Application of cyclotriphosphazene-terpyridyl fluorescent probe to detection of flavonoid substances

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to application of a cyclotriphosphazene-terpyridyl fluorescent probe to detection of flavonoid substances. The invention provides a fluorescent probe for detecting flavonoid substances. The fluorescent probe belongs to cyclotriphosphazene-terpyridyl compounds. The fluorescent probe has a fluorescence red shift phenomenon after being combined with a flavonoid substance, realizes ratio type fluorescence detection, has the advantages of quick response, high selectivity, interference resistance and low cost, provides a new direction for detection of the flavonoid substance, and has important application value in the fields of environmental science, biological science and the like.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the application of cyclotriphosphazene-terpyridine fluorescent probes in the detection of flavonoids. Background Technology

[0002] Quercetin (3,3',4',5,7-pentahydroxyflavone, Qr) is one of the most studied flavonols in nature, widely found in various vegetables, fruits, and traditional Chinese medicines, and has received considerable attention in the biomedical field. For example, quercetin has been found to effectively treat various diseases and disorders, including chronic bronchitis, hypertension, diabetes, and eye diseases caused by cataracts. Due to the pharmacological and biological activities of Qr, it is necessary to establish a simple and sensitive detection method. Currently, various analytical methods, such as capillary electrophoresis, ultraviolet light, and HPLC, combined with various sensing systems, are used to detect and track Qr.

[0003] Fluorescent probes, possessing the desired properties of high sensitivity and real-time imaging, have been reported for the detection of Or, including small molecule fluorescent probes, metal nanomaterials, and carbon quantum dot-based micro / nano probes. However, most fluorescence detection methods are based on the fluorescence quenching detection of probes by Qr, which may affect the accuracy of the determination. Since the content of Qr in many plants is very low, typically at the micromolar level, it is of great significance to design fluorescence-enhancing probes for the concentration detection of Qr.

[0004] Therefore, developing rapid, sensitive, and specific methods for detecting Qr is of great research significance in the field of bioscience. Summary of the Invention

[0005] This invention provides a cyclotriphosphazene-terpyridine fluorescent probe, which enables the qualitative or quantitative detection of flavonoids in a simple and efficient manner. It exhibits excellent specificity for quercetin, and can be used to detect Qr in fruits (apples), foods (honey), and tobacco (cigarettes). It has important application value in environmental science and biological science.

[0006] The technical solution of the present invention is a cyclotriphosphazene-terpyridine fluorescent probe, which is any one or any combination of three ligands: Cpz-2TPY, Cpz-4TPY and Cpz-6TPY.

[0007] This invention also provides a method for preparing the above-mentioned cyclotriphosphazene-terpyridine fluorescent probe, the steps of which include:

[0008] a. Under the catalysis of anhydrous cesium carbonate, hexachlorocyclotriphosphazene (Cpz) and biphenyl hydroquinone were subjected to a heating and reflux reaction in anhydrous tetrahydrofuran solution to obtain cyclotriphosphazene ligands (Cpz-2Cl and / or Cpz-4Cl);

[0009] b. Under anhydrous cesium carbonate catalysis, terpyridine (TPY) ligands and cyclotriphosphazene ligands (Cpz-2Cl and / or Cpz-4Cl) are reacted under reflux in anhydrous tetrahydrofuran solution to yield cyclotriphosphazene-terpyridine (Cpz-2TPY and / or Cpz-4TPY) compounds; or,

[0010] c. Under the catalysis of anhydrous cesium carbonate, terpyridine (TPY) ligand and hexachlorocyclotriphosphazene (Cpz) are reacted under reflux in anhydrous tetrahydrofuran solution to obtain the cyclotriphosphazene-terpyridine (Cpz-6TPY) compound.

[0011] Step a: After adding anhydrous cesium carbonate, the system is evacuated and protected with nitrogen. Under low temperature conditions, hexachlorocyclotriphosphazene (Cpz) is added, followed by dehydrogenation and the addition of biphenyl hydroquinone.

[0012] In step a, the molar ratio of hexachlorocyclotriphosphazene (Cpz) to biphenyl is 1:1-5, preferably 1:1-3; the concentration of biphenyl is 0.01-1.5 mmol / mL, preferably 0.03-0.07 mmol / mL. Further, in the Cpz-2Cl ligand formation system, the molar ratio of hexachlorocyclotriphosphazene (Cpz) to biphenyl is 1:1.5-3, preferably 1:2-2.5; the concentration of biphenyl is 0.01-1.5 mmol / mL, preferably 0.06-0.07 mmol / mL; in the Cpz-4Cl ligand formation system, the molar ratio of hexachlorocyclotriphosphazene (Cpz) to biphenyl is 1:1-2, preferably 1:1-1.5; the concentration of biphenyl is 0.01-1 mmol / mL, preferably 0.03-0.04 mmol / mL.

[0013] In step a, the molar ratio of anhydrous cesium carbonate to hexachlorocyclotriphosphazene (Cpz) is 1-10:1, preferably 2-8:1. Further, in the Cpz-2Cl cyclotriphosphazene ligand formation system, the molar ratio of anhydrous cesium carbonate to hexachlorocyclotriphosphazene (Cpz) is 4-8:1, preferably 5:1; in the Cpz-4Cl cyclotriphosphazene ligand formation system, the molar ratio of anhydrous cesium carbonate to hexachlorocyclotriphosphazene (Cpz) is 1-4:1, preferably 2-2.5:1.

[0014] Step a: Reflux the mixture for 12-14 hours, preferably reflux at 50°C for 12 hours. After the reflux reaction is complete, centrifuge and collect the supernatant, concentrate to obtain a white solid. Purify the white solid by column chromatography, using a 2:1 (v / v) mixture of PE (petroleum ether):EA (ethyl acetate) as the eluent.

[0015] Step b: After adding anhydrous cesium carbonate, the system is evacuated and protected with nitrogen. Then, terpyridine (TPY) ligand is added. After dehydrogenation at low temperature, cyclotriphosphazene ligands (Cpz-2Cl and / or Cpz-4Cl) are added.

[0016] In step b, the molar ratio of cyclotriphosphazene ligand (Cpz-2Cl and / or Cpz-4Cl) to terpyridine (TPY) ligand is 1:1.5-10, preferably 1:2-8; the concentration of cyclotriphosphazene ligand (Cpz-2Cl and / or Cpz-4Cl) is 0.01-0.5 mmol / mL. Furthermore, in the formation system of cyclotriphosphazene-terpyridine Cpz-2TPY, the molar ratio of cyclotriphosphazene ligand (Cpz-2Cl) to terpyridine (TPY) ligand is 1:1.5-3, preferably 1:2-2.5, and the concentration of cyclotriphosphazene ligand (Cpz-2Cl) is 0.01-1 mmol / mL, preferably 0.03-0.04 mmol / mL; in the formation system of cyclotriphosphazene-terpyridine Cpz-4TPY, the molar ratio of cyclotriphosphazene ligand (Cpz-4Cl) to terpyridine (TPY) ligand is 1:3.5-6, preferably 1:4.3-5, and the concentration of cyclotriphosphazene ligand (Cpz-2Cl) is 0.08-0.5 mmol / mL, preferably 0.09-0.2 mmol / mL.

[0017] In step b, the molar ratio of anhydrous cesium carbonate to terpyridine (TPY) is 1-6:1, preferably 1-4:1. Further, in the cyclotriphosphazene-terpyridine Cpz-2TPY formation system, the molar ratio of anhydrous cesium carbonate to terpyridine (TPY) is 1-4:1, preferably 2-2.5:1; in the cyclotriphosphazene-terpyridine Cpz-4TPY formation system, the molar ratio of anhydrous cesium carbonate to terpyridine (TPY) is 1-3:1, preferably 1-1.5:1.

[0018] Step b: reflux the mixture for 20-26 hours, preferably reflux at 50°C for 24 hours. After the reflux reaction is complete, centrifuge and collect the supernatant. Add 1M sodium hydroxide solution and stir to precipitate a brown solid.

[0019] Step c: After adding anhydrous cesium carbonate, the system is evacuated and protected with nitrogen. Then, terpyridine (TPY) ligand is added, followed by dehydrogenation at low temperature, and then cyclotriphosphazene ligand (Cpz) is added.

[0020] In step c, the molar ratio of cyclotriphosphazene ligand (Cpz) to terpyridine (TPY) ligand is 1:5.5-9, preferably 1:6.5-7; the concentration of cyclotriphosphazene ligand (Cpz) is 0.01-1 mmol / mL, preferably 0.01-0.02 mmol / mL.

[0021] In step c, the molar ratio of anhydrous cesium carbonate to terpyridine (TPY) ligand is 1:1-2, preferably 1:1.4-1.5.

[0022] Step c: reflux reaction for 33-38 hours, preferably reflux reaction at 50°C for 36 hours.

[0023] Step c: After the reflux reaction is complete, centrifuge to collect the supernatant, add 1M sodium hydroxide solution, and stir to precipitate a brown solid.

[0024] The preparation method of the terpyridine (TPY) includes: reacting 2-acetylpyridine with 4-hydroxybenzaldehyde in an aqueous ethanol solution containing an alkaline substance. After the reaction is complete, acetic acid is added to adjust the pH to about 4, and a yellow-green flocculent solid precipitates out.

[0025] The molar ratio of 2-acetylpyridine to 4-hydroxybenzaldehyde is 2-4:1, preferably 2:1; the concentration of 2-acetylpyridine is 0.1-1 mmol / mL, 0.4-0.5 mmol / mL.

[0026] The ethanol-water solution containing alkaline substances is composed of sodium hydroxide, ethanol, water and ammonia in a ratio of 5-5.5 mmol:2 mL:1 mL:6 mL.

[0027] The cyclotriphosphazene-terpyridine fluorescent probe (Cpz-2TPY, and / or Cpz-4TPY, and / or Cpz-6TPY) provided by the present invention, through its interaction with Al 3+ The metal complex probes (Cpz-2TPY-Al, and / or Cpz-4TPY-Al, and / or Cpz-6TPY-Al) formed by complexation enable ratiometric detection of quercetin (Qr), exhibiting rapid response, good photostability, high selectivity, and strong anti-interference performance. The metal complex probes (Cpz-2TPY-Al, and / or Cpz-4TPY-Al, and / or Cpz-6TPY-Al) generate excellent fluorescence properties after binding with Qr, making the cyclotriphosphazene-terpyridine fluorescent probe and its metal complex (especially its Al) provided in this invention... 3+ The complex can be used for the detection of flavonoids, especially Qr. This detection method is non-invasive, simple to operate, low in cost, fast and efficient, providing a new detection probe and detection method for the detection of flavonoids, especially Qr.

[0028] The cyclotriphosphazene-terpyridine fluorescent probe and / or its metal complex provided by the present invention can be used for the detection of flavonoids, and to prepare products for the detection of flavonoids. These products include, but are not limited to, detection reagents, kits, components, devices, or equipment. Reagents include, but are not limited to, solutions containing the cyclotriphosphazene-terpyridine fluorescent probe and / or its metal complex described above. Components include, but are not limited to, magnetic beads, panels, chips, channels, and pathways loaded with the cyclotriphosphazene-terpyridine fluorescent probe and / or its metal complex described above. Devices or equipment are equipped with the aforementioned components. Flavonoids refer to a general term for a series of compounds consisting of two benzene rings connected by three carbon atoms, i.e., a class of compounds with a C6-C3-C6 structure, including but not limited to flavones, flavonols, flavanones, flavanone alcohols, isoflavones, isoflavonones, chalcones, dihydrochalcones, orange ketones, flavans, flavanols, etc., such as quercetin. The metal ions used for the metal complex include, but are not limited to, Al. 3+ Na + K + Mg 2+ Ca 2+ Cu 2+ Zn 2+ Fe 2+ Fe 3+ Co 2+ Cd 2+ Ni 2 + Yb 3+ Eu 3+ and Tb 3+ Any one or any combination thereof, preferably Al 3+ K + Zn 2+ Fe 3+ Yb 3+ Eu 3+ and Tb 3+ Any one or any combination thereof, as an implementation method, is A1 3+ .

[0029] This invention provides a product for detecting flavonoids, containing a cyclotriphosphazene-terpyridine fluorescent probe and / or its metal complex provided by the present invention. The product includes, but is not limited to, detection reagents, kits, components, devices, or equipment. Reagents include, but are not limited to, solutions containing the cyclotriphosphazene-terpyridine fluorescent probe and / or its metal complex provided by the present invention. Components include, but are not limited to, magnetic beads, panels, chips, channels, and pathways loaded with the cyclotriphosphazene-terpyridine fluorescent probe and / or its metal complex provided by the present invention. Devices or equipment are equipped with the aforementioned components. The flavonoids refer to a general term for a series of compounds consisting of two benzene rings linked by three carbon atoms, i.e., a class of compounds with a C6-C3-C6 structure, including, but not limited to, flavones, flavonols, flavanones, flavanone alcohols, isoflavones, isoflavonones, chalcones, dihydrochalcones, orange ketones, flavans, flavanols, etc. For example, the metal ion used in the metal complex of quercetin, as one embodiment, includes, but is not limited to, Al. 3+ Na + K + Mg 2+ Ca 2+ Cu 2+ Zn 2+ Fe 2+ Fe 3 + Co 2+ Cd 2+ Ni 2+ Yb 3+ Eu 3+ and Tb 3+ Any one or any combination thereof, preferably Al 3+ K + Zn 2+ Fe 3+ Yb 3+ Eu 3 + and Tb 3+ Any one or any combination thereof, as an implementation method, is A1 3+ .

[0030] This invention provides a method for detecting flavonoids, comprising the steps of: using a cyclotriphosphazene-terpyridine fluorescent probe Cpz-2TPY and / or its metal complex (especially Al) provided above. 3+ (complexes), Cpz-4TPY and / or its metal complexes (especially Al) 3+ (complexes), Cpz-6TPY and / or its metal complexes (especially Al) 3+At least one of the complexes is used as a probe, which is mixed with the sample to form a detection system. Under 280nm excitation light, fluorescence in the range of 300nm to 650nm is collected to perform qualitative and / or quantitative detection of flavonoids in the sample.

[0031] Furthermore, when using at least one of the cyclotriphosphazene-terpyridine fluorescent probes Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY provided by the present invention as a probe, metal ions need to be added to the detection system to form a metal complex.

[0032] Metal complexes use metal ions including but not limited to Al 3+ Na + K + Mg 2+ Ca 2+ Cu 2+ Zn 2+ Fe 2+ Fe 3+ Co 2+ Cd 2+ Ni 2+ Yb 3+ Eu 3+ and Tb 3+ Any one or any combination thereof, preferably Al 3+ K + Zn 2+ Fe 3+ Yb 3+ Eu 3+ and Tb 3+ Any one or any combination thereof, as an implementation method, is A1 3+ .

[0033] Furthermore, using the fluorescence intensity ratio of the metal complex of the cyclotriphosphazene-terpyridine fluorescent probe provided by the present invention as the ordinate and the concentration of flavonoids (especially quercetin) as the x and y axes, the qualitative and / or quantitative determination of boron-containing compounds in the sample is performed using the external standard method, internal standard method, or standard addition method. Specifically, Cpz-2TPY and / or its metal complex (especially Al) is used. 3+ When the complex is used as a probe, take I 489nm / I 351nm The fluorescence intensity ratio, in terms of Cpz-4TPY and / or its metal complexes (especially Al) 3+ When the complex is used as a probe, take I 487nm / I 358nm The fluorescence intensity ratio, in terms of Cpz-6TPY and / or its metal complexes (especially Al) 3+ When the complex is used as a probe, take I 487nm / I 375nm The ratio of fluorescence intensity.

[0034] Furthermore, the solvent used in the detection system can be any one or any combination of toluene, dichloromethane, tetrahydrofuran, ethyl acetate, ethanol, N,N-dimethylformamide, acetonitrile, dimethyl sulfoxide, water, HEPES buffer solution, and PBS buffer solution, preferably any one or any combination of ethanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, water, HEPES buffer solution, and PBS buffer solution, preferably ethanol.

[0035] Furthermore, the flavonoids include, but are not limited to, flavones, flavonols, flavanones, flavanone alcohols, isoflavones, isoflavonones, chalcones, dihydrochalcones, orangeones, flavans, flavanols, etc., such as quercetin as one embodiment.

[0036] Furthermore, the sources of the samples to be tested include, but are not limited to, food, plants, plant organs, plant tissues, plant cells, health products, pharmaceuticals, animal (human) tissues, animal (human) organs, animal (human) body fluids, animal (human) metabolites, environmental water resources, environmental soil resources, tobacco and its products, daily chemical products, etc., such as honey, red wine and other foods, cigarettes and other tobacco. Any sample containing flavonoids (especially quercetin) regardless of its form can be detected by the cyclotriphosphazene-terpyridine fluorescent probe provided above in this invention.

[0037] Verification through examples shows that the three metal complex probes (Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al) provided by this invention achieve selective detection of the flavonoid compound Qr in ethanol (EtOH) solution, with detection limits of 0.43 μM, 0.20 μM, and 61.76 nM, respectively. After binding to Qr, the three metal complex probes exhibit a red shift in fluorescence, achieving ratiometric detection of Qr in food (honey, red wine) and tobacco (cigarettes). The fluorescent probes of this invention possess advantages such as rapid response, high selectivity, anti-interference, and low cost, providing a new approach and method for the detection of flavonoids, and have significant application value in environmental science and biological sciences. Attached Figure Description

[0038] Figure 1 The UV absorption spectra of fluorescent ligands Cpz-2TPY(A), Cpz-4TPY(C), and Cpz-6TPY(E) in different solvents; the fluorescence emission spectra of fluorescent ligands Cpz-2TPY(B), Cpz-4TPY(D), and Cpz-6TPY(F) in different solvents.

[0039] Figure 2The bar chart shows the ratio of the highest fluorescence point of Cpz-2TPY(A), Cpz-4TPY(B), and Cpz-6TPY(C) in ethanol solution to different metal ions and after the addition of Qr. Figure 2 In (A) / (B) / (C), the horizontal coordinates from bottom to top (or from left to right) are Na. + Mg 2+ Ca 2+ Al 3+ Ni 2+ Co 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Fe 3+ Ru 3+ Eu 3+ 、Tb 3+ Yb 3+ .

[0040] Figure 3 The bar chart shows the ratio of the highest fluorescence points for Qr detection for Cpz-2TPY-Al(A), Cpz-4TPY-Al(B), and Cpz-6TPY-Al(C) in different solution systems. Figure 3 In (A) / (B) / (C), the horizontal axis from bottom to top (or from left to right) consists of toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EA), ethanol (EtOH), acetonitrile (ACN), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water (H2O), HEPES, and PBS.

[0041] Figure 4 For Cpz-2TPY(A), Cpz-4TPY(B), Cpz-6TPY(C) and Al 3+ The Job's plot.

[0042] Figure 5 The kinetics of Cpz-2TPY-Al(A), Cpz-4TPY-Al(B), Cpz-6TPY-Al(C) and Qr were studied.

[0043] Figure 6The fluorescence spectra of Cpz-2TPY-Al(A), Cpz-4TPY-Al(C), and Cpz-6TPY-Al(E) with different organic compounds (50 μM), cations (50 μM), anions (50 μM), and Qr (10 μM) are shown. The fluorescence intensity of Cpz-2TPY-Al(B), Cpz-4TPY-Al(D), and Cpz-6TPY-Al(F) with various organic compounds (50 μM), cations (50 μM), and anions (50 μM), as well as the ratio of the highest fluorescence emission intensity after the addition of Qr (10 μM), are shown in the fluorescence bar chart.

[0044] Figure 7 Fluorescence spectra of Cpz-2TPY-Al (A), Cpz-4TPY-Al (C), and Cpz-6TPY-Al (E) with Qr concentrations in their respective working ranges; linear relationship between fluorescence intensity and Qr concentration for Cpz-2TPY-Al (B), Cpz-4TPY-Al (D), and Cpz-6TPY-Al (F).

[0045] Figure 8 The fluorescent probe Cpz-6TPY-Al, a metal complex, was used in red wine. Figure 8 A) Honey Figure 8 B) and tobacco ( Figure 8 C) I in the three samples 487nm / I 375nm Working curves showing the relationship between intensity and different Qr concentrations (0-10 μM).

[0046] Figure 9 For Cpz-2TPY 1 1H NMR spectrum (solvent: CDCl3).

[0047] Figure 10 For Cpz-2TPY 1 A magnified view of the H NMR spectrum in the 6-9 ppm range.

[0048] Figure 11 For Cpz-2TPY 13 C10 NMR spectrum (solvent: CDCl3).

[0049] Figure 12 For Cpz-4TPY 1 1H NMR spectrum (solvent: CDCl3).

[0050] Figure 13 For Cpz-4TPY 1 A magnified view of the 1H NMR spectrum in the range of 7-9.5 ppm.

[0051] Figure 14For Cpz-4TPY 13 C10 NMR spectrum (solvent: CDCl3).

[0052] Figure 15 For Cpz-6TPY 1 1H NMR spectrum (solvent: CDCl3).

[0053] Figure 16 For Cpz-6TPY 1 A magnified view of the 1H NMR spectrum in the range of 7-9.5 ppm.

[0054] Figure 17 For Cpz-6TPY 13 C10 NMR spectrum (solvent: CDCl3). Detailed Implementation

[0055] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0056] Example 1: Synthesis of the fluorescent ligand TPY

[0057] As shown in the synthetic route (1), 2-acetylpyridine (2.25 mL, 20.0 mmol), 4-hydroxybenzaldehyde (1.225 g, 10.0 mmol), and sodium hydroxide (1.458 g, 26.0 mmol) were accurately measured and dissolved in a mixed solution of 10 mL ethanol, 5 mL deionized water, and 30 mL ammonia in a three-necked flask. After stirring at room temperature for 24 h, the solution changed from wine red to dark brown, and the reaction was stopped by thin-layer chromatography. The pH of the reaction solution was adjusted to about 4 with acetic acid, and a precipitate was observed. After filtration, a yellow-green flocculent solid TPY (2.74 g, yield 84.31%) was obtained. 1 H NMR(400MHz,Chloroform-d)δppm 6.98(d,J=8.19Hz,2H)7.53(t,J=1.00Hz,2H)7.80(d,J=8.19Hz,2H)8.04(d,J=6.72Hz,2H)8.67(br.s.,4H)8.77(d,J=1.00Hz,2H)9.87(bs.s.1H).

[0058]

[0059] Example 2: Synthesis of the fluorescent ligand Cpz-2Cl

[0060] As shown in the synthetic route (2), anhydrous cesium carbonate (468 mg, 1.44 mmol) was accurately weighed into a three-necked flask, and an N2 ball was inserted for protection after evacuation. Under low temperature conditions, a THF solution (5 mL) of hexachlorocyclotriphosphazene (100 mg, 0.288 mmol) was added. After dehydrogenation for 15 min, a THF solution (5 mL) of biphenyl hydroquinone (117.8 mg, 0.632 mmol) was added. After stirring for another 10 min, the ice bath was removed, and the reaction solution was heated to 50 °C and refluxed. 31 The reaction was monitored by P NMR until the P signal of hexachlorocyclotriphosphazene disappeared, at which point the reaction was stopped. Salts in the reaction solution were separated by centrifugation, discarded, and the supernatant was concentrated to obtain a white solid. The solid was purified by column chromatography using a PE:EA solvent (volume ratio of 2:1) to finally obtain a white solid Cpz-2Cl (760 mg, yield 92%). 1 H NMR(400MHz,Chloroform-d)δppm 7.34-7.43(m,8H)7.44-7.52(m,4H)7.57(d,J=7.58Hz,4H)13C NMR (101MHz, CDCl3) δ121.83,126.49,128.58,129.74,129.98,147.78.

[0061]

[0062] Example 3: Synthesis of the fluorescent ligand Cpz-4Cl

[0063] As shown in the synthetic route (3), anhydrous cesium carbonate (206 mg, 0.63 mmol) was accurately weighed into a three-necked flask, and an N2 ball was inserted for protection after evacuation. Under low temperature conditions, a THF solution (5 mL) of hexachlorocyclotriphosphazene (100 mg, 0.288 mmol) was added. After dehydrogenation for 15 min, a THF solution (5 mL) of biphenyl hydroquinone (59 mg, 0.32 mmol) was added. After stirring for another 10 min, the ice bath was removed, and the reaction solution was heated to 50 °C and refluxed. 31 The reaction was monitored by P NMR until the P signal of hexachlorocyclotriphosphazene disappeared, at which point the reaction was stopped. Salts in the reaction solution were separated by centrifugation, discarded, and the supernatant was concentrated to obtain a white solid. The solid was purified by column chromatography using PE:DCM at a solvent volume ratio of 10:1 to finally obtain a white solid Cpz-4Cl (450 mg, yield 75%). 1H NMR (400MHz, Chloroform-d) δppm 7.33 (dt, J=8.01, 1.25Hz, 2H) 7.40-7.46 (m, 2H) 7.48-7.54 (m, 2H) 7.59 (dd, J=7.64, 1.65Hz, 2H). 13 C NMR (101MHz, CDCl3) δ128.30,128.32,129.88,130.21,147.30,147.49.

[0064]

[0065] Example 4: Synthesis of the fluorescent ligand Cpz-2TPY

[0066] As shown in the synthetic route (4), anhydrous cesium carbonate (284 mg, 0.87 mmol) was accurately weighed into a three-necked flask and protected with an N2 ball after vacuuming. TPY (125 mg, 0.384 mmol) was then dissolved in 5 mL of anhydrous THF. After dehydrogenation for 30 min at low temperature, an anhydrous THF solution of Cpz-2Cl (100 mg, 0.174 mmol) (5 mL) was added. After stirring for another 10 min, the ice bath was removed, and the reaction mixture was heated to 50 °C and refluxed. 31 The reaction was monitored by P NMR until the P signal of Cpz-2Cl disappeared, at which point the reaction was stopped. The salts in the reaction solution were separated by centrifugation and discarded, yielding the supernatant. 0.81 g of sodium hydroxide was weighed and dissolved in 20 mL of deionized water to prepare a 1 M sodium hydroxide solution. The supernatant was stirred, and the sodium hydroxide solution was added dropwise during stirring. A precipitate was observed to form, which was then filtered, dried, and the resulting brown solid Cpz-2TPY (265 mg, yield 84.87%) was obtained. 1 H NMR (400MHz, Chloroform- d )δppm 7.16(d,J=7.95Hz,4H)7.30-7.45(m,12H)7.51-7.63(m,8H)7.91(d,J=1.59Hz,4H)8.01(d,J=8.44Hz)8.70(d,J=7.95Hz,4H)8.73-8.83(m,8H). 13CNMR(101MHz, CDCl3)δ118.78,121.36,121.73,121.79,121.93,123.84,126.06,128.67,128.76,129.59,129.79,135.66 ,136.84,148.13,148.17,149.15,149.41,151.66,151.73,156.07,156.21.[M+H]+m / z:Cald.1174.263;Found:1174.269.

[0067]

[0068] Example 5: Synthesis of the fluorescent ligand Cpz-4TPY

[0069] As shown in the synthetic route (5), anhydrous cesium carbonate (353 mg, 1.085 mmol) was accurately weighed into a three-necked flask and protected with an N2 ball after vacuuming. TPY (311 mg, 0.955 mmol) was then dissolved in 5 mL of anhydrous THF. After dehydrogenation for 30 min at low temperature, an anhydrous THF solution of Cpz-4Cl (100 mg, 0.217 mmol) (5 mL) was added. After stirring for another 10 min, the ice bath was removed, and the reaction mixture was heated to 50 °C and refluxed. 31 The reaction was monitored by P NMR until the P signal of Cpz-4Cl disappeared, at which point the reaction was stopped. The salts in the reaction solution were separated by centrifugation and discarded, yielding the supernatant. 0.81 g of sodium hydroxide was dissolved in 20 mL of deionized water to prepare a 1 M sodium hydroxide solution. The supernatant was stirred, and the sodium hydroxide solution was added dropwise during stirring. A precipitate was observed to form, which was then filtered, dried, and the resulting brown solid Cpz-4TPY (282 mg, yield 80.34%) was obtained. 1 H NMR(400MHz,Chloroform-d)δppm 7.08(d,J=7.95Hz,2H)7.23(dd,J=6.66,5.20Hz,8H)7.35-7.44(m,10H)7.48(m,2H)7.59(d,J=7.46H z,2H)7.77(td,J=7.61,1.28Hz,8H)7.86(d,J=8.56Hz,8H)8.49(d,J=7.95Hz,8H)8.56-8.62(m,16H). 13C NMR (101MHz, CDCl3) δ118.48,121.10,121.52,123.51,126.16,128.56,128.75,129.65,129.95,135 .49,136.50,148.95,151.36,151.40,155.66,155.99.[M+H]+m / z:Cald.1638.438; Found:1638.459.

[0070]

[0071] Example 6: Synthesis of the fluorescent ligand Cpz-6TPY

[0072] As shown in the synthetic route (6), anhydrous cesium carbonate (234 mg, 0.723 mmol) was accurately weighed into a three-necked flask and protected with an N2 ball after vacuuming. TPY (337 mg, 1.03 mmol) was then dissolved in 5 mL of anhydrous THF. After dehydrogenation for 30 min at low temperature, an anhydrous THF solution of Cpz (50 mg, 0.144 mmol) (5 mL) was added. After stirring for another 10 min, the ice bath was removed, and the reaction mixture was heated to 50 °C and refluxed. 31 The reaction was monitored by P NMR until the P signal of Cpz disappeared, at which point the reaction was stopped. Salts in the reaction solution were separated by centrifugation and discarded, yielding the supernatant. 0.81 g of sodium hydroxide was weighed and dissolved in 20 mL of deionized water to prepare a 1 M sodium hydroxide solution. The supernatant was stirred, and the sodium hydroxide solution was added dropwise during stirring. A precipitate was observed to form, which was then filtered, dried, and the brown solid Cpz-6TPY (150 mg, yield 70.82%) was obtained. 1 H NMR(400MHz,Chloroform-d)δppm 7.18(d,J=5.38Hz,12H)7.34(d,J=8.31Hz,12H)7.72(br.s.,12H)7.82(d,J=8.44Hz,12H)8.41(d,J=7.82Hz,12H)8.53(s,24H). 13 CNMR(101MHz, CDCl3)δ118.47,121.00,121.28,123.38,128.60,135.41,136.37,148.90,155.49,155.92.[M+Na]+m / z: Cald.2102.613; Found:2102.647.

[0073]

[0074] Example 7: Study on UV absorption and fluorescence spectra of fluorescent ligands Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY in solvents of different polarities

[0075] (1) Accurately weigh the specified mass of Cpz-2TPY, Cpz-4TPY and Cpz-6TPY using an analytical balance and dissolve them in 5 mL of tetrahydrofuran (THF) solution to prepare Cpz-2TPY, Cpz-4TPY and Cpz-6TPY test stock solutions (1 mM). The stock solutions are stored in a refrigerator at 4 °C away from light and have a shelf life of 3 months.

[0076] (2) Add 2 mL of toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), ethanol (EtOH), N,N-dimethylformamide (DMF), acetonitrile (ACN), and dimethyl sulfoxide (DMSO) to a 3.5 mL four-sided transparent quartz cuvette, and add 20 μL of probe stock solution to obtain a test solution with a concentration of 10 μM.

[0077] (3) Place the working solution into the UV-Vis absorption spectrum of the pre-scanned baseline, set the scanning range to 250–450 nm, and obtain the UV-Vis absorption spectra of Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY in different solutions. Read the absorbance at the maximum absorption peak of Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY in different solvents; the spectra are shown below. Figure 1 As shown in A, C, and E.

[0078] (4) Place the working solution in a fluorescence spectrometer, set the excitation wavelength to 280 nm, and the scanning range to 300 nm–650 nm. Obtain the fluorescence spectra of Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY in different solutions. The spectra are shown below. Figure 1 As shown in B, D, and F. Relevant absorption and emission parameters are shown in Table 1-3.

[0079] Table 1 Solvent effects of the fluorescent ligand Cpz-2TPY

[0080]

[0081] Table 2 Solvent effects of the fluorescent ligand Cpz-4TPY

[0082]

[0083] Table 3 Solvent effects of the fluorescent ligand Cpz-6TPY

[0084]

[0085] Example 8: Metal ion selectivity of fluorescent ligands Cpz-2TPY, Cpz-4TPY and Cpz-6TPY and their sensing ability for Qr

[0086] (1) Accurately weigh the specified mass of the metal cation salt compound using an analytical balance, and use 10 mL of deionized water to separately weigh Al 3+ Na + K + Mg 2+ Ca 2+ Cu 2+ Zn 2+ Fe 2+ Fe 3+ Co 2+ Cd 2+ Ni 2+ Yb 3+ Eu 3+ and Tb 3+ Prepare a 10 mM metal cation stock solution. Store the stock solution in a refrigerator at 4°C away from light. The shelf life is 3 months.

[0087] (2) 2 mL of ethanol solution was added to a 3.5 mL four-sided transparent quartz cuvette, followed by the addition of 20 μL stock solutions of Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY, respectively, to prepare a detection concentration of 10 mM for the fluorescent probe molecules. Subsequently, stock solutions covering a variety of metal ions, specifically including Al, were introduced into the solutions of these three fluorescent ligands. 3+ Na + K + Mg 2 + Ca 2+ Cu 2+ Zn 2+ Fe 2+ Fe 3+ Co 2+ Cd 2+ Ni 2+ Yb 3+ Eu 3+ and Tb 3+ There are a total of 15 different metal ions.

[0088] (3) Place the working solution in a fluorescence spectrometer, set the excitation wavelength to 280 nm, and the scanning range to 300 nm–650 nm. Record the fluorescence of the three fluorescent ligands Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY at I0. 489nm / I 351nm I 487nm / I 358nm I487nm / I 375nm The fluorescence intensity when the sample binds to different metal ions. The results are as follows: Figure 2 As shown.

[0089] (4) Figure 2 Visually, among the metal ions examined, Al... 3+ The ion-synthesized Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al metal complexes all exhibited the most significant response to quercetin. Therefore, it can be confirmed that Al... 3+ It is an ideal ion choice for constructing Cpz-nTPY-Al metal complexes for quercetin detection.

[0090] Example 9: Study on the detection system of metal complex Cpz-nTPY-Al for quercetin (Qr)

[0091] To determine the optimal detection system for Qr using the metal complex Cpz-nTPY-Al, the changes in fluorescence intensity of Cpz-nTPY-Al and Qr in different solvents were investigated. The specific procedures are as follows:

[0092] (1) Weigh an appropriate amount of quercetin dihydrate solid, dissolve it in 10 mL of ethanol, and prepare a 1 mM Qr stock solution for immediate use.

[0093] (2) Add 2 mL of toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EA), ethanol (EtOH), N,N-dimethylformamide (DMF), acetonitrile (ACN), dimethyl sulfoxide (DMSO), water (H2O), and buffer solutions (HEPES and PBS) to 11 3.5 mL four-sided transparent quartz cuvettes respectively. Then add 20 μL of metal complex Cpz-nTPY-Al stock solution to obtain test solutions with a probe concentration of 10 μM.

[0094] (3) Add 20 μL of Qr stock solution to each of the above cuvettes to obtain a test solution with a Qr concentration of 10 μM.

[0095] (4) Place the working solution in a fluorescence spectrometer, set the excitation wavelength to 280 nm, and the scanning range to 300–650 nm. Record the fluorescence wavelengths of the three fluorescent metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al at I0. 489nm / I 351nm I 487nm / I 358nm I 487nm / I 375nmThe fluorescence intensity when the Qr molecule binds to the target. The results are as follows: Figure 3 As shown.

[0096] (5) Figure 3 As shown, when ethanol is used as the solvent system, Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al exhibit the most significant fluorescence enhancement effect for the detection of Qr, with fluorescence enhancement factors reaching their respective maximum values ​​of 0.437, 4.432, and 5.223 times, respectively. Therefore, we determine that ethanol solution is the optimal solution system for the detection of Cpz-nTPY-Al metal complexes in Qr.

[0097] Example 10: Probe ligands Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY bind to Al 3+ Job's Plot Curve Study

[0098] By measuring the binding of probe ligands Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY to Al... 3+ Job's Plot working curves were used to determine the probe ligands Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY and Al. 3+ The complexation ratio. The specific operation is as follows:

[0099] (1) Accurately weigh the specified mass of Cpz-2TPY, Cpz-4TPY and Cpz-6TPY using an analytical balance and dissolve them in 5 mL of tetrahydrofuran (THF) solution to prepare Cpz-2TPY, Cpz-4TPY and Cpz-6TPY test stock solutions (1 mM). The stock solutions are stored in a refrigerator at 4 °C away from light and have a shelf life of 3 months.

[0100] (2) Different concentrations of probe ligands Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY and detector Qr were added to 2 mL of ethanol solution in 11 3.5 mL four-sided transparent quartz cuvettes, respectively, while maintaining a total concentration of probe ligands and Qr at 50 μM. The proportions of fluorescent probe ligands and Qr were varied. The cuvettes were placed in a fluorescence spectrometer. The excitation wavelength was set to 280 nm, and the detection wavelength range was 300 nm to 650 nm. The fluorescence spectra of the probes were obtained.

[0101] (3) The fluorescence spectra of the above ligands Cpz-2TPY, Cpz-4TPY and Cpz-6TPY were recorded at 489 nm and 351 nm, 487 nm and 358 nm, and 487 nm and 375 nm, respectively, to obtain a series of I values ​​related to Qr concentration. 489nm / I 351nm I 487nm / I358nm I 487nm / I 375nm The ratio. Using this ratio as the ordinate and [Qr] / ([Qr]+[Cpz-nTPY]) as the abscissa, a Job's Plot curve is obtained, as shown below. Figure 4 As shown in AC.

[0102] (4) Figure 4 As shown in Figure A, the two regression lines for Cpz-2TPY intersect at a concentration ratio of approximately 0.67, a result that strongly supports the interaction between Cpz-2TPY and Al. 3+ The resulting complex follows a stoichiometric ratio of 1:2. Similarly, the two regression lines for Cpz-4TPY intersect at a concentration ratio of approximately 0.8, revealing the interaction between Cpz-4TPY and Al. 3+ Complexation follows a stoichiometric ratio of 1:4 (see...) Figure 4 B). Finally, the two regression lines of Cpz-6TPY intersected at a concentration ratio of approximately 0.86, indicating that Cpz-6TPY interacts with Al. 3+ Upon binding, a stoichiometric complex with a ratio of 1:6 was formed (see...) Figure 4 C).

[0103] Example 11 Kinetic Study of Quercetin (Qr) Detection by Metal Complex Cpz-nTPY-Al

[0104] To determine the optimal kinetic response time for the detection of Qr by the genus complex Cpz-nTPY-Al, the change in fluorescence intensity of the genus complex Cpz-nTPY-Al and Qr in the ethanol system over time was investigated. The specific procedures are as follows:

[0105] (1) Accurately weigh the specified mass of Cpz-2TPY, Cpz-4TPY and Cpz-6TPY using an analytical balance and dissolve them in 5 mL of tetrahydrofuran (THF) solution to prepare Cpz-2TPY, Cpz-4TPY and Cpz-6TPY test stock solutions (1 mM). The stock solutions are stored in a refrigerator at 4 °C away from light and have a shelf life of 3 months.

[0106] (2) Add 2 mL of ethanol solution to each of the following 3.5 mL four-sided transparent quartz cuvettes: 20 μL of probe ligand Cpz-2TPY stock solution and 4 μL of Al. 3+ Stock solution, 20 μL of probe ligand Cpz-4TPY stock solution and 8 μL of Al 3+ Stock solution, 20 μL of probe ligand Cpz-6TPY stock solution and 12 μL of Al 3+Prepare a stock solution and a 20 μL stock solution of the analyte Qr. Place the cuvette in the fluorescence spectrometer. Set the excitation wavelength to 280 nm and the detection wavelength range to 300 nm–650 nm. Obtain the fluorescence spectrum of the probe.

[0107] (3) The fluorescence intensities of the above metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al with Qr were recorded at 489 nm and 351 nm, 487 nm and 358 nm, and 487 nm and 375 nm, respectively, to obtain a series of time-dependent I... 489nm / I 351nm I 487nm / I 358nm I 487nm / I 375nm The fluorescence ratio data were used. Fluorescence curves of the metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al binding to Qr were fitted with this ratio on the ordinate and reaction time on the abscissa, as shown below. Figure 5 As shown in AC.

[0108] (4) From Figure 5 It can be clearly observed that when Qr is introduced into the system, the fluorescence intensity of these three metal complexes reaches saturation within 390s, 420s, and 120s, respectively. Notably, compared to Cpz-2TPY-Al and Cpz-4TPY-Al, which contain only 2 and 4 terpyridine units respectively, Cpz-6TPY-Al, with 6 terpyridine units, exhibits a much faster Qr complexation ability, enabling effective detection of Qr in an extremely short time (only 120 seconds).

[0109] Example 12 Study on the selectivity and anti-interference properties of metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al and Cpz-6TPY-Al for Qr

[0110] To test the selectivity of the metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al for Qr, the fluorescence response of the probe molecules to different types of metal ions, anions, and homologues was tested. The tested targets included Na. + K + Ca 2+ Mg 2 + Cl - CO3 2- SO4 2- H2PO4 - HPO4 2- PO4 3-AcO - The following substances were included: catechin, salicylic acid (SA), arginine (Arg), L-cysteine ​​(Cys), phenylalanine (PHE), luteolin, apigenin, and genistein. The detection concentration of the metal ions was 50 μM, and the working concentration of the probe molecules was 10 μM. The specific operating procedure is as follows:

[0111] (1) Prepare a 10 mM metal ion stock solution. Weigh the corresponding amount of the substance and put it into a 5 mL volumetric flask. Add about 4 mL of deionized water to dissolve it, and then add more deionized water to the mark of the volumetric flask. Shake well and store in the refrigerator for later use.

[0112] (2) Place 2 mL of ethanol solution into a 3.5 mL four-sided transparent quartz cuvette. Add 20 μL of metal complex probe stock solution to the cuvette and mix well. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 280 nm and the detection wavelength range to 300–650 nm. Obtain the initial fluorescence spectrum of the probe, and record the fluorescence intensity at 489 nm and 351 nm, 487 nm and 358 nm, and 487 nm and 375 nm, respectively. Calculate the fluorescence intensity using IL. 489nm / I 351nm I 487nm / I 358nm I 487nm / I 375nm The ratio of is taken as I0.

[0113] (3) Add 10 μL of each of the various detection molecules and ions to the cuvettes in (2) to obtain a series of test solutions containing 50 μM of detection molecules and ions. Test the fluorescence spectrum of the above test solutions.

[0114] (4) Record the fluorescence intensity at 489 nm and 351 nm, 487 nm and 358 nm, and 487 nm and 375 nm in the above fluorescence spectra to obtain the Ig of the metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al and Cpz-6TPY-Al in various interfering environments. 489nm / I 351nm I 487nm / I 358nm I 487nm / I 375nm The ratio (I) is calculated. The fluorescence intensity value is then compared to I0 in (2). A bar chart is plotted using the I / I0 values ​​for each sample, and the results are as follows: Figure 6 As shown.

[0115] (5) The experimental results clearly show that even at concentrations of interfering substances up to five times the concentration of Qr, the metal complexes (Cpz-2TPY-Al, Cpz-4TPY-Al, Cpz-6TPY-Al) can still induce significant fluorescence enhancement in the detection of Qr (e.g., Figure 6 (As shown in B, D, and F). This series of anti-interference experiments fully demonstrates the robustness of the metal complexes for Qr detection, indicating that they are not affected by other substances. Therefore, we have reason to believe that these metal complexes are promising candidates for Qr detection in complex systems such as honey, tobacco, and red wine.

[0116] Example 13: Study on the calculation of the detection limit of Qr for metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al and Cpz-6TPY-Al

[0117] To test the fluorescence response characteristics of metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al to different concentrations of Qr, the experimental procedure is described in detail using an ethanol solution as an example.

[0118] (1) Place 2 mL of ethanol solution into a 3.5 mL four-sided transparent quartz cuvette. Add 20 μL of stock solutions (1 mM) of metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al to the cuvette respectively, and shake well. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 280 nm and the detection wavelength range to 300 nm–650 nm. Obtain the initial fluorescence spectrum of the probe.

[0119] (2) Add 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μL of Qr stock solution (1 mM) to cuvettes containing the three metal complexes, respectively, to obtain test solutions containing 2, 4, 6, 8, 10, and 20 μM Qr. Measure the fluorescence spectra of the above test solutions (e.g., Figure 7 (As shown in A, C, and E).

[0120] (3) Record the fluorescence intensity of the three metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al and Cpz-6TPY-Al at 489 nm and 351 nm, 487 nm and 358 nm, and 487 nm and 375 nm, respectively, and obtain a series of I values ​​related to Qr concentration. 489nm / I 351nm I 487nm / I 358nm I 487nm / I 375nm The ratio. A scatter plot of this ratio against the Qr concentration, such as... Figure 7 As shown in B, D, and F.

[0121] (4) Linear fitting was performed on the 7B linear interval (0-10 μM), the 7D linear interval (0-6 μM), and the 7F linear interval (0-5 μM), and the detection limits of the three metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al and Cpz-6TPY-Al for Qr in ethanol solution were determined according to 3σ / k.

[0122] (5) The calculation results are as follows: the detection limit of Cpz-2TPY-Al for Qr is 0.427 μM, and its linear response range is between 0 and 8 μM (e.g., Figure 7 (As shown in AB); Cpz-4TPY-Al has a lower detection limit for Qr, at 0.049 μM, and a linear range of 0-6 μM (as shown in AB). Figure 7 (as shown in CD); while the detection limit of Cpz-6TPY-Al is even lower at 61.76 nM, and its linear range is between 0-5 μM (as shown in CD). Figure 7 (As shown in EF). Compared to probes containing only a single terpyridine ligand (TPY) (with a detection limit (LOD) of 0.464 μM), the three metal complexes Cpz-2TPY-Al, Cpz-4TPY-Al, and Cpz-6TPY-Al successfully achieved intermolecular synergistic effects by ingeniously introducing multiple terpyridine recognition sites. This unique effect significantly enhances the probe's ability to recognize Qr, thereby greatly improving the sensitivity and selectivity of detection.

[0123] Example 14: Detection of Qr in real samples of red wine, honey, and tobacco using the metal complex probe Cpz-6TPY-Al.

[0124] To verify the effectiveness of the metal complex in practical applications, we used a spiked recovery method to perform quantitative analysis of the Qr of the metal complex Cpz-6TPY-Al in three different food matrices: red wine, honey, and tobacco. The specific procedures are as follows:

[0125] (1) Add 2 mL of ethanol solution to each of the 3.5 mL four-sided transparent quartz cuvettes, and then add 20 μL of the actual sample diluted 10 times to each. Add 20 μL of the fluorescent ligand Cpz-6TPY (1 mM) and 12 μL of Al to the cuvettes. 3+ Prepare the stock solution (10 mM) and shake well. Place the cuvette in the fluorescence spectrometer. Set the excitation wavelength to 280 nm and the detection wavelength to 300–650 nm. Obtain the initial fluorescence intensities of the metal complex probe Cpz-6TPY-Al at 487 nm and 375 nm in three different real samples.

[0126] (2) Add 2, 4, 8, 12 and 16 μL of Qr stock solution (1 mM) to cuvettes to obtain test solutions of 1, 2, 4, 6 and 8 μM Qr. Test the fluorescence intensity of the above test solutions at 487 nm and 375 nm.

[0127] (3) Record the fluorescence intensity at 487 nm and 375 nm in the above fluorescence spectrum, respectively, and calculate I. 487nm / I 375nm A series of I values ​​related to Qr concentration were obtained. 487nm / I 375nm Linear relationship. (Using I) 487nm / I 375nm A scatter plot is created with Qr concentration as the x-axis and Qr concentration as the y-axis, as shown below. Figure 8 As shown in AC.

[0128] (4) Figure 8 Linear fitting was performed on the linear interval (0–8 μM) to obtain the standard curve equation y = 0.38214x + 0.998, R0. 2 =0.998; for Figure 8 Linear fitting was performed on the linear interval (0–8 μM) to obtain the standard curve equation y = 0.50163x + 0.67335, R0. 2 =0.999; for Figure 8 Linear fitting was performed on the linear interval (0–8 μM) to obtain the standard curve equation y = 0.47253x + 0.61195, R0. 2 =0.998.

[0129] (5) Add known concentrations of BPA standard solutions (1, 3, and 5 μM) to the actual samples diluted 10-fold, record the fluorescence intensity of the probe, and calculate I. 487nm / I 375nm The ratio was used to determine the content of Qr in the actual sample, and the result was obtained by applying the standard linear equation. This step was repeated three times. The test results are shown in Table 4.

[0130] (6) Table 4 lists the experimental results of the standard additive method. Analysis of the data shows that our proposed method not only has a stable recovery rate ranging from 96.53% to 105.63%, but also that the RSD value is within an acceptable range, fluctuating between 0.96% and 2.58%. In summary, based on Al... 3+ The regulated cyclotriphosphazene-terpyridine derivative Cpz-nTPY-Al demonstrated high accuracy and excellent selectivity for Qr in real sample analysis, providing a reliable and efficient method for the quantitative analysis of Qr in food.

[0131] Table 4 shows the detection of Qr (n=3) in actual samples.

[0132]

[0133] Finally, it should be noted that this invention is not limited to the above-described embodiments. Any improvements made by those skilled in the art based on the disclosure of this invention without departing from the scope of this invention should be within the protection scope of this invention.

Claims

1. A cyclotriphosphazene-terpyridine fluorescent probe, characterized in that, As shown in structural formula (Ⅰ), any one or any combination of Cpz-2TPY, Cpz-4TPY, and Cpz-6TPY:

2. The cyclotriphosphazene-terpyridine fluorescent probe and / or its metal complex as described in claim 1 for the detection of flavonoids and / or the preparation of products for the detection of flavonoids.

3. A product for detecting flavonoids, characterized in that, It contains a cyclotriphosphazene-terpyridine fluorescent probe as described in claim 1 and / or its metal complex.

4. The product for detecting flavonoids according to claim 3, characterized in that, The product is at least one of the following: a test reagent, a kit, a component, a device, or a device.

5. The product for detecting flavonoids according to claim 3, characterized in that, The metal ion Al 3 + Na + K + Mg 2+ Ca 2+ Cu 2+ Zn 2+ Fe 2+ Fe 3+ Co 2+ Cd 2+ Ni 2+ Yb 3+ Eu 3+ 、Tb 3+ Any one or any combination thereof.

6. A method for detecting flavonoids, characterized in that the steps include... include: Using the cyclotriphosphazene-terpyridine fluorescent probe and / or its metal complex as described in claim 1 as a probe, a detection system is formed by mixing it with the sample to be tested. Under 280nm excitation light, fluorescence in the range of 300nm to 650nm is collected to perform qualitative and / or quantitative detection of flavonoids in the sample.

7. The detection method according to claim 6, characterized in that, Using the cyclotriphosphazene-terpyridine fluorescent probe of claim 1 as the probe, metal ions need to be added to the detection system to form a metal complex.

8. The detection method according to claim 6 or 7, characterized in that, Using the fluorescence intensity ratio of the metal complex of the cyclotriphosphazene-terpyridine fluorescent probe described in claim 1 as the ordinate and the concentration of flavonoids as the x and y axes, the qualitative and / or quantitative determination of boron-containing compounds in the sample is carried out using the external standard method, internal standard method, or standard addition method.

9. The detection method according to claim 6, characterized in that, The solvent used in the detection system can be any one or any combination of toluene, dichloromethane, tetrahydrofuran, ethyl acetate, ethanol, N,N-dimethylformamide, acetonitrile, dimethyl sulfoxide, water, HEPES buffer solution, and PBS buffer solution.

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