge, and dry to obtain the fluorescence-off probe CYCU-3.

2. The preparation method of a fluorescence-off probe for detecting CTC according to claim 1, characterized in that, In step S11, the molar ratio of H2SDC to anhydrous AlCl3 is 0.5 - 1.5:0.5 - 1.5, and the concentration of H2SDC in DMF is 20 - 30 mM.

3. The preparation method of a fluorescence-off probe for detecting CTC according to claim 1, characterized in that, In step S12, the molar dosage relationship between CH3COOH and H2SDC is 5:1 - 2.

4. The preparation method of a fluorescence-off probe for detecting CTC according to claim 1, characterized in that, In step S12, the reaction is carried out at 150 - 200 °C for 24 - 72 h.

5. The preparation method of a fluorescence-off probe for detecting CTC according to claim 1, characterized in that, In step S14, the ultrasonication time is 10 - 20 min, the centrifugation is at 8000 - 10000 rpm for 5 - 20 min; the drying is vacuum drying at 50 - 100 °C for 2 - 24 h.

6. Use of a fluorescence-off probe for detecting CTCs, characterized in that, It includes: S21. Disperse the fluorescence-off probe CYCU-3 according to any one of claims 1 - 5 in HEPES buffer to obtain a CYCU-3 colloidal suspension; S22. Adjust the pH value of the CYCU-3 colloidal suspension to neutral; S23. Take the sample to be tested, add it to the CYCU-3 colloidal suspension, mix well, and detect CTC at a fixed excitation wavelength.

7. Use of a fluorescence-off probe for detecting CTC according to claim 6, characterized in that, In step S21, the concentration of the CYCU-3 colloidal suspension is 0.1 - 1 mg / mL.

8. Use of a fluorescence-off probe for detecting CTC according to claim 6, characterized in that, In step S23, the volume ratio of the sample to be tested to the CYCU-3 solution is 1:

99.

9. Use of a fluorescence-off probe for detecting CTC according to claim 6, characterized in that, In step S23, the fixed excitation wavelength is 254 - 365 nm.

10. Use of a fluorescence-off probe for detecting CTC according to claim 6, characterized in that, In step S23, the detection time is 2 - 10 min.

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