A method for simultaneously detecting Cu in mitochondria of cells + and Cu 2+ Surface enhanced Raman spectroscopy probe and its preparation and application

Abstract

The invention discloses a method for Cu in cell mitochondria. + and Cu 2+ A surface-enhanced Raman spectroscopy probe for simultaneous detection and its preparation method were designed and synthesized, and a Cu 2+ functionalized with an alkynyl group and having a specific recognition group was prepared. + Ligand and Cu 2+ Ligands; then the nano-Venus with good enhancement ability was prepared by the seed growth method; finally, the two ligands were co-modified with the reference molecule and the mitochondrial targeting molecule onto the nano-Venus through the gold acetylene bond to obtain a highly selective Cu + and Cu 2+ The present invention also discloses the application of the probe in detection in living cells. The surface enhanced Raman spectroscopy probe of the present invention has the advantage of being able to analyze multiple substances simultaneously, and can be used to analyze Cu in cell mitochondria. + and Cu 2+ Simultaneous detection and imaging to solve the problem of Cu + and Cu 2+ The surface enhanced Raman spectroscopy probe of the present invention plays an important role in realizing the simultaneous detection of multiple redox active substances in cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and relates to a method for simultaneously detecting Cu + and Cu 2+ Surface enhanced Raman spectroscopy probe and preparation method and application thereof. Background Art

[0002] Copper is an essential trace element that is vital for maintaining normal physiological functions of living organisms. Copper is mainly present in the living system in the form of reduced cuprous ions (Cu + ) and oxidized copper ions (Cu 2+ ) exists in the form of Cu 2+ / Cu + The reduction potential of the system is relatively high, and copper-containing enzymes act as oxidants in many key redox reactions. Mitochondria are the main site of redox reactions in cells and are also copper-rich organelles. Copper acts as a cofactor for cytochrome C oxidase to reduce oxygen to water in the last step of aerobic respiration, and is also involved in the formation of superoxide dismutase to resist oxidative stress. On the other hand, incomplete reduction of oxygen in mitochondria will produce ROS, and Cu + and Cu 2+ Abnormal homeostasis of mitochondrial copper may cause increased ROS levels, damage cells and ultimately lead to neurodegenerative diseases. Therefore, mitochondrial copper homeostasis is essential for maintaining oxygen metabolism in cells. + and Cu 2+ It is of great significance to correctly understand the pathological mechanism of neurodegenerative diseases. It is necessary to establish a method that can simultaneously detect Cu in cell mitochondria. + and Cu 2+ 's analysis strategy.

[0003] Currently, some fluorescent sensors have been reported for Cu + Cu 2+ However, the fluorescence spectrum peak is broad, and crosstalk may occur when multiple substances are detected simultaneously, so it is difficult to detect Cu by fluorescence method. + and Cu 2+ To achieve simultaneous detection, it is necessary to develop a method that can simultaneously detect Cu in cell mitochondria. + and Cu 2+ new method. Summary of the invention

[0004] In order to solve the shortcomings of the prior art, the present invention aims to provide a method for Cu + and Cu 2+ The surface enhanced Raman spectroscopy probe for simultaneous detection of Cu +Recognition ligand, Cu 2+ Recognition ligands, reference molecules and mitochondrial targeting molecules can be stably bound to nano-Venus with surface-enhanced Raman effect to prepare a nanostructured ... + and Cu 2+ The surface enhanced Raman spectroscopy probe of the present invention can be used to detect Cu in mitochondria of cells. + and Cu 2+ simultaneous determination.

[0005] The present invention innovatively proposes the inclusion of "Cu + Recognition ligand, Cu 2+ Surface-enhanced Raman spectroscopy probes with "ligand recognition" to simultaneously detect Cu + and Cu 2+ The technical concept is not easy to come up with, because it is impossible to directly obtain Cu + or Cu 2+ Raman spectrum, so it is necessary to use ligands to identify ions to complete the detection, but Cu + The recognition group of the ion has multiple sulfur atoms, which can easily lead to the aggregation of gold nanoparticles, so a stable modification interface needs to be constructed.

[0006] The present invention provides a method for simultaneously detecting Cu in cell mitochondria. + and Cu 2+ The surface-enhanced Raman spectroscopy probe can be used to detect Cu in mitochondria. + and Cu 2+ A highly selective, accurate and simultaneously quantitative surface-enhanced Raman spectroscopy probe comprising a nano-Volume 240 with a surface-enhanced Raman effect and a Cu ions co-modified and stably bound to the nano-Volume 240 through a gold-acetylene bond + Recognition ligand, Cu 2+ Identify ligands, reference molecules, and mitochondrial targeting molecules.

[0007] Among them, the Cu + Recognition ligand, Cu 2+ The recognition ligand is an alkyne-functionalized Cu with a specific recognition group. + Recognition ligand, Cu 2+ Identify ligands.

[0008] Among them, the Cu + The recognition ligands include but are not limited to Cu + The recognition ligand is one or more of ETPB, bathocuproine, and o-phenanthroline; preferably, ETPB.

[0009] Among them, the Cu 2+ The recognition ligands include but are not limited to Cu 2+The recognition ligand is one or more of PHDA, ethylenediaminetetraacetic acid, and bipyridine; preferably, PHDA.

[0010] The reference molecule includes, but is not limited to, one or more of 4-(triisopropylsilylethynyl)benzyne (TIPP), 4-ethynylbenzonitrile, and 4-mercaptobenzonitrile; preferably, TIPP.

[0011] The mitochondrial targeting molecule includes, but is not limited to, one or more of propargyltriphenylphosphonium bromide (PTPB), triphenylphosphine, and triphenylmethylphosphonium chloride; preferably, it is PTPB.

[0012] The probe of the present invention utilizes the specific recognition function of the ligand and the ligand binding Cu + and Cu 2+ The change of the fingerprint spectrum of surface enhanced Raman spectroscopy realizes the + and Cu 2+ The probe of the present invention can detect Cu + Cu 2+ It exhibits good linear response when detecting Cu + and Cu 2+ The probe of the present invention also has the characteristics of good selectivity and fast response speed. Combined with the surface enhanced Raman spectroscopy signal of the reference molecule, it can realize the detection of Cu + and Cu 2+ Accurate quantitative analysis.

[0013] The present invention also provides a method for preparing the surface enhanced Raman spectroscopy probe, firstly preparing a nano-Venus with surface enhanced Raman effect, synthesizing an alkyne-functionalized Cu with a specific recognition group, + Recognition ligand, Cu 2+ Identify ligands, screen reference molecules, and transfer the Cu + Recognition ligand, Cu 2+ The recognition ligand, reference molecule and mitochondrial targeting molecule are co-modified onto the nano-Volume 24 by gold-acetylene bonds to prepare a highly selective Cu + and Cu 2+ The present invention designs and synthesizes highly selective Cu + The recognition ligand, Cu 2+ The recognition ligand is identified, a stable internal reference molecule is screened out, and the aforementioned three molecules are co-modified with the mitochondrial targeting molecule onto the nano Venus to form the probe.

[0014] In the method for preparing a surface enhanced Raman spectroscopy probe of the present invention, Cu is added to the nano-gold stars. + Recognition ligand, Cu 2+The recognition ligand, the reference molecule and the mitochondrial targeting molecule are mixed in an ethanol solution and stirred for reaction, wherein the Cu + Recognition ligand, Cu 2+ The concentrations of recognition ligand, reference molecule and mitochondrial targeting molecule are 1-100 μM; + Recognition ligand, Cu 2+ The volumes of the recognition ligand, reference molecule and mitochondrial targeting molecule are 1-10 μL; the reaction temperature is 0-100° C.; and the stirring time is 1-5 hours.

[0015] Specifically, the preparation of the nano-gold stars with surface enhanced Raman effect comprises the following steps:

[0016] Step (1): stirring a sodium citrate solution and a chloroauric acid solution to obtain a gold seed solution;

[0017] Step (2): Mix and stir the gold seed solution, chloroauric acid solution, silver nitrate solution, ascorbic acid solution and dilute hydrochloric acid prepared in step (1) to obtain the nano-gold stars with surface enhanced Raman effect.

[0018] Specifically, the Cu + Preparation of recognition ligand: First, bis(2-((2-(2-(ethylthio)ethyl)thio)ethyl)amine and methyl 4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoate were synthesized separately, and then the two were connected by halogenation reaction, and finally reacted with propargylamine to obtain Cu + Identify ligands.

[0019] Specifically, the Cu 2+ Preparation of recognition ligand: First, 3-alkyne-1-butanol and bromopropyne are coupled to obtain 3,6-diyne-1-heptanol, then the hydroxyl group is replaced by a bromine atom, and finally a halogenation reaction is carried out with dimethylpyridiniumamine to obtain Cu 2+ Identify ligands.

[0020] In particular, the reference molecule is a commercial molecule.

[0021] Specifically, the mitochondrial targeting molecule is a commercially available molecule.

[0022] The present invention also provides a Cu-containing compound prepared by the method for use in cell mitochondria. + and Cu 2+ Simultaneous detection of surface-enhanced Raman spectroscopy probes.

[0023] The present invention also proposes to use the surface enhanced Raman spectroscopy probe to detect Cu in cell mitochondria. + and Cu 2+ The method of simultaneous detection. Specifically, the Cu+ , Cu 2+ The specific detection steps are:

[0024] The surface enhanced Raman spectroscopy probe prepared in the above steps was dispersed into 1 mL of phosphate buffer, and 1 μL of gradient concentrations of Cu + Cu 2+ solution, and then detect the surface enhanced Raman spectroscopy of the probe: With the addition of Cu + As the volume of the solution increases, the surface enhanced Raman spectrum at 2215 cm -1 The peak intensity of 2155cm -1 The peak intensity of Cu + Concentration and peak intensity ratio I 2215 / I 2155 It exhibits good linearity in the range of 0.5-14 μM, and the linear equation is I 2215 / I 2155 =-0.055*[Cu + ]+2.130, the detection limit was 0.32μM; with the addition of Cu 2+ As the volume of the solution increases, the surface enhanced Raman spectrum at 2236 cm -1 The peak intensity of 2155cm -1 The peak intensity of Cu 2+ Concentration and peak intensity ratio I 2236 / I 2155 It exhibits good linearity in the range of 0.5-14 μM, and the linear equation is I 2236 / I 2155 =-0.022*[Cu 2 + ] + 0.984, and the detection limit was 0.44 μM. The surface enhanced Raman spectroscopy probe prepared in the above steps was then dispersed into 1 mL of phosphate buffer, and 1 μL of gradient concentrations of Cu + and Cu 2+ solution, and then detect the surface enhanced Raman spectroscopy of the probe: With the addition of Cu + and Cu 2+ As the volume of the solution increases, the surface enhanced Raman spectrum at 2215 cm -1 and 2236cm -1 The peak intensity of 2155cm -1 The peak intensity of Cu + and Cu 2+ Concentration and peak intensity ratio I 2215 / I 2155 ,I 2236 / I 2155It shows good linearity in the range of 0.5-14 μM, and the slope coefficient of the linear curve is consistent with that of the single detection, indicating that the surface enhanced Raman spectroscopy probe is effective in detecting Cu + and Cu 2+ There is no crosstalk. Can be used for Cu + and Cu 2+ simultaneous detection.

[0025] The present invention also provides the surface enhanced Raman spectroscopy probe in exogenous Cu 2+ Detection of Cu in mitochondria during stimulation + and Cu 2+ Application of concentration changes.

[0026] The present invention also proposes that the surface enhanced Raman spectroscopy probe is used to detect Cu in cell mitochondria. + and Cu 2+ Simultaneous detection and / or monitoring of mitochondrial Cu in cells for non-diagnostic purposes + and Cu 2+ concentration changes, and / or Cu in mitochondria of cells for non-diagnostic purposes + and Cu 2+ Quantitative analysis of exogenous Cu for non-diagnostic purposes 2+ Real-time detection of Cu in mitochondria in a stimulation model + and Cu 2+ application.

[0027] The present invention also provides a Cu + A method for preparing the recognition ligand ETPB, the preparation method comprising the following steps:

[0028] Step (1), 2-methylmercaptoethanol, thiourea and hydrobromic acid are mixed and stirred to obtain 2-methylmercaptoethanethiol; the reaction process is shown in reaction formula (1):

[0029]

[0030] Step (2), mixing the 2-methylthioethanethiol prepared in step (1) with bis(2-chloroethyl)amine in anhydrous ethanol to obtain bis(2-((2-(2-(ethylthio)ethyl)thio)ethyl)amine; the reaction process is shown in reaction formula (2):

[0031]

[0032] Step (3), methyl 4-ethynylbenzoate, 1-(chloromethyl)-4-ethynylbenzene, cuprous chloride and tetramethylethylenediamine are mixed and stirred in acetone to obtain methyl 4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoate; the reaction process is shown in reaction formula (3):

[0033]

[0034] Step (4), stirring the bis(2-((2-(2-(ethylthio)ethyl)thio)ethyl)amine prepared in step (2), the 4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoic acid methyl ester prepared in step (3) and potassium carbonate in acetonitrile to obtain 4-((4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)phenyl)butane-1,3-diyn-1-yl)benzoic acid methyl ester; the reaction process is shown in reaction formula (4):

[0035]

[0036] Step (5), mixing the methyl 4-((4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)phenyl)butane-1,3-diyn-1-yl)benzoate prepared in step (4), propargylamine and trimethylaluminum in dichloromethane to obtain Cu + Ligand ETPB; the reaction process is shown in reaction formula (5):

[0037]

[0038] The preparation of Cu + The overall reaction formula for recognizing the ligand ETPB is shown in the following formula (A):

[0039]

[0040] In step (1), the molar ratio of 2-methylmercaptoethanol, thiourea and hydrobromic acid is (0.5-2): (0.5-2): (0.5-2); preferably, it is 1:1:2.

[0041] In step (1), the reaction temperature is 100-200°C; preferably, 100°C.

[0042] In step (1), the reaction time is 12-24 hours; preferably, 12 hours.

[0043] In step (1), a post-treatment process is also included after the reaction: after the reaction is completed, dichloromethane is extracted and then dichloromethane is evaporated to remove.

[0044] In step (2), the molar ratio of 2-methylmercaptoethanethiol to bis(2-chloroethyl)amine is (0.5-5):(0.5-2); preferably, it is 3:1.

[0045] In step (2), the amount of anhydrous ethanol added is 50-100 mL; preferably, 100 mL.

[0046] In step (2), the reaction temperature is 50-100°C; preferably, 80°C.

[0047] In step (2), the reaction time is 12-24 hours; preferably, 24 hours.

[0048] In step (3), the molar ratio of methyl 4-ethynylbenzoate, 1-(chloromethyl)-4-ethynylbenzene, cuprous chloride and tetramethylethylenediamine is (0.5-2):(0.5-2):(0.5-2):(0.5-2); preferably, it is 2:1:1:2.

[0049] In step (3), the amount of acetone added is 20-50 mL; preferably, 50 mL.

[0050] In step (3), the reaction temperature is 0-50°C; preferably, 0°C.

[0051] In step (3), the reaction time is 2-4 hours; preferably, 2 hours.

[0052] In step (4), the molar ratio of bis(2-((2-(2-(ethylthio)ethyl)thio)ethyl)amine, methyl 4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoate and potassium carbonate is (0.5-2):(0.5-2):(0.5-2); preferably, it is 1:1.2:2.

[0053] In step (4), the amount of acetonitrile added is 20-50 mL; preferably, 50 mL.

[0054] In step (4), the reaction temperature is 50-100°C; preferably, 80°C.

[0055] In step (4), the reaction time is 12-24 hours; preferably, 24 hours.

[0056] In step (5), the molar ratio of methyl 4-((4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)phenyl)but-1,3-diyn-1-yl)benzoate, propargylamine and trimethylaluminum is (0.5-2):(0.5-2):(0.5-2); preferably, it is 1:2:2.

[0057] In step (5), the amount of dichloromethane added is 10-20 mL; preferably, 10 mL.

[0058] In step (5), the reaction temperature is 0-50°C; preferably, 0°C.

[0059] In step (5), the reaction time is 1-2 hours; preferably, 1 hour.

[0060] The present invention also provides a Cu 2+ A method for preparing a recognition ligand PHDA, the preparation method comprising the following steps:

[0061] Step (1), 3-alkyne-1-butanol, propyne bromide, potassium iodide, cesium carbonate and cuprous iodide are mixed and stirred in N,N-dimethylformamide to obtain 3,6-diyne-1-heptanol; the reaction process is shown in reaction formula (1):

[0062]

[0063] Step (2), mixing the 3,6-diyne-1-heptanol, triphenylphosphine and carbon tetrabromide prepared in step (1) in dichloromethane to obtain 7-bromo-1,4-heptadiyne; the reaction process is shown in reaction formula (2):

[0064]

[0065] Step (3), mixing the 7-bromo-1,4-heptadiyne, dimethylpyridinamine and potassium carbonate prepared in step (2) in acetonitrile and stirring to obtain Cu 2+ Recognize the ligand PHDA; the reaction process is shown in reaction formula (3):

[0066]

[0067]

[0068] The preparation of Cu 2+ The overall reaction formula for recognizing the ligand PHDA is shown in the following formula (B):

[0069]

[0070] In step (1), the molar ratio of 3-alkyne-1-butanol, propyne bromide, potassium iodide, cesium carbonate and cuprous iodide is (0.5-2):(0.5-2):(0.5-2):(0.5-2):(0.5-2); preferably, it is 1:1:1:1:1.

[0071] In step (1), the amount of N,N-dimethylformamide added is 20-50 mL; preferably, 20 mL.

[0072] In step (1), the reaction temperature is 20-50°C; preferably, 20°C.

[0073] In step (1), the reaction time is 12-24 hours; preferably, 12 hours.

[0074] In step (2), the molar ratio of 3,6-diyne-1-heptanol, triphenylphosphine and carbon tetrabromide is (0.5-2):(0.5-2):(0.5-2); preferably, it is 1:1:1.

[0075] In step (2), the amount of dichloromethane added is 20-50 mL; preferably, 50 mL.

[0076] In step (2), the reaction temperature is 0-50°C; preferably, 0°C.

[0077] In step (2), the reaction time is 2-4 hours; preferably, 2 hours.

[0078] In step (3), the molar ratio of 7-bromo-1,4-heptadiyne, dimethylpyridinamine and potassium carbonate is (0.5-2):(0.5-2):(0.5-2); preferably, it is 1:1.2:2.

[0079] In step (3), the amount of acetonitrile added is 20-50 mL; preferably, 50 mL.

[0080] In step (3), the reaction temperature is 50-100°C; preferably, 80°C.

[0081] In step (3), the reaction time is 12-24 hours; preferably, 24 hours.

[0082] The present invention also provides a method for preparing nano-gold stars, the preparation method comprising the following steps:

[0083] (1) stirring a sodium citrate solution and a chloroauric acid solution to obtain a gold seed solution;

[0084] (2) Mixing and stirring the gold seed solution, chloroauric acid solution, silver nitrate solution, ascorbic acid solution and dilute hydrochloric acid prepared in step (1) to obtain nano-gold stars.

[0085] In step (1), the concentration of the sodium citrate solution is 1%-5%; preferably, 1%.

[0086] In step (1), the volume of the sodium citrate solution is 5-15 mL; preferably, 15 mL.

[0087] In step (1), the concentration of the chloroauric acid solution is 0.1-0.5 mM; preferably, 0.25 mM.

[0088] In step (1), the volume of the chloroauric acid solution is 50-200 mL; preferably, 100 mL.

[0089] In step (1), the reaction temperature is 50-100°C; preferably, 100°C.

[0090] In step (1), the reaction time is 0.5-2 hours; preferably, 0.5 hours.

[0091] In step (2), the volume of the gold seed solution is 0.1-0.5 mL; preferably, 0.5 mL.

[0092] In step (2), the concentration of the chloroauric acid solution is 0.1-0.25 mM; preferably, 0.25 mM.

[0093] In step (2), the volume of the chloroauric acid solution is 50-200 mL; preferably, 50 mL.

[0094] In step (2), the concentration of the silver nitrate solution is 1-5 mM; preferably, 2 mM.

[0095] In step (2), the volume of the silver nitrate solution is 0.1-0.5 mL; preferably, 0.5 mL.

[0096] In step (2), the concentration of the ascorbic acid solution is 0.1-0.5M; preferably, 0.1M.

[0097] In step (2), the volume of ascorbic acid is 0.1-0.5 mL; preferably, 0.25 mL.

[0098] In step (2), the concentration of the dilute hydrochloric acid is 0.1-1M; preferably, 1M.

[0099] In step (2), the volume of the dilute hydrochloric acid is 10-100 μL; preferably, 30 μL.

[0100] In step (2), the reaction temperature is 0-50°C; preferably, 25°C.

[0101] In step (2), the reaction time is 1-10 minutes; preferably, 2 minutes.

[0102] The present invention also provides a method for using Cu in cell mitochondria. + and Cu 2+ A method for preparing a surface enhanced Raman spectroscopy probe for simultaneous detection, wherein the preparation method comprises adding Cu to nano-gold stars under nitrogen protection conditions. + Recognition ligands ETPB, Cu 2+The recognition ligand PHDA, the reference molecule TIPP and the mitochondrial targeting molecule PTPB are prepared by stirring an ethanol solution.

[0103] Among them, the Cu + Recognition ligands ETPB, Cu 2+ The concentrations of the recognition ligand PHDA, the reference molecule TIPP and the mitochondrial targeting molecule PTPB are 1-100 μM, respectively; preferably, 1 μM.

[0104] Among them, the Cu + Recognition ligands ETPB, Cu 2+ The volumes of the recognition ligand PHDA, the reference molecule TIPP and the mitochondrial targeting molecule PTPB are 1-10 μL, respectively; preferably, 1 μL.

[0105] Wherein, the reaction temperature is 0-100°C; preferably, 60°C.

[0106] The stirring time is 1-5 hours; preferably, 2 hours.

[0107] The present invention also provides Cu prepared by the above preparation method + Recognition of the ligand ETPB.

[0108] The present invention also provides Cu prepared by the above preparation method 2+ Recognizes the ligand PHDA.

[0109] The present invention also provides the Cu + Recognition ligands ETPB and Cu 2+ The recognition ligand PHDA was prepared and simultaneously detected Cu + and Cu 2+ Application in sensors and / or surface enhanced Raman spectroscopy probes.

[0110] The beneficial effects of the present invention include: the present invention discloses a method for Cu + and Cu 2+ A surface-enhanced Raman spectroscopy probe for simultaneous detection was designed and synthesized, and a Cu 2+ functionalized with an alkyne group and a specific recognition group was designed and synthesized. + Ligand and Cu 2 + Ligands; then the nano-Venus with good enhancement ability was prepared by the seed growth method; finally, the two ligands were co-modified with the reference molecule and the mitochondrial targeting molecule onto the nano-Venus through the gold acetylene bond to obtain a highly selective Cu + and Cu 2+ The present invention firstly prepares a nano-gold star with surface enhanced Raman effect, and then synthesizes the specific Cu +Recognition ligands ETPB and Cu 2+ Recognizes the ligand PHDA by specific Cu + Recognition ligands ETPB, Cu 2+ The recognition ligand PHDA, the reference molecule TIPP and the mitochondrial targeting molecule were modified onto nano-Venus to construct a nanostructured molecule that can simultaneously detect Cu + and Cu 2+ The probe construction proposed by the present invention and its application in the simultaneous detection of Cu + and Cu 2+ The present invention also discloses the application of the probe in detection of living cells. + and Cu 2+ The surface-enhanced Raman spectroscopy probe has the advantage of being able to analyze multiple substances simultaneously and can be used to analyze Cu in cell mitochondria. + and Cu 2+ Simultaneous detection and imaging can also be used for exogenous Cu 2+ Detection of Cu in cell mitochondria under stimulation + and Cu 2+ concentration changes, and can also solve the problem of Cu + and Cu 2+ The surface enhanced Raman spectroscopy probe and the detection method and application thereof of the present invention play an important role in realizing the simultaneous detection of multiple redox active substances in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 This is a transmission electron microscope picture of the nano-gold stars prepared in the present invention.

[0112] Figure 2 The Cu prepared by the present invention + and Cu 2+ Surface enhanced Raman spectra of the identification ligands ETPB (I), PHDA (II), reference molecule TIPP (III), and mitochondrial targeting molecule PTPB (IV) modified individually and together (V) on nano-Venus.

[0113] Figure 3 The surface enhanced Raman spectroscopy probe prepared by the present invention is used for Cu + When detected separately, different Cu + Surface enhanced Raman spectra of Cu 2+ When detected separately, different Cu 2+ Surface enhanced Raman spectra under different concentrations. The small figure is the linear fitting curve of the concentration response curve (B); Cu +and Cu 2+ Simultaneous detection of different Cu + , Cu 2+ Surface enhanced Raman spectroscopy (C) and different Cu + Concentration, Cu 2+ Concentration to peak intensity ratio I 2215 / I 2155 ,I 2236 / I 2155 The linear fitting curve (D).

[0114] Figure 4 The surface enhanced Raman spectroscopy probe prepared by the present invention is used for Cu + and Cu 2+ The selectivity and competition experiments of common metal ions (Na + , Fe 2+ , Fe 3+ 、Zn 2+ ), amino acids (Glu, Cys), neurotransmitters (DA), metabolites (GSH), and common reactive oxygen species (O2 ·- , H2O2) alone (A) and with Cu + , Cu 2+ Interference when they coexist (B).

[0115] Figure 5 This is a transmission electron microscope picture of the surface enhanced Raman spectroscopy probe prepared by the present invention entering into the mitochondria of cells.

[0116] Figure 6 The surface enhanced Raman spectroscopy probe prepared by the present invention is exposed to different concentrations of exogenous Cu 2+ (A: 0 μM; B: 100 μM) Surface enhanced Raman spectroscopy imaging and ratio change under stimulation (C). DETAILED DESCRIPTION

[0117] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0118] Example 1

[0119] Cu +Preparation of recognition ligand ETPB: 2-methylthioethanol (4.25 g, 40 mmol), thiourea (3.05 g, 40 mmol), and 48% hydrobromic acid (8.5 mL, 75 mmol) were mixed and stirred at 100°C for 12 hours to obtain 2-methylthioethanethiol (3.91 g) with a yield of 80%. 2-Methylthioethanethiol (3.91 g, 32 mmol) and bis(2-chloroethyl)amine (1.90 g, 11 mmol) were mixed in 100 mL of anhydrous ethanol and stirred at 80°C for 24 hours to obtain bis(2-((2-(ethylthio)ethyl)thio)ethyl)amine (2.69 g) with a yield of 78%. CuCl (1.00 g, 10 mmol), tetramethylethylenediamine (3.0 mL, 20 mmol), Methyl 4-ethynylbenzoate (3.20 g, 20 mmol) and 1-(chloromethyl)-4-ethynylbenzene (1.51 g, 10 mmol) were mixed in 50 mL of acetone and stirred at 0°C for 2 hours to give methyl 4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoate (0.99 g) with a yield of 32%. The mixture was stirred at 80 °C for 24 hours to give 4-((4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)phenyl)but-1,3-diyn-1-yl)benzoic acid. Methyl ester (1.08 g), yield 71%. Propargylamine (0.20 g, 3.6 mmol), trimethylaluminum (3.6 mL, 1 M in n-hexane) and methyl 4-((4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)phenyl)butane-1,3-diyn-1-yl)benzoate (1.08 g, 1.8 mmol) were mixed in 10 mL of dichloromethane and stirred at 0°C for 1 hour to obtain Cu + The recognition ligand ETPB (0.74 g) was obtained with a yield of 68%.

[0120] 1H NMR (500MHz, CDCl3) δ (ppm): 7.76 (d, J = 8.0Hz, 2H), 7.57 (d, J = 8.0Hz, 2H), 7.48 (d, J = 7.8Hz, 2H), 7.34 (d, J = 7.8Hz, 2H), 6.43 (t, J = 5.2Hz ,1H),4.24(dd,J=5.2,2.5Hz,2H),3.65(s,2H),2.80-2.61(m,16H),2.54(q,J=7.7Hz,4H),2.28(t,J=2.6Hz,1H),1.25(t,J=7.4Hz,6H).

[0121] 13 C NMR(500MHz, CDCl3)δ(ppm):166.14,133.79,132.64,132.60,128.81,127.13,125 .47,80.32,79.27,76.46,72.07,58.40,53.96,32.42,31.78,29.88,26.09,14.83.

[0122] Example 2

[0123] Cu 2+ Preparation of recognition ligand PHDA: 3-butyn-1-ol (0.70 g, 10 mmol), CuI (1.90 g, 10 mmol), KI (1.66 g, 10 mmol), Cs2CO3 (3.26 g, 10 mmol) and bromopropyne (1.19 g, 10 mmol) were mixed in 20 mL of N, N-dimethylformamide and stirred at 20°C for 12 hours to obtain 3,6-diyne-1-heptanol (0.70 g) with a yield of 65%. 3,6-diyne-1-heptanol (0.70 g, 6.5 mmol), triphenylphosphine (1.70 g, 6.5 mmol) and carbon tetrabromide (2.15 g, 6.5 mmol) were mixed in 50 mL of dichloromethane and stirred at 0°C for 2 hours to obtain 7-bromo-1,4-heptadiyne (0.88 g) with a yield of 80%. 7-Bromo-1,4-heptadiyne (0.88 g, 5.2 mmol), dimethylpyridinamine (1.23 g, 6.2 mmol) and K2CO3 (1.44 g, 10.4 mmol) were mixed in 50 mL of acetonitrile and stirred at 80 °C for 24 hours to obtain Cu 2+ Recognition ligand PHDA (0.65 g), yield 43%.

[0124] 1H NMR (500MHz, CDCl3) δ (ppm): 8.50 (d, J = 4.9Hz, 2H), 7.65 (t, J = 8.4Hz, 2H), 7.60 (d, J = 8.1Hz, 2H), 7.13 (t, J = 6.8Hz, 2H), 3.86 (s, 4H), 3.12 (q, J = 2.5Hz, 2H), 2.77 (t, J = 7.2Hz, 2H), 2.40 (tt, J = 7.3, 2.4Hz, 2H), 2.05 (t, J = 2.7Hz, 1H).

[0125] 13 C NMR (500MHz, CDCl3) δ (ppm): 159.43, 148.94, 136.51, 136.49, 122.98, 122.95, 122.04, 79.39, 78.67, 74.04, 68.55, 60.09, 52.86, 17.36, 9.61.

[0126] Example 3

[0127] Preparation of nano-Volume stars: 100 mL of 0.25 mM HAuCl4·3H2O and 15 mL of 1% sodium citrate solution were mixed and stirred at 100°C for 0.5 h to obtain a gold seed solution. 50 mL of 0.25 mM HAuCl4·3H2O solution, 0.5 mL of the above gold seeds, 30 μL of 1 M HCl solution, 0.5 mL of 2 mM silver nitrate solution and 0.25 mL of 0.1 M ascorbic acid solution were mixed and stirred at 25°C for 2 min to obtain a nano-Volume star solution.

[0128] Figure 1 This is a transmission electron microscope image of the nano-gold stars prepared in Example 3 of the present invention.

[0129] Example 4

[0130] 1 μL of the 1 μM ETPB solution prepared in Example 1 of the present invention, the PHDA solution, TIPP solution and PTPB solution prepared in Example 2 of the present invention were added to 1 mL of the nano-Venus solution prepared in Example 3 of the present invention, and the dissolved oxygen in the mixed solution was discharged by nitrogen bubbling for 30 minutes. Then, under nitrogen protection, the mixture was stirred at 60° C. for 2 hours, and the resulting solution was centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded to remove excess molecules in the solution, thereby obtaining a surface enhanced Raman spectroscopy probe.

[0131] Figure 2 The Cu prepared by the present invention + and Cu 2+Surface enhanced Raman spectra of the identification ligands ETPB, PHDA, reference molecule TIPP, and mitochondrial targeting molecule PTPB modified individually or together on nano-Venus.

[0132] Example 5

[0133] Surface-enhanced Raman spectroscopy probes for Cu + and Cu 2+ Simultaneous detection: The surface enhanced Raman spectroscopy probe prepared in Example 4 of the present invention was dispersed into 1 mL of phosphate buffer, and 1 μL of gradient concentrations of Cu + Cu 2+ solution, and then detect the surface enhanced Raman spectroscopy of the probe: With the addition of Cu + As the volume of the solution increases, the surface enhanced Raman spectrum at 2215 cm -1 The peak intensity of 2155cm -1 The peak intensity of Cu + Concentration and peak intensity ratio I 2215 / I 2155 It showed good linearity in the range of 0.5-14μM, with a detection limit of 0.32μM. 2+ As the volume of the solution increases, the surface enhanced Raman spectrum at 2236 cm -1 The peak intensity of 2155cm -1 The peak intensity of Cu 2+ Concentration and peak intensity ratio I 2236 / I 2155 It showed good linearity in the range of 0.5-14 μM, and the detection limit was 0.44 μM. The above surface enhanced Raman spectroscopy probe was then dispersed into 1 mL of phosphate buffer, and 1 μL of gradient concentrations of Cu + and Cu 2+ solution, and then detect the surface enhanced Raman spectroscopy of the probe: With the addition of Cu + and Cu 2+ As the volume of the solution increases, the surface enhanced Raman spectrum at 2215 cm -1 and 2236cm -1 The peak intensity of 2155cm -1 The peak intensity of Cu + and Cu 2+ Concentration and peak intensity ratio I 2215 / I 2155 ,I 2236 / I 2155 It exhibited good linearity in the range of 0.5-14 μM, and the slope coefficient of the linear curve was consistent with that of the single test.

[0134] Figure 3 The surface enhanced Raman spectroscopy probe prepared in Example 4 of the present invention is used for Cu + When detected separately, different Cu + Surface enhanced Raman spectra of Cu + Concentration and peak intensity ratio I 2215 / I 2155 It exhibits good linearity in the range of 0.5-14 μM, and the linear equation is I 2215 / I 2155 =-0.055*[Cu + ]+2.130, detection limit is 0.32μM; Cu 2+ When detected separately, different Cu 2+ concentration (B), the final Cu 2+ Concentration and peak intensity ratio I 2236 / I 2155 It exhibits good linearity in the range of 0.5-14 μM, and the linear equation is I 2236 / I 2155 =-0.022*[Cu 2+ ]+0.984, detection limit is 0.44μM; Cu + and Cu 2+ Simultaneous detection of different Cu + , Cu 2+ Surface enhanced Raman spectroscopy (C) and different Cu + Concentration, Cu 2+ Concentration to peak intensity ratio I 2215 / I 2155 ,I 2236 / I 2155 Linear fitting curve of Cu + and Cu 2+ There is no significant difference in the slope coefficient of the linear fitting curve when detecting simultaneously and when detecting separately, which proves that the surface enhanced Raman spectroscopy probe prepared in Example 4 of the present invention is effective in detecting Cu + and Cu 2+ There is no crosstalk and it can be used for simultaneous detection.

[0135] Example 6

[0136] Study on the selectivity of surface enhanced Raman spectroscopy probes: In order to explore the effects of common interferences on the simultaneous detection of Cu + and Cu 2+ The influence of surface enhanced Raman spectroscopy probes on common metal ions (Na + , Fe 2+ , Fe 3+ 、Zn 2 +), amino acids (Glu, Cys), neurotransmitters (DA), metabolites (GSH), and common reactive oxygen species (O2 ·- , H2O2) alone and with Cu + , Cu 2+ Interference when they coexist. + The concentration of is 100mM, the concentration of DA is 1μM, and the concentration of other substances is 10μM. Figure 4 It can be seen that A indicates that when the interference exists alone, the interference has an effect on Cu + and Cu 2+ The interference is less than 5.7%, B indicates that the interference is related to Cu + and Cu 2+ When coexisting, the interference + and Cu 2+ The interference is less than 4.3%. Figure 4 The dark gray block in A represents the interference ability of the current interferer when it exists alone. Figure 4 The dark gray blocks in B represent the current interference and Cu + Cu 2+ The ability to interfere when they exist simultaneously. Figure 6 The percentages in A represent the I obtained when the interferent is added alone 2215 / I 2155 Compared with adding Cu + Obtained I 2215 / I 2155 or the I obtained when the interferent is added alone 2236 / I 2155 Compared with adding Cu 2+ Obtained I 2236 / I 2155 ratio; Figure 6 The percentage in B represents the simultaneous addition of interferents and Cu + I 2215 / I 2155 Compared with adding Cu + Obtained I 2215 / I 2155 ratio, or adding interference and Cu 2+ I 2236 / I 2155 Compared with adding Cu 2+ Obtained I 2236 / I 2155 Ratio.

[0137] Example 7

[0138] Simultaneous detection of Cu + and Cu 2+ Surface-enhanced Raman spectroscopy probe for exogenous Cu2+ Cu in mitochondria stimulated by + and Cu 2+ Simultaneous detection: All animal experiments were approved by the Experimental Animal Ethics Committee of East China Normal University. C57BL / 6 mice used in the experiment were purchased from Shanghai Biological Model Biotechnology Development Co., Ltd. Newborn C57BL / 6 wild-type mice were anesthetized with halothane, and the brain tissue was quickly removed and immersed in Hank's balanced salt solution to separate the cortex part, digest and blow the tissue, and place it in ice water to obtain a suspension containing neurons. The obtained neuronal suspension was centrifuged and the supernatant was discarded. MEM culture medium was added to resuspend the neurons, and the neurons were inoculated into a glass-bottomed cell culture dish and placed in a cell culture incubator for 4 hours. The neurons adhered to the wall for about 4 hours, and then the plating liquid was discarded, and an appropriate amount of Neurobasal culture medium was added, and the dish was returned to the cell culture incubator for continued culture. The culture medium was replaced every 3 days, and the growth status of the neurons was observed under a microscope. Subsequent imaging experiments were performed 5 days after inoculation. Before the Raman confocal imaging experiment, the neurons were incubated with a culture medium containing 0.46 mg / mL of surface-enhanced Raman spectroscopy probe for 4 hours. Then the culture medium was discarded and the cells were washed three times with PBS to remove the probes that did not enter the cells. Finally, the culture dish was placed in a micro-incubator and placed under the objective lens of a confocal Raman spectrometer for cell imaging experiments.

[0139] Figure 5 This is a transmission electron microscope picture of the surface enhanced Raman spectroscopy probe prepared by the present invention entering into the mitochondria of cells.

[0140] Figure 6 The surface enhanced Raman spectroscopy probe prepared by the present invention is exposed to different concentrations of exogenous Cu 2+ (A: 0 μM; B: 100 μM) surface enhanced Raman spectroscopy imaging and ratio change (C) under stimulation. The results show that the surface enhanced Raman spectroscopy probe prepared in Example 4 of the present invention can accurately detect different concentrations of exogenous Cu 2+ Cu in mitochondria after stimulation + and Cu 2+ Proceed to imaging.

[0141] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A method for the detection of Cu in mitochondria + and Cu 2+ The surface enhanced Raman spectroscopy probe for simultaneous detection is characterized in that The invention comprises a nano-gold star having a surface enhanced Raman effect, and a Cu bonded to the nano-gold star through a gold acetylene bond co-modification. + Recognition ligand, Cu 2+ Identification ligand, reference molecule and mitochondrial targeting molecule; the Cu + Recognition ligand, Cu 2+ The recognition ligand is an alkyne-functionalized Cu with a specific recognition group. + Recognition ligand, Cu 2+ Recognizes ligands; wherein, The Cu + The recognition ligand is Cu + Recognizes the ligand ETPB; The Cu + Preparation of recognition ligand ETPB: First, bis(2-((2-(2-(ethylthio)ethyl)thio)ethyl)amine and methyl 4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoate were synthesized separately, and then the two were connected by halogenation reaction, and finally reacted with propargylamine to obtain Cu + Recognition ligand; the Cu + The structure of the recognition ligand ETPB is as follows: The Cu 2+ The recognition ligand is Cu 2+ Recognizes the ligand PHDA; The Cu 2+ Preparation of recognition ligand PHDA: First, 3-alkyne-1-butanol and bromopropyne were coupled to obtain 3,6-diyne-1-heptanol, and then the hydroxyl group was replaced by a bromine atom, and finally a halogenation reaction with dimethylpyridiniumamine was carried out to obtain Cu 2+ Recognition ligand; the Cu 2+ The structure of the recognition ligand PHDA is as follows: The reference molecule includes one or more of 4-(triisopropylsilylethynyl)benzylene TIPP, 4-ethynylbenzonitrile, and 4-mercaptobenzonitrile; The mitochondrial targeting molecule includes one or more of propargyltriphenylphosphonium bromide PTPB, triphenylphosphine, and triphenylmethylphosphonium chloride.

2. The method for preparing a surface enhanced Raman spectroscopy probe according to claim 1, characterized in that: The preparation method comprises: preparing nano-Venus with surface enhanced Raman effect by seed growth method, synthesizing Cu functionalized with alkyne group and having specific recognition group + Recognition ligand, Cu 2+ Identify ligands, screen reference molecules, and transfer the Cu + Recognition ligand, Cu 2+ The recognition ligand, reference molecule and mitochondrial targeting molecule are co-modified onto the nano-Volume 24 by gold-acetylene bonds to prepare a highly selective Cu + and Cu 2+ Simultaneously and accurately quantitate the probe.

3. The preparation method according to claim 2, characterized in that: In the method, Cu is added to the nano-gold stars. + Recognition ligand, Cu 2+ The recognition ligand, the reference molecule and the mitochondrial targeting molecule are mixed in an ethanol solution and stirred to react, wherein: The Cu + Recognition ligand, Cu 2+ The concentrations of recognition ligand, reference molecule, and mitochondrial targeting molecule were 1–100 μM; The Cu + Recognition ligand, Cu 2+ The volumes of recognition ligand, reference molecule, and mitochondrial targeting molecule were 1–10 μL, respectively; The reaction temperature is 0-100°C; The stirring time is 1-5 hours.

4. The method for preparing Cu in cell mitochondria obtained by the method of claim 2 or 3 + and Cu 2+ Simultaneous detection of surface-enhanced Raman spectroscopy probes.

5. A method for detecting Cu in mitochondria of cells using the surface enhanced Raman spectroscopy probe as claimed in claim 1 or 4 + and Cu 2+ Simultaneous detection method.

6. The method according to claim 5, characterized in that The detection method comprises the following steps: firstly, adding a culture medium containing a surface enhanced Raman spectroscopy probe into a culture dish incubating neuronal cells, and placing the dish in a cell culture incubator for incubation for 4 hours; then discarding the culture medium, washing the dish three times with a phosphate buffer to remove the probe that has not entered the neuron; and finally, placing the dish in a micro-culture incubator and placing it under the objective lens of a confocal Raman spectrometer to perform a cell imaging experiment.

7. A kind of Cu + The preparation method of the recognition ligand ETPB is characterized in that: The preparation method comprises the following steps: Step (1): mixing 2-methylmercaptoethanol, thiourea and hydrobromic acid to obtain a product, 12-methylmercaptoethanethiol; Step (2): mixing the 2-methylthioethanethiol prepared in step (1) with bis(2-chloroethyl)amine in anhydrous ethanol to obtain a product, 2-bis(2-((2-(2-(ethylthio)ethyl)thio)ethyl)amine; Step (3): Mixing methyl 4-ethynylbenzoate, 1-(chloromethyl)-4-ethynylbenzene, cuprous chloride and tetramethylethylenediamine in acetone and stirring to obtain the product 3-4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoate; Step (4): stirring the bis(2-((2-(2-(ethylthio)ethyl)thio)ethyl)amine prepared in step (2), 4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoic acid methyl ester prepared in step (3) and potassium carbonate in acetonitrile to obtain the product 4-((4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)phenyl)butane-1,3-diyn-1-yl)benzoic acid methyl ester; Step (5): Mix the methyl 4-((4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)phenyl)but-1,3-diyn-1-yl)benzoate prepared in step (4), propargylamine and trimethylaluminum in dichloromethane and stir to obtain Cu + Ligand ETPB; The reaction process of the preparation method is shown in reaction formula (A):

8. The preparation method according to claim 7, characterized in that: In the step (1), the molar ratio of 2-methylmercaptoethanol, thiourea and hydrobromic acid is (0.5-2): (0.5-2): (0.5-2); the reaction temperature is 100-200° C.; the reaction time is 12-24 hours; And / or, in the step (2), the molar ratio of 2-methylmercaptoethanethiol and bis(2-chloroethyl)amine is (0.5-5):(0.5-2); the amount of anhydrous ethanol added is 50-100 mL; the reaction temperature is 50-100° C.; and the reaction time is 12-24 hours; And / or, in the step (3), the molar ratio of methyl 4-ethynylbenzoate, 1-(chloromethyl)-4-ethynylbenzene, cuprous chloride and tetramethylethylenediamine is (0.5-2):(0.5-2):(0.5-2):(0.5-2); the amount of acetone added is 20-50 mL; the reaction temperature is 0-50° C.; and the reaction time is 2-4 hours; And / or, in the step (4), the molar ratio of bis(2-((2-(2-(ethylthio)ethyl)thio)ethyl)amine, methyl 4-((4-(chloromethyl)phenyl)butane-1,3-diyn-1-yl)benzoate and potassium carbonate is (0.5-2):(0.5-2):(0.5-2); the amount of acetonitrile added is 20-50 mL; the reaction temperature is 50-100° C.; and the reaction time is 12-24 hours; And / or, in the step (5), the molar ratio of methyl 4-((4-((bis(2-((2-(ethylthio)ethyl)thio)ethyl)amino)methyl)phenyl)but-1,3-diyn-1-yl)benzoate, propargylamine and trimethylaluminum is (0.5-2):(0.5-2):(0.5-2); the amount of dichloromethane added is 10-20 mL; the reaction temperature is 0-50° C.; and the reaction time is 1-2 hours.

9. Cu prepared by the method as claimed in claim 7 or 8 + Recognition of the ligand ETPB.

10. A kind of Cu 2+ The preparation method of recognition ligand PHDA is characterized in that: The preparation method comprises the following steps: Step (1): 3-alkyne-1-butanol, propyne bromide, potassium iodide, cesium carbonate and cuprous iodide are mixed and stirred in N,N-dimethylformamide to obtain a product 13,6-diyne-1-heptanol; Step (2): mixing the 3,6-diyne-1-heptanol, triphenylphosphine and carbon tetrabromide prepared in step (1) in dichloromethane and stirring to obtain the product 27-bromo-1,4-heptadiyne; Step (3): Mix the 7-bromo-1,4-heptadiyne, dimethylpyridinamine and potassium carbonate prepared in step (2) in acetonitrile and stir to obtain Cu 2+ Recognizes the ligand PHDA; The reaction process of the preparation method is shown in reaction formula (B):

11. The preparation method according to claim 10, characterized in that: In the step (1), the molar ratio of 3-alkyn-1-butanol, propyne bromide, potassium iodide, cesium carbonate and cuprous iodide is (0.5-2): (0.5-2): (0.5-2): (0.5-2): (0.5-2); the amount of N,N-dimethylformamide added is 20-50 mL; the reaction temperature is 20-50° C.; and the reaction time is 12-24 hours; And / or, in the step (2), the molar ratio of the 3,6-diyne-1-heptanol, triphenylphosphine and carbon tetrabromide is (0.5-2): (0.5-2): (0.5-2); the amount of dichloromethane added is 20-50 mL; the reaction temperature is 0-50° C.; the reaction time is 2-4 hours; And / or, in the step (3), the molar ratio of 7-bromo-1,4-heptadiyne, dimethylpyridinamine and potassium carbonate is (0.5-2): (0.5-2): (0.5-2); the amount of acetonitrile added is 20-50 mL; the reaction temperature is 50-100° C.; and the reaction time is 12-24 hours.

12. Cu prepared by the method according to claim 10 or 11 2+ Recognizes the ligand PHDA.

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