Application of triphenylamine-terpyridyl fluorescent probe to detection of nucleotide and / or chain-like polyphosphoric acid

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to application of a triphenylamine-terpyridyl fluorescent probe to detection of nucleotide and / or chain-shaped polyphosphoric acid. The fluorescent ligand TPA-3TPY provided by the invention is chelated with # imgabs0 # ions in situ to successfully prepare the metal complex, so that the fluorescence emission wavelength moves towards the long wave direction. When the nucleotide and the derivative thereof are detected, the # imgabs 1 # ion is separated from the fluorescent ligand, and the fluorescence emission wavelength is recovered, so that the effective detection of the nucleotide and the derivative thereof is realized. The method has the advantages of quick response, high selectivity, interference resistance and low cost, and has important application value in the fields of food science, environmental science, biological science and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and particularly relates to the application of triphenylamine-terpyridine fluorescent probes in detecting nucleotides and / or chain polyphosphates. Background Art

[0002] Adenosine triphosphate (ATP) is the primary energy source within cells. It releases energy through hydrolysis reactions, which fuels various cellular activities. ATP plays a key role in cell signaling. In the nervous system, for example, it serves not only as a neurotransmitter but also as a neuromodulator, playing a crucial role in information transmission between synapses. ATP also acts as an intercellular signaling molecule, transmitting information by activating specific receptors (such as P2 receptors), thereby regulating cellular function and behavior. Furthermore, abnormal ATP concentrations can be associated with a variety of diseases or physiological conditions. For example, excessive ATP intake can cause central nervous system hyperexcitability, cardiac arrhythmias, and hypercalcemia. ATP is also a key indicator for rapid, efficient, and accurate testing of food safety and preservation, playing an indispensable role in every link of the food supply chain—from production and processing to distribution and catering services. ATP also plays an irreplaceable role in aquatic environments, such as water quality testing and water treatment process monitoring (microbial growth testing, activated sludge testing, and water treatment equipment operational status monitoring). ATP detection technology has shown broad application space and significant practical value in many fields such as food safety, water quality monitoring, and medical and health care.

[0003] Currently, ATP detection mainly adopts a variety of methods such as high-performance liquid chromatography (HPLC), spectrophotometry, electrophoresis and fluorescent probe method. Among them, the fluorescent probe method has made breakthrough progress in recent years. Researchers have successfully developed a number of excellent fluorescent probes. These probes are not only highly sensitive and selective, but can also effectively penetrate the cell membrane and realize real-time monitoring of ATP concentration in living cells. Thanks to their excellent fluorescence properties and biocompatibility, this type of probe has demonstrated reliable ATP detection capabilities in complex biological systems. For example, a study reported a triphenylamine-based two-photon fluorescent probe (MP), which can selectively localize to the cell nucleus and emit fluorescence after binding to ATP. The probe exhibits excellent cell membrane penetration ability and precise selectivity, responding only to ATP and having almost no reaction to other biological molecules. Researchers have successfully applied it to live cell imaging experiments, realizing real-time monitoring of ATP levels in the cell nucleus. At the same time, Professor Li Yulong's team at Peking University developed the revolutionary genetically encoded ATP probe GRABATP1.0. This technology has achieved breakthrough high-precision spatiotemporal observation of dynamic changes in extracellular ATP in living animals and in vitro experiments. This research provides a powerful tool for deciphering the function of purinergic signaling and is expected to play an important role in fields such as neuroscience and immunology.

[0004] Therefore, developing a rapid, sensitive and specific method for detecting ATP has important research significance in the field of biological sciences. Summary of the Invention

[0005] The present invention provides a triphenylamine-terpyridine fluorescent probe, which is used for detecting nucleotides or their derivatives, chain polyphosphates or their derivatives, especially ATP, and has the advantages of simple operation, fast response, high selectivity, anti-interference, low cost, high efficiency, and the like.

[0006] The technical solution of the present invention is a triphenylamine-terpyridine fluorescent probe, the structural formula of which is shown in formula (I):

[0007]

[0008] The present invention provides a method for preparing a triphenylamine-terpyridine fluorescent probe, comprising the following steps:

[0009] a. Triphenylamine (TPA) and phosphorus oxychloride (POCl3) are heated under reflux in DMF to obtain triphenylamine-dialdehyde (TPA-2CHO); triphenylamine-dialdehyde (TPA-2CHO) is then heated under reflux with phosphorus oxychloride (POCl3) in DMF to obtain triphenylamine-trialdehyde (TPA-3CHO), as shown in Scheme 1;

[0010]

[0011] b. Triphenylamine-trialdehyde (TPA-3CHO) reacts with 2-acetylpyridine in ethanol with the addition of sodium hydroxide and ammonia to obtain triphenylamine-terpyridine ligand (TPA-3TPY), as shown in Scheme 2.

[0012]

[0013] In step a, phosphorus oxychloride (POCl3) was added to DMF under nitrogen protection at 0°C.

[0014] In step a, the molar ratio of triphenylamine (TPA) to phosphorus oxychloride (POCl3) is 1:20-30, preferably 1:25; the molar concentration of triphenylamine (TPA) is 0.05-1mmol / mL, preferably 0.2-0.4mmol / mL; the molar ratio of triphenylamine-dialdehyde (TPA-2CHO) to phosphorus oxychloride (POCl3) is 1:10-25, preferably 1:15-20; the concentration of phosphorus oxychloride (POCl3) is 1-15mmol / mL, preferably 6-6.5mmol / mL.

[0015] In step a, triphenylamine (TPA) and phosphorus oxychloride (POCl3) are refluxed at 95°C for 1-5 hours, preferably for 1-1.5 hours. After the reaction is completed, the mixture is cooled to room temperature, ice water is added, and then a 1M NaOH solution is added to adjust the mixture to alkalinity. The organic phase is extracted with dichloromethane, washed with water, dried over anhydrous sodium sulfate, filtered, and the organic solvent is removed under reduced pressure to obtain triphenylamine-dialdehyde (TPA-2CHO).

[0016] In step a, triphenylamine-dialdehyde (TPA-2CHO) and phosphorus oxychloride (POCl3) are refluxed at 95°C for 1.5-2 hours. After the reaction, the mixture is cooled to room temperature, ice water is added, and then a 1M NaOH solution is added to adjust the mixture to alkalinity. The organic phase is extracted with dichloromethane, washed with water, dried over anhydrous sodium sulfate, filtered, and the organic solvent is removed under reduced pressure to obtain triphenylamine-trialdehyde (TPA-3CHO).

[0017] In step b, the molar ratio of triphenylamine-trialdehyde (TPA-3CHO) to 2-acetylpyridine is 1:8-12, preferably 1:9; the molar concentration of triphenylamine-trialdehyde (TPA-3CHO) is 0.005-0.1 mmol / mL, preferably 0.015-0.03 mmol / mL; the volume ratio of ethanol to aqueous ammonia is 1:0.5-5, preferably 1:1; and the concentration of sodium hydroxide is 0.05-0.5 mmol / mL, preferably 0.12-0.13 mmol / mL.

[0018] In step b, triphenylamine-trialdehyde (TPA-3CHO) is reacted with 2-acetylpyridine at room temperature (25°C) for 70-75 hours. After the reaction, the precipitate is filtered and washed with ether. The precipitate is dissolved in ethanol and recrystallized to obtain triphenylamine-terpyridine ligand (TPA-3TPY).

[0019] The triphenylamine-terpyridine fluorescent probe provided by the present invention relies on the strong affinity between terpyridine (TPY) and metal ions. Through experiments, we found that the fluorescent ligand TPA-3TPY and Zn 2+ The metal complex was successfully prepared by in-situ chelation of ions. This reaction process caused the fluorescence emission wavelength of the ligand to shift significantly to the long-wave direction, from green fluorescence to orange fluorescence. 2+ The strong binding ability between them induces Zn 2+ The ions are detached from the fluorescent ligand, which allows the fluorescence signal of the TPA-3TPY ligand to recover, changing from orange fluorescence back to green fluorescence, thereby achieving effective detection of ATP.

[0020] Based on the above principles, the triphenylamine-terpyridine fluorescent probe and / or its metal complex provided above of the present invention can be used to detect nucleotides, and / or nucleotide derivatives, and / or chain polyphosphates, and / or chain polyphosphate derivatives, and is used to prepare products for detecting nucleotides, and / or nucleotide derivatives, and / or chain polyphosphates, and / or chain polyphosphate derivatives. The nucleotides include but are not limited to any one or any combination of nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates, such as ATP. The chain polyphosphate includes but is not limited to any one or any combination of pyrophosphate and triphosphate. The derivative refers to a derivative obtained by replacing one or more atoms or groups in a nucleotide or chain polyphosphate with other atoms or groups, or refers to a derivative with similar properties obtained by adjusting the structure of a nucleotide or chain polyphosphate without changing the position and type of the original substituent. The products include but are not limited to detection reagents, kits, components, devices or equipment, etc. The reagents include but are not limited to solutions containing the triphenylamine-terpyridine fluorescent probe and / or its metal complex provided by the present invention, the components include but are not limited to magnetic beads, panels, chips, pipes, channels, etc. loaded with the triphenylamine-terpyridine fluorescent probe and / or its metal complex provided by the present invention, and the devices or equipment are equipped with the above-mentioned components. The metal ions used for the metal complex include but are not limited to Na + , K + Mg 2+ , Ca 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Al3+ 、Co 2+ 、Cd 2+ 、Ni 2+ , Pb 2 + 、Ba 2+ 、Yb 3+ 、Eu 3+ , Tb 3+ Any one or any combination thereof, preferably Cu 2+ 、Zn 2+ 、Co 2+ 、Cd 2+ Any one or any combination thereof, more preferably Zn 2+ 、Cd 2+ Any one or any combination thereof, as an embodiment, Zn 2+ .

[0021] The following products provided by the present invention contain the triphenylamine-terpyridine fluorescent probe and / or its metal complex provided above:

[0022] (1) Products for detecting nucleotides and / or their derivatives;

[0023] (2) Products for chain polyphosphoric acid and / or its derivatives.

[0024] The nucleotides include, but are not limited to, any one or any combination of nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates, such as ATP. The chain polyphosphates include, but are not limited to any one or any combination of pyrophosphates and triphosphates. The derivatives refer to derivatives obtained by replacing one or more atoms or groups in a nucleotide or chain polyphosphate with other atoms or groups, or derivatives with similar properties obtained by adjusting the structure of a nucleotide or chain polyphosphate without changing the position or type of the original substituents.

[0025] The products include but are not limited to detection reagents, kits, components, devices or equipment, etc. The reagents include but are not limited to solutions containing the triphenylamine-terpyridine fluorescent probe and / or its metal complex provided by the present invention, and the components include but are not limited to magnetic beads, panels, chips, pipes, passages, etc. loaded with the triphenylamine-terpyridine fluorescent probe and / or its metal complex provided by the present invention, and the above-mentioned components are configured in the device or equipment.

[0026] The metal ions used in the metal complex include but are not limited to Na + , K + Mg 2+ , Ca 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Fe3+ 、Al 3+ 、Co 2+ 、Cd 2+ 、Ni 2+ , Pb 2+ 、Ba 2+ 、Yb 3+ 、Eu 3+ , Tb 3+ Any one or any combination thereof, preferably Cu 2+ 、Zn 2+ 、Co 2 + 、Cd 2+ Any one or any combination thereof, more preferably Zn 2+ 、Cd 2+ Any one or any combination thereof, as an embodiment, Zn 2+ .

[0027] The present invention provides a detection method for detecting nucleotides and / or their derivatives and / or chain polyphosphates and / or their derivatives. The method comprises the following steps: using the triphenylamine-terpyridine fluorescent probe and / or its metal complex provided by the present invention as a probe, mixing it with a sample to be tested to form a detection system; collecting fluorescence within a range of 395 nm to 750 nm under 375 nm excitation light, and performing qualitative and / or quantitative detection of the nucleotides and / or their derivatives and / or chain polyphosphates and / or their derivatives in the sample.

[0028] Furthermore, when the triphenylamine-terpyridine fluorescent probe provided by the present invention is used as a probe, metal ions need to be added to the detection system to form a metal complex.

[0029] The metal ions used in the metal complex include but are not limited to Na + , K + Mg 2+ , Ca 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ 、Co 2+ 、Cd 2+ 、Ni 2+ , Pb 2+ 、Ba 2+ 、Yb 3+ 、Eu 3+ , Tb 3+ Any one or any combination thereof, preferably Cu 2+ 、Zn 2+ 、Co 2 + 、Cd2+ Any one or any combination thereof, more preferably Zn 2+ 、Cd 2+ Any one or any combination thereof, as an embodiment, Zn 2+ .

[0030] Furthermore, I 510nm The fluorescence intensity or I 496nm / I 610nm The fluorescence intensity ratio of the sample and the concentration of the nucleotides and / or their derivatives and / or chain polyphosphates and / or their derivatives are used as the horizontal and vertical coordinates, and the external standard method, internal standard method or standard addition method are used to qualitatively and / or quantitatively determine the boron-containing compounds in the sample.

[0031] Furthermore, I 510nm , I 496nm with I 610nm The fluorescence intensity value at each fluorescence wavelength refers to the fluorescence intensity value obtained by deducting the initial fluorescence intensity of the triphenylamine-terpyridine fluorescent probe metal complex obtained before adding the sample to be tested from the fluorescence intensity obtained after adding the sample to the detection system.

[0032] Furthermore, the solvent used in the detection system includes any one or any combination of toluene, dichloromethane, tetrahydrofuran, ethanol, N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide, such as dimethyl sulfoxide as an embodiment.

[0033] Furthermore, a buffer solution is added to the detection system, and the volume ratio of the solvent to the buffer solution used in the detection system is 8-10:1, preferably 9:1; the buffer solution is a HEPES buffer solution.

[0034] The nucleotides include, but are not limited to, any one or any combination of nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates, such as ATP. The chain polyphosphates include, but are not limited to any one or any combination of pyrophosphates and triphosphates. The derivatives refer to derivatives obtained by replacing one or more atoms or groups in a nucleotide or chain polyphosphate with other atoms or groups, or derivatives with similar properties obtained by adjusting the structure of a nucleotide or chain polyphosphate without changing the position or type of the original substituents.

[0035] 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, medicines, 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 human body fluids (urine). All samples containing nucleotides and / or their derivatives, and / or chain polyphosphates and / or their derivatives, regardless of their form, can be detected using the triphenylamine-terpyridine fluorescent probe provided by the present invention.

[0036] The present invention provides a triphenylamine-terpyridine fluorescent probe and metal (especially Zn 2+ ) can be used to detect nucleotides and / or their derivatives, and / or chain polyphosphates and / or their derivatives, particularly ATP. It offers advantages such as good selectivity, interference resistance, and rapid response. The detection limit for ATP is 0.2 μM, and the linear response range is 40-70 μM. This provides a new method for the detection of nucleotides and / or their derivatives, and / or chain polyphosphates and / or their derivatives, particularly ATP, and holds significant application potential and value in a variety of fields, including food safety, water quality monitoring, and healthcare. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 UV absorption spectra of the fluorescent ligand TPA-3TPY in toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EA), ethanol (EtOH), N,N-dimethylformamide (DMF), acetonitrile (ACN) and dimethyl sulfoxide (DMSO).

[0038] Figure 2 Fluorescence spectra of the fluorescent ligand TPA-3TPY in toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EA), ethanol (EtOH), N,N-dimethylformamide (DMF), acetonitrile (ACN) and dimethyl sulfoxide (DMSO).

[0039] Figure 3 The figure shows the response of the fluorescent ligand TPA-3TPY (10 μM) to different metal ions (30 μM) in DMSO / Hepes (10 mM, 9 / 1, v / v) solution at room temperature and the fluorescence color of the fluorescent ligand solution when different metal ions are added under 365 nm UV light.

[0040] Figure 4 In DMSO / Hepes (10 mM, 9 / 1, v / v) solution, the fluorescent ligand TPA-3TPY (10 μM) reacted with different metal ions (30 μM) to form TPA-3TPY-M in situ.n+ The complex responded to ATP (50 μM).

[0041] Figure 5 In DMSO / Hepes (10 mM, 9 / 1, v / v) solution, TPA-3TPY and Zn 2+ ([TPA-3TPY]+[Zn 2+ ]=20μM) working curve analysis. ex =375nm.

[0042] Figure 6 In DMSO / Hepes (10 mM, 9 / 1, v / v) solution, TPA-3TPY-Zn 2+ (TPA-3TPY: 10 μM) was used to detect the kinetics of ATP, and the observation wavelength was 510 nm.

[0043] Figure 7 In DMSO / Hepes (10 mM, 9 / 1, v / v) solution, TPA-3TPY-Zn 2+ The complex probe shows (a) selectivity for common anions; (b) selectivity for homologues; and (c) anti-interference properties towards related substances (50 μM ATP, 50 μM homologues, 100 μM related anions). ex =375nm.

[0044] Figure 8 (a) Fluorescence spectra of the system after adding ATP (0-60 μM) to DMSO / Hepes (10 mM, 9 / 1, v / v) solution. (b) Fluorescence spectra of TPA-3TPY-Zn at 510 nm. 2+ The emission intensity of the complex probe is compared with the standard calibration curve of different ATP concentrations. ex =375nm. (c) shows the reaction of TPA-3TPY with Zn under a handheld 365nm UV lamp. 2+ Diagram showing the changes in the formation of in situ complexes upon addition of ATP.

[0045] Figure 9 The metal complex fluorescent probe TPA-3TPY in urine samples 496nm / I 510nm Working curve between intensity and different ATP concentrations (20-60 μM).

[0046] Figure 10 For the fluorescent ligand TPA-3CHO 1 H NMR spectrum, using CDCl3 as solvent.

[0047] Figure 11 For the fluorescent ligand TPA-3TPY1 H NMR spectrum, using CDCl3 as solvent.

[0048] Figure 12 For the fluorescent ligand TPA-3TPY 13 C NMR spectrum, the solvent used was CDCl3. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Example 1: Synthesis of triphenylamine-trialdehyde ligand (TPA-3CHO)

[0051] As shown in Synthesis Route 1, under nitrogen, POCl3 (9.5 mL, 101.9 mmol) was added dropwise to DMF (7.26 mL, 93.8 mmol) at 0 ° C, and the reaction mixture was stirred for 1 hour. Triphenylamine (1.0 g, 4.08 mmol) was added to the reaction system and stirred for 4 hours at 95 ° C. After the reaction solution was cooled to room temperature, it was poured into 200 mL of ice water and adjusted to alkalinity with 1 M NaOH solution. It was then extracted with 200 mL of dichloromethane, and the organic phase was washed three times with 50 mL of water, finally dried over anhydrous sodium sulfate and filtered.

[0052] The solvent was then removed under reduced pressure and a mixture of cooled POCl (7.6 mL, 81.5 mmol) and DMF (5.78 mL, 74.6 mmol) was added. The reaction mixture was stirred at 95° C. for 1.5 hours, and after naturally cooling to room temperature, it was slowly poured into 200 mL of ice water. 1 M sodium hydroxide solution was subsequently used to adjust the mixture to alkalinity. Extraction and separation were performed with 200 mL of dichloromethane, and the organic phase was washed three times with 50 mL of water and then dried over anhydrous sodium sulfate. After solvent evaporation, the crude product was purified by column chromatography to obtain TPA-3CHO (0.860 g, 95%) as a yellow solid. 1 H NMR (400MHz, Chloroform-d) δppm: 7.27 (d, J = 8.56 Hz, 6H) 7.87 (d, J = 8.44 Hz, 6H) 9.98 (s, 6H).

[0053]

[0054] Example 2: Synthesis of triphenylamine-terpyridine ligand (TPA-3TPY)

[0055] As shown in Synthesis Route 2, 0.20 g (0.6 mmol) of TPA-3CHO and 0.61 g (5.4 mmol) of 2-acetylpyridine were thoroughly stirred and mixed in 20 mL of ethanol. Subsequently, 0.20 g (4.9 mmol) of sodium hydroxide powder and 20 mL of ammonia solution were added to the mixed system. At this time, the solution showed an orange-yellow color and was continuously stirred for 72 hours at 25 ° C to ensure sufficient reaction. After the reaction was completed, the precipitate was separated from the solution by filtration. The precipitate was then washed with ether to remove impurities remaining on the surface. In order to further improve the purity of the product, the washed precipitate was dissolved in ethanol and purified by recrystallization. Finally, the fluorescent ligand TPA-3TPY was obtained, the mass of which was 76% of the calculated theoretical yield, showing a high synthesis efficiency. 1 H NMR(400MHz,Chloroform-d)sppm 7.32-7.44(m,12H)7.84-7.98(m,12H)8.65-8.83(m,18H). 13 C NMR (101MHz, CDCl3) δ118.39,118.51,121.36,123.75,124.53,128.25,128.49,136.86,148.52,149.14,149.73,155.89,156.40.

[0056]

[0057] Example 3 Study on UV Absorption and Fluorescence Spectra of Fluorescent Ligand TPA-3TPY in Solvents of Different Polarities

[0058] (1) Use an analytical balance to accurately weigh the specified mass of TPA-3TPY and dissolve it in 5 mL of dimethyl sulfoxide (DMSO) solution to prepare the TPA-3TPY test stock solution (1 mM). The stock solution should be stored in a refrigerator at 4°C away from light. The shelf life is 3 months.

[0059] (2) In a 3.5 mL four-sided light-transmitting quartz cuvette, 2 mL of toluene (Tol), dichloromethane (DCM), tetrahydrofuran (THF), ethanol (EtOH), N,N-dimethylformamide (DMF), acetonitrile (ACN), and dimethyl sulfoxide (DMSO) were added, and 20 μL of the probe stock solution was added to obtain a test solution with a concentration of 10 μM.

[0060] (3) Place the working solution in a UV-visible absorption spectrum with a scanned baseline, set the scan range to 250-550 nm, and obtain the UV-visible absorption spectra of TPA-3TPY in different solutions. Read the absorbance at the maximum absorption peak of TPA-3TPY in different solvents. The spectra are as follows: Figure 1 shown.

[0061] (4) Place the working solution in a fluorescence spectrometer, set the excitation wavelength to 375 nm, and scan the range from 395 nm to 750 nm to obtain the fluorescence spectra of TPA-3TPY in different solutions. Figure 2 The relevant absorption and emission parameters are shown in Table 1.

[0062] Table 1 Solvent effects of fluorescent ligand TPA-3TPY

[0063]

[0064]

[0065] Example 4 Study on Metal Ion Selectivity of Fluorescent Ligand TPA-3TPY

[0066] (1) Use an analytical balance to accurately weigh the specified mass of the metal cation salt compound, and use 10 mL of deionized water to mix Na + , K + Mg 2+ , Ca 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ 、Co 2+ 、Cd 2+ 、Ni 2+ , Pb 2+ 、Ba 2+ 、Yb 3+ 、Eu 3+ and Tb 3+ The metal cation stock solution was prepared to have a concentration of 10 mM. The stock solution was stored in a refrigerator at 4°C away from light and had a shelf life of 3 months.

[0067] (2) 2 mL of DMSO / HEPES (10 mM, 9 / 1, v / v) solution was added to a 3.5 mL four-sided transparent quartz cuvette, followed by 20 μL of TPA-3TPY stock solution to prepare a fluorescent probe molecule with a detection concentration of 10 mM. Subsequently, stock solutions of various metal ions, including Na + , K + Mg 2+, Ca 2+ 、Cu 2 + 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ 、Co 2+ 、Cd 2+ 、Ni 2+ , Pb 2+ 、Ba 2+ 、Yb 3+ 、Eu 3+ and Tb 3+ , a total of 17 different metal ions.

[0068] (3) Place the working solution in a fluorescence spectrometer, set the excitation wavelength to 375 nm, scan range to 395 nm to 800 nm, and record the fluorescence spectrum of the TPA-3TPY fluorescent ligand and the fluorescence spectrum under ultraviolet light. Figure 3 shown.

[0069] (4) From Figure 3 It can be observed intuitively that Cu 2+ 、Zn 2+ 、Co 2+ and Cd 2+ ions significantly weakened the fluorescence intensity of TPA-3TPY, especially Cu 2+ The addition of ions completely quenches the ligand fluorescence. 2+ The addition of not only led to a weakening of the fluorescence intensity, but also triggered an obvious wavelength red shift. The positive charge carried by the metal ions probably promoted the intramolecular charge transfer (ICT) between TPA and TPY, leading to this phenomenon.

[0070] Example 5 Study on the responsiveness of the fluorescent ligand TPA-3TPY to ATP

[0071] (1) Use an analytical balance to accurately weigh the specified mass of the metal cation salt compound, and use 10 mL of deionized water to mix Na + , K + Mg 2+ , Ca 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ 、Co 2+ 、Cd 2+ 、Ni 2+ , Pb 2+ 、Ba 2+ 、Yb 3+、Eu 3+ and Tb 3+ The metal cation stock solution was prepared to have a concentration of 10 mM. The stock solution was stored in a refrigerator at 4°C away from light and had a shelf life of 3 months.

[0072] (2) 2 mL of DMSO / HEPES (10 mM, 9 / 1, v / v) solution was added to a 3.5 mL four-sided transparent quartz cuvette, followed by 20 μL of TPA-3TPY stock solution to prepare a detection concentration of 10 mM fluorescent ligand molecules. Subsequently, stock solutions of various metal ions, including Na + , K + Mg 2+ , Ca 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ 、Co 2+ 、Cd 2+ 、Ni 2+ , Pb 2+ 、Ba 2+ 、Yb 3+ 、Eu 3+ and Tb 3+ , a total of 17 different metal ions.

[0073] (3) Place the working solution in a fluorescence spectrometer, set the excitation wavelength to 375 nm, scan range to 395 nm to 800 nm, and record the fluorescence of TPA-3TPY-M n+ Fluorescence intensity of the metal complex probe at 510 nm.

[0074] (4) Subsequently, 50 μM ATP stock solution was added to the above solutions containing various metal ions. The working solution was placed in a fluorescence spectrometer, the excitation wavelength was set to 375 nm, the scanning range was 395 nm to 800 nm, and the TPA-3TPY-M n+ +The fluorescence intensity of ATP fluorescent ligand at 510nm. Figure 4 shown.

[0075] (5) Figure 4 As revealed, Cu 2+ 、Zn 2+ 、Co 2+ and Cd 2+ ions interacted with the TPA-3TPY ligand, especially Cu 2+ The addition of ions almost completely quenched the fluorescence of the ligand.2+ 、Cd 2+ 、Co 2+ The introduction of TPA-3TPY-Zn 2+ 、TPA-3TPY-Cd 2+ and TPA-TPY-Zn 2+ The metal complexes all showed fluorescence quenching at 510 nm. As expected, ATP and Zn 2+ The strong affinity between TPA-3TPY-Zn 2+ Zn in the complex 2+ The ions are replaced, which frees the fluorescent ligand and restores the fluorescence signal, thus achieving specific fluorescence detection of ATP. Therefore, our subsequent research mainly focuses on TPA-3TPY-Zn 2+ complex.

[0076] Example 6 Probe ligand TPA-3TPY binds to Zn 2+ Job's Plot Curve Study

[0077] By measuring the probe ligand TPA-3TPY binding to Zn 2+ Job's Plot working curve to determine the probe ligand TPA-3TPY and Zn 2+ The specific operation is as follows:

[0078] (1) Use an analytical balance to accurately weigh the specified mass of TPA-3TPY and dissolve it in 5 mL of dimethyl sulfoxide (DMSO) solution to prepare the TPA-3TPY test stock solution (1 mM). The stock solution should be stored in a refrigerator at 4°C away from light. The shelf life is 3 months.

[0079] (2) Add 2 mL of DMSO solution to 11 3.5 mL four-sided transparent quartz cuvettes and add different concentrations of probe ligand TPA-3TPY and detection substance Zn 2+ , keeping the total concentration of probe ligand and ATP at 20 μM, changing the concentration of fluorescent probe ligand and Zn 2+ Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 375 nm and the detection wavelength range to 395 nm to 750 nm. Obtain the fluorescence spectrum of the probe.

[0080] (3) Record the fluorescence intensity of the above ligand TPA-3TPY fluorescence spectrum at 510nm to obtain a series of 2+ The fluorescence intensity is related to the concentration. The ratio is the vertical axis, and [Zn 2+ ] / ([Zn 2+]+[TPA-3TPY]) as the horizontal axis, and the Job's Plot curve is obtained by fitting, such as Figure 5 shown.

[0081] (4) Figure 5 As clearly shown, when Zn 2+ The concentration ratio of ions ([Zn 2+ ] / ([Zn 2+ ]+[TPA-3TPY])) reaches 0.75, the fluorescence intensity drops to the lowest point. This key finding strongly proves that the fluorescent ligand TPA-3TPY and Zn 2+ The binding stoichiometric ratio between ions is 1:3. In view of this, we decided to use TPA-3TPY and Zn in the subsequent ATP sensing study. 2+ ions in situ formed TPA-3TPY-Zn in a 1:3 ratio 2+ The complex serves as a probe.

[0082] Example 7 Kinetic Study on Detection of Adenosine Triphosphate (ATP) by Metal Complex TPA-3TPY-Zn

[0083] To test the optimal kinetic response time of the metal complex TPA-3TPY-Zn for detecting ATP, the fluorescence intensity changes of the metal complex TPA-3TPY-Zn and ATP in a DMSO / HEPES (10mM, 9 / 1, v / v) system over time were studied. The specific operation is as follows:

[0084] (1) Use an analytical balance to accurately weigh the specified mass of TPA-3TPY and dissolve it in 5 mL of DMSO solution to prepare the TPA-3TPY test stock solution (1 mM). The stock solution should be stored in a refrigerator at 4°C away from light. The shelf life is 3 months.

[0085] (2) Add 2 mL of DMSO / HEPES (10 mM, 9 / 1, v / v) solution to a 3.5 mL four-sided transparent quartz cuvette, add 20 μL of probe ligand TPA-3TPY stock solution and 6 μL of Zn 2+ Add 50 μL of the probe's stock solution and 50 μL of the ATP stock solution. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 375 nm and the detection wavelength range to 395 nm to 750 nm. Obtain the probe's fluorescence spectrum.

[0086] (3) The fluorescence intensity of the metal complex TPA-3TPY-Zn at 510 nm during the reaction time when it binds to ATP is recorded, and a series of fluorescence intensity data related to time is obtained. The ratio is used as the ordinate and the reaction time is used as the abscissa to fit the fluorescence curve of the metal complex TPA-3TPY-Zn binding to ATP along the response time, as shown in FIG. Figure 6 shown.

[0087] (4) Figure 6 As shown in the figure, with the addition of ATP, the fluorescence quickly recovered within 30 seconds. The entire sensing process took less than 40 seconds, demonstrating extremely high efficiency.

[0088] Example 8 Study on the Selectivity and Anti-interference of Metal Complex TPA-3TPY-Zn to ATP

[0089] In order to test the selectivity and anti-interference of the metal complex TPA-3TPY-Zn to ATP, the fluorescence response of the probe molecule to different types of anions and homologues was tested. The test objects included anions (F - 、Cl - , I - 、AcO - 、NO2 - 、HCO3 - 、CO3 2- 、SO4 2- 、HPO4 2- 、H2PO4 - PO4 3- ) and homologues (ADP, AMP, CTP, GTP, UTP, PPi). The detection concentration of anions is 100 μM, the detection concentration of homologues and ATP is 50 μM, and the working concentration of the probe molecule is 10 μM. The specific operation process is as follows:

[0090] (1) Prepare a 10 mM metal ion stock solution. Weigh the appropriate amount of material into a 5 mL volumetric flask. Dissolve the solution in approximately 4 mL of deionized water. Continue adding deionized water to the mark on the flask. Shake well and store in a refrigerator until ready for use.

[0091] (2) Place 2 mL of DMSO / HEPES (10 mM, 9 / 1, v / v) solution in a 3.5 mL four-sided transparent quartz cuvette. Add 20 μL of TPA-3TPY stock solution (1 mM) and 6 μL of Zn 2+ Prepare the stock solution (10 mM) and shake well. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 375 nm and the detection wavelength range to 395–750 nm. Obtain the initial fluorescence spectrum of the probe, recording the fluorescence intensity and fluorescence spectrum at 510 nm.

[0092] (3) Add 100 μM of various anions and 50 μM of homologues to the cuvettes in (2) to obtain a series of test solutions for detecting anions and homologues. Test the fluorescence spectra of the above test solutions and record the fluorescence spectra, such as Figure 7 As shown in AB.

[0093] (4) Record the fluorescence intensity at 510 nm in the above fluorescence spectrum to obtain the fluorescence intensity at 510 nm of the metal complex TPA-3TPY-Zn in various interference environments. Use the fluorescence intensity as the ordinate and the different ions as the abscissa to draw an anti-interference graph, as shown in Figure 2. Figure 7 As shown in C.

[0094] (5) Figure 7 As shown in A, TPA-3TPY-Zn 2+ The complex is inert to all anions except ATP, and its fluorescence spectrum does not change. Only ATP molecules can restore the fluorescence characteristics of the TPA-3TPY ligand. This finding confirms that TPA-3TPY-Zn 2+ The complex has excellent selective response to ATP in the context of common anions. 2+ The ability of metal complexes to recognize ATP in the environment of homologues (ADP, AMP, CTP, UTP, GTP, PPi). Figure 7 As shown in B, given the high similarity of the structures of ATP and its derivatives, TPA-3TPY-Zn 2+ In addition, in order to verify the TPA-3TPY-Zn 2+ We conducted a fluorescence competition experiment to investigate the anti-interference performance of the complex in response to ATP. Figure 7 As shown in C, even in the presence of other common anion interferents, TPA-3TPY-Zn 2+ The complex probe's ability to sense ATP remains robust and is hardly affected by these potential interferents. 2+ The complex probe has excellent selective recognition ability for ATP, can maintain stable detection performance under the coexistence of multiple anions, and exhibits excellent anti-interference properties.

[0095] Example 9 Calculation of the detection limit of ATP by the metal complex TPA-3TPY

[0096] In order to test the fluorescence response characteristics of the metal complex TPA-3TPY to different concentrations of ATP, the experimental process was described in detail using DMSO / HEPES (10 mM, 9 / 1, v / v) solution as an example.

[0097] (1) Place 2 mL of DMSO / HEPES (10 mM, 9 / 1, v / v) solution in a 3.5 mL four-sided transparent quartz cuvette. Add 20 μL of the metal complex TPA-3TPY stock solution (1 mM), 6 μL of Zn2+ Prepare the stock solution (1 mM) and shake well. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 375 nm and the detection wavelength range to 395 nm to 750 nm. Obtain the initial fluorescence spectrum of the metal complex probe.

[0098] (2) 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, and 12 μL of ATP stock solution (10 mM) were added to the cuvette containing the metal complex TPA-3TPY to obtain test solutions containing 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, and 60 μM ATP. The fluorescence spectra of the above test solutions were tested (e.g. Figure 8 (as shown in A).

[0099] (3) Record the fluorescence intensity of the metal complex TPA-3TPY-Zn at 510 nm to obtain a series of fluorescence data related to ATP concentration. Use this value to make a scatter plot of ATP concentration, as shown in Figure 2. Figure 8 As shown in B.

[0100] (4) Linear fitting was performed on the linear range of 10B (5-40 μM), and the detection limit of the metal complex TPA-3TPY for ATP in DMSO / HEPES (10 mM, 9 / 1, v / v) solution was calculated based on 3σ / k.

[0101] (5) Figure 8 Figure A shows the change in fluorescence intensity of the system when the ATP concentration increases from 0 to 50 μM. It can be clearly seen that with the continuous addition of ATP, the fluorescence emission intensity at 510 nm continues to increase. It is particularly noteworthy that there is a good linear correlation between ATP concentration and fluorescence intensity, and its linear equation is y = 752.8571x-1541.2855, and R 2 =0.993(see Figure 8 B). Based on the calculation formula of LOD=3σ / k, we found that the minimum detection limit of TPA-3TPY-Zn complex for ATP can reach 0.20μM, and its linear response range is between 5 and 40μM. This research result fully demonstrates that TPA-3TPY-Zn 2+ The complex probe can achieve accurate quantitative detection of ATP and exhibits excellent sensitivity.

[0102] The ligand molecule was used at a working concentration of 10 μM in DMSO / HEPES (10 mM, 9 / 1, v / v) solution and 30 μM metal Zn 2+The ion complex formed a metal complex probe (TPA-3TPY). In an environment containing 0-50 μM ATP, the test solution was detected to change from green fluorescence to orange fluorescence, indicating the formation of the metal complex probe (TPA-3TPY); with the addition of ATP, the test solution was detected to change from orange fluorescence back to green fluorescence, indicating that Zn 2+ The metal complex probe complexed with ATP and detached from the original ligand, thus successfully detecting ATP. When using a DMSO / HEPES (10mM, 9 / 1, v / v) solution as the working medium, the fluorescence emission wavelength displayed by the metal complex probe when recognizing ATP gradually shifted from 610nm to 510nm. The linear response range for ATP was 5-40μM, and the detection limit was 0.2μM.

[0103] Example 10 Detection of ATP in actual human urine samples using the metal complex probe TPA-3TPY

[0104] To verify the effectiveness of metal complexes in practical applications, we used a spike-recovery method to quantitatively analyze ATP in human urine using the metal complex TPA-3TPY. The specific steps are as follows:

[0105] (1) Add 2 mL of DMSO / HEPES (10 mM, 9 / 1, v / v) solution to a 3.5 mL four-sided transparent quartz cuvette, and add 20 μL of the actual sample diluted 10 times. Add 20 μL of fluorescent ligand TPA-3TPY (1 mM) and 6 μL of Zn 2+ Prepare the stock solution (10 mM) and shake well. Place the cuvette in a fluorescence spectrometer. Set the excitation wavelength to 375 nm and the detection wavelength to 395–750 nm. Obtain the initial fluorescence intensity of the metal complex probe TPY-3TPY-Zn at 525 nm for the three actual samples.

[0106] (2) Add 2, 4, 6, 8, 10, and 12 μL of ATP stock solution (10 mM) to the cuvette to obtain test solutions with 10, 20, 30, 40, 50, and 60 μM ATP. Measure the fluorescence intensity of the above test solutions at 496 nm and 610 nm, and calculate I 496nm / I 610nm .

[0107] (3) Record the fluorescence intensity at 496 nm and 610 nm in the above fluorescence spectrum respectively, and calculate I 496nm / I 610nm Use the data as the vertical axis and ATP concentration as the horizontal axis to make a scatter plot, such as Figure 9 shown.

[0108] (4) Yes Figure 9 Linear fitting was performed in the linear range (20-60 μM) to obtain the standard curve equation.

[0109] (5) ATP standard solutions of known concentrations (25, 35, and 45 μM) were added to the actual sample diluted 10-fold. The fluorescence intensity of the probe was recorded and the ATP content in the actual sample was calculated according to the standard linear equation. This step was repeated three times. The test results are shown in Table 2.

[0110] (6) As can be seen from Table 2, the recovery rate of ATP is between 98.82% and 103.10%, and the relative standard deviation (RSD) is between 0.08% and 2.05%, which are significantly lower than the critical value of 4.0%. These data indicate that TPA-3TPY-Zn 2+ The error of metal complex detection of ATP is small, which confirms its reliability in detecting ATP in actual human urine.

[0111] Table 2 Detection of ATP in actual samples (n=3)

[0112]

[0113] Finally, it should be noted that the present invention is not limited to the above-mentioned embodiments. Any improvements made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. The triphenylamine-terpyridine fluorescent probe and / or its metal complex represented by the structural formula (I) has at least one of the following applications: (1) Detection of nucleotides and / or their derivatives; (2) Detection of chain polyphosphate and / or its derivatives; (3) Products for detecting nucleotides and / or their derivatives; (4) Products for detecting chain polyphosphoric acid and / or its derivatives; 2. At least one of the following products contains the triphenylamine-terpyridine fluorescent probe represented by the structural formula (I) and / or its metal complex: (1) Products for detecting nucleotides and / or their derivatives; (2) Products for detecting chain polyphosphoric acid and / or its derivatives.

3. The product according to claim 2, characterized in that The nucleotides include any one or any combination of nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates; the chain polyphosphates include any one or any combination of pyrophosphates and triphosphates; and the derivatives refer to derivatives obtained by replacing one or more atoms or groups in a nucleotide or chain polyphosphate with other atoms or groups, or derivatives with similar properties obtained by adjusting the structure of a nucleotide or chain polyphosphate without changing the position and type of the original substituents.

4. The product according to claim 2, characterized in that Metal complexes with metal ions include Na + , K + Mg 2+ , Ca 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ 、Co 2+ 、Cd 2+ 、Ni 2+ , Pb 2+ 、Ba 2+ 、Yb 3+ 、Eu 3+ 、Tb 3+ Any one or any combination of .

5. The product according to claim 2, characterized in that The product includes any one or any combination of detection reagents, kits, components, devices or equipment.

6. A detection method for detecting nucleotides and / or their derivatives and / or chain polyphosphates and / or their derivatives, characterized in that the steps include: The triphenylamine-terpyridine fluorescent probe and / or its metal complex represented by the structural formula (I) is used as a probe and mixed with the sample to be tested to form a detection system. Under 375nm excitation light, fluorescence in the range of 395nm to 750nm is collected to perform qualitative and / or quantitative detection of nucleotides and / or their derivatives and / or chain polyphosphates and / or their derivatives in the sample.

7. A detection method according to claim 6, characterized in that, When the triphenylamine-terpyridine fluorescent probe represented by the structural formula (I) is used as a probe, metal ions need to be added to the detection system to form a metal complex.

8. A detection method according to claim 6, characterized in that: Take I 510nm The fluorescence intensity or I 496nm / I 610nm The fluorescence intensity ratio of the sample and the concentration of the nucleotides and / or their derivatives and / or chain polyphosphates and / or their derivatives are used as the horizontal and vertical coordinates, and the external standard method, internal standard method or standard addition method are used to qualitatively and / or quantitatively determine the boron-containing compounds in the sample.

9. A detection method according to claim 6, characterized in that: The solvent used in the detection system includes any one or any combination of toluene, dichloromethane, tetrahydrofuran, ethanol, N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide.

10. A detection method according to claim 9, characterized in that: A buffer solution is also added to the detection system.

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