A dual-emission metal ruthenium complex probe and its preparation method and application

Abstract

The present invention discloses a dual-emission metal ruthenium complex probe and its preparation method and application. The metal ruthenium complex probe has the characteristics of simple synthesis method and high yield. The metal ruthenium complex can not only detect the ratio luminescence of the β-amyloid protein fibrillation process in real time, but also perform ratio imaging analysis of β-amyloid protein fibers. The detection process has the characteristics of high reliability and accurate results. Compared with commercial single-emission probes, the detection accuracy of the metal ruthenium complex probe is significantly improved, demonstrating that the dual-emission metal ruthenium complex probe has good application prospects in the early detection of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of luminescent probe detection, and in particular to a dual-emission metal ruthenium complex probe, its preparation, and its application in ratiometric detection of beta-amyloid protein fibrillation process. Background Art

[0002] Alzheimer's disease (AD) is a degenerative condition of the central nervous system that primarily occurs in the elderly or pre-elderly. Its clinical manifestations are primarily progressive memory and cognitive impairment, as well as behavioral and personality abnormalities. The incidence of AD increases with age. As the aging population intensifies, the number of AD patients is projected to increase by approximately 40% to 78 million by 2030 and to 139 million by 2050, making it a major threat to humanity. Currently, there are no effective drugs or treatments for AD, making the development of detection methods and related drugs crucial.

[0003] Although the exact pathogenesis of AD remains unclear, it is generally believed that the fibrillization of β-amyloid protein is one of the important causes of AD. Therefore, research and development of detection methods for the process of β-amyloid protein fibrillization will not only provide a molecular understanding of the pathogenesis of AD, but also provide important evidence for early diagnosis of AD and the development of related drugs.

[0004] Based on current research on detection methods for β-amyloid fibrils, luminescent probe detection has become one of the preferred detection methods due to its simplicity, high sensitivity, and real-time imaging capabilities. Compared to currently commercially available single-emission probes, the development of ratiometric luminescent probes based on dual-emission metal complexes can effectively reduce the impact of factors such as instrument performance, probe concentration, and photobleaching on detection, thereby significantly improving the accuracy of β-amyloid fibril detection. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a dual-emission metal ruthenium complex probe suitable for ratiometric detection of amyloid fibrillation, which has the characteristics of high detection reliability and accurate detection results.

[0006] The present invention also aims to provide a method for preparing the dual-emission metal ruthenium complex probe.

[0007] The present invention also aims to provide the use of the dual-emission metal ruthenium complex probe in real-time detection of amyloid protein fibrillation process.

[0008] The present invention also aims to provide the use of the dual-emission metal ruthenium complex probe in ratiometric imaging detection of amyloid protein fibers.

[0009] In order to achieve the above object, the solution of the present invention is:

[0010] A dual-emission metal ruthenium complex probe, the general structural formula of which is shown in I,

[0011]

[0012] Wherein, R1 and R2 are hydrogen atoms or methoxy groups, They are different bidentate polypyridine ligands.

[0013] R1 and R2 are selected from one of a hydrogen atom and a methoxy group.

[0014] Selected from bidentate polypyridyl ligands as well as One of them.

[0015] Selected from as well as One of them.

[0016] The following lists of preferred compounds further illustrate a dual-emission metal ruthenium complex probe of the present invention, which should not be construed as limiting the present invention in any way.

[0017]

[0018]

[0019] A method for preparing a dual-emission ruthenium complex probe comprises the following steps:

[0020] (1) Weigh compound A, p-cymene ruthenium dichloride dimer, and bidentate polypyridine ligand compound B in a molar ratio of 1:2-2.2:2-2.2. and bidentate polypyridine ligand compound C

[0021] (2) Under a nitrogen atmosphere, a solvent and a bidentate polypyridine ligand compound B are added to compound A, the reaction temperature is controlled at 80°C to 90°C for metal coordination reaction, and the heating reaction is carried out for 3 to 7 hours. The volume ratio of the solvent to the amount of compound A is 8 to 16 mL: 0.1 mmol;

[0022] (3) Then, the bidentate polypyridine ligand compound C is added to the reaction system of step (2), and the reaction temperature is controlled at 130°C to 150°C to carry out the metal coordination reaction, and the reaction is refluxed for 10 to 15 hours, cooled, filtered, and washed to obtain compound D.

[0023] (4) Weigh compound D and bidentate ligand compound E in a molar ratio of 1:1.1-1.5.

[0024] (5) Adding bidentate ligand compound E to compound D, and then adding a solvent to carry out a metal coordination reaction to obtain the dual-emission metal ruthenium complex probe, wherein the ratio of the volume of the solvent to the amount of the compound D is 8-12 mL:0.1 mmol.

[0025] In step (2), the solvent is one of 1,4-dioxane, acetonitrile and N,N-dimethylformamide.

[0026] In step (5), the solvent is one of tert-butyl alcohol, ethylene glycol and ethylene glycol dimethyl ether.

[0027] The synthesis route of the dual-emission ruthenium complex probe is as follows:

[0028]

[0029] That is, the metal ruthenium complex A undergoes chelation reaction with different bidentate polypyridine ligands in steps under the action of a solvent.

[0030] A dual-emission ruthenium complex probe is used in real-time ratiometric luminescence detection of β-amyloid protein fibrillation process, and the detection method comprises the following steps:

[0031] (1) preparing the dual-emission metal ruthenium complex probe;

[0032] (2) adding the β-amyloid protein sample to a buffer solution and incubating at 37°C to form a β-amyloid protein aggregate solution;

[0033] (3) The dual-emission metal ruthenium complex probe prepared in step (1) and the β-amyloid protein aggregate solution prepared in step (2) were respectively aspirated at intervals of 5 to 10 minutes, mixed, and the change in the ratio of the dual emission intensities of the metal ruthenium complex probe was detected by fluorescence spectrometer.

[0034] In step (2), the buffer solution is 300 mM NaCl / 100 mM Tris, and the pH of the buffer solution is 7.5.

[0035] In step (3), the parameters of the fluorescence spectrometer are set as follows: the excitation and emission slit widths are both 10 nm; the delay time is 1 s; the detection voltage is 700 V; the excitation wavelength is 340 nm; the gain is 1; and the sensitivity is 8.0 s.

[0036] An application of a dual-emission metal ruthenium complex probe to ratiometric imaging detection of β-amyloid protein fibers, wherein the detection method comprises the following steps:

[0037] (1) preparing the dual-emission metal ruthenium complex probe;

[0038] (2) adding the β-amyloid protein sample to a buffer solution and incubating at 37°C for 150 minutes to form β-amyloid protein fibers;

[0039] (3) The dual-emission metal ruthenium complex probe prepared in step (1) and the β-amyloid protein fiber prepared in step (2) are respectively aspirated, mixed, and the ratio imaging change of the metal ruthenium complex probe is detected using a laser confocal microscope.

[0040] In step (2), the buffer solution is 300 mM NaCl / 100 mM Tris, and the pH of the buffer solution is 7.5.

[0041] In step (3), the parameters of the laser confocal microscope are set as follows: excitation wavelengths of 405 and 488 nm; signal detection ranges of 425-525 nm and 600-700 nm, and a detection voltage of 600 V.

[0042] After adopting the above technical solution, the present invention provides a dual-emission metal ruthenium complex probe having a general structural formula as shown in I. The dual-emission metal ruthenium complex probe has the characteristics of simple synthesis method and high yield. Using fluorescence spectroscopy testing method, the metal ruthenium complex can detect the development of β-amyloid protein fibrillation process in real time by ratiometric luminescence; using laser confocal microscopy testing technology, the metal ruthenium complex can also perform ratiometric imaging analysis of β-amyloid protein fibers. Compared with commercial single-emission probes Thioflavin T Compared with the conventional method, the reliability and accuracy of the detection process are significantly improved, which proves that the dual-emission ruthenium complex probe has a good application prospect in the early detection of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The present invention will be further described below with reference to the accompanying drawings and examples.

[0044] Figure 1 This is the synthetic route of compound 13 of the present invention;

[0045] Figure 2 is the H NMR spectrum of compound 13 of the present invention;

[0046] Figure 3 is the C NMR spectrum of compound 13 of the present invention;

[0047] Figure 4 Compound 13 of the present invention is effective against Aβ40 Ratiometric luminescence detection of fibrosis;

[0048] Figure 5 Compound 13 of the present invention is effective against Aβ 42 Ratiometric luminescence detection of fibrosis;

[0049] Figure 6 Compound 13 of the present invention is effective against Aβ 40 Ratio imaging detection diagram of fibers;

[0050] Figure 7 Compound 13 of the present invention is effective against Aβ 42 Ratio imaging detection of fibers. DETAILED DESCRIPTION

[0051] In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments.

[0052] 1. Preparation of Dual-Emission Ruthenium Complex Probe

[0053] Example 1

[0054] Synthesis of compound 2:

[0055] Under nitrogen atmosphere, 92 mg (0.15 mmol) of p-cymene dichloride ruthenium dimer (compound A) and 54 mg (0.3 mmol) of 1,10-phenanthroline (compound B) were added to a pressure-resistant reaction bottle. After evacuating the gas twice, 10 mL of N,N-dimethylformamide was heated at 90°C for 5 h. 85 mg (0.3 mmol) of dipyrido[3,2-a:2',3'-c]phenazine (Compound C) was then added to the reaction flask. The temperature was raised to 130°C and refluxed for 12 h before stopping the reaction. The reaction was cooled to room temperature, filtered, and washed to obtain 150 mg of a black solid (Compound D) (80% yield). 128 mg (0.2 mmol) of the resulting black solid and 37 mg (0.2 mmol) of 2-pyrazinyl-2-pyridylmethylamine (Compound E) were weighed into a microwave reaction flask, 8 mL of ethylene glycol was added, and the microwave power was set to 325 W for 30 min. The reaction was cooled, saturated potassium hexafluorophosphate solution was added, and the crude product was filtered. The resulting crude product was passed through a silica gel column to obtain 160 mg of a yellow solid (Compound 2) (76% yield).

[0056] Example 2

[0057] The synthesis of compound 13 is as follows Figure 1 As shown:

[0058] Under nitrogen atmosphere, 92 mg (0.15 mmol) of p-cymene dichloride ruthenium dimer (compound A) and 59 mg (0.33 mmol) of 1,10-phenanthroline (compound B) were added to a pressure-resistant reaction bottle. After evacuating the gas twice, 10 mL of N,N-dimethylformamide was heated at 85°C for 6 h. Then, 94 mg (0.33 mmol) of dipyrido[3,2-a:2',3'-c]phenazine (Compound C) was added to the reaction flask. The temperature was raised to 150°C and refluxed for 15 h before stopping the reaction. The reaction was cooled to room temperature, filtered, and washed to obtain 160 mg (85% yield) of a black solid (Compound D). 58 mg (0.09 mmol) of the obtained black solid and 20 mg (0.09 mmol) of 2-(5-methoxypyrazinyl)-2-pyridylmethylamine (Compound E) were weighed into a microwave reaction flask. 10 mL of ethylene glycol was added. The microwave power was set to 325 W and the reaction time was 40 min. The reaction was cooled, saturated potassium hexafluorophosphate solution was added, and the crude product was filtered. The resulting crude product was passed through a silica gel column to obtain 50 mg of Compound 13 as a yellow solid in a 52% yield.

[0059] Example 3

[0060] Synthesis of compound 21:

[0061] Under nitrogen atmosphere, 92 mg (0.15 mmol) of p-cymene dichloride ruthenium dimer (compound A) and 72 mg (0.4 mmol) of 1,10-phenanthroline (compound B) were added to a pressure-resistant reaction bottle. After evacuating the gas twice, 10 mL of N,N-dimethylformamide was heated at 85°C for 4 h. 113 mg (0.4 mmol) of dipyrido[3,2-a:2',3'-c]phenazine (Compound C) was then added to the reaction flask. The temperature was raised to 130°C and refluxed for 14 h before the reaction was stopped. The reaction was cooled to room temperature, filtered, and washed to obtain 165 mg of a black solid (Compound D) (88% yield). 128 mg (0.2 mmol) of the resulting black solid and 43 mg (0.2 mmol) of 2-(pyrazinyl)-2-(4-methoxypyridinyl)methylamine (Compound E) were weighed into a microwave reaction flask. 8 mL of ethylene glycol was added. The microwave power was set to 325 W and the reaction time was 30 min. The reaction was cooled, saturated potassium hexafluorophosphate solution was added, and the crude product was filtered. The resulting crude product was passed through a silica gel column to obtain 160 mg of Compound 21 as a yellow solid (75% yield).

[0062] 2. Structural Analysis

[0063] The H NMR spectrum of compound 13 is shown in Figure 2As shown, 1HNMR (400MHz, CD3CN): δ [ppm]: 3.57 (s, 3H), 3.74 (s, 3H), 6.74-6.80 (m, 1H), 7.03 (s, 1H), 7.19 (s, 1H), 7.36-7.45 (m, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.80-7.86 (m, 2H), 7.96 (dd, J = 4.0 and 8.0Hz,1H),8.06(t,J=8.0Hz,1H),8.12-8.21(m,4H),8.24(d,J=8.0Hz,1H),8.29-8.32(m,1H),8.40(s,1H),8.49(d,J=8.0Hz,2H) ,8.81(dd,J=4.0and8.0Hz,1H),8.89(dd,J=4.0and8.0Hz,1H),8.97(t,J=8.0Hz,1H),9.47(d,J=8.0Hz,1H),9.80(t,J=8.0Hz,1H).

[0064] The NMR carbon spectrum of compound 13 is as follows Figure 3 As shown, 13C NMR (100 MHz, CDCl3): δ [ppm]: 41.2, 54.9, 116.9, 121.4, 126.7, 128.0, 128.0, 128.9, 129.2, 130.6, 131.8, 131.9, 132.4, 133.4, 134.3, 135.0, 137.8, 138.6, 140.5, 140.9, 143.7, 148.5, 148.8, 149.0, 149.5, 151.5, 151.9, 152.0, 153.9, 154.3, 154.9, 155.1, 156.1, 158.5, 159.7.

[0065] Compound 13 Flight 780.0 [M-2PF6] 2+ , calculated value 780.1.

[0066] Elemental analysis of compound 13: C, 45.45; H, 2.80; N, 12.67; calculated value: C, 45.27; H, 2.97; N, 12.88.

[0067] The results showed that the H-NMR spectrum, C-NMR spectrum, time-of-flight mass spectrometry and elemental analysis were consistent with the structural formula of compound 13.

[0068] 3. Ratiometric luminescence real-time detection of β-amyloid protein fibrils

[0069] Example 4

[0070] The application of the dual-emission metal ruthenium complex probe 13 in real-time detection of the β-amyloid protein fibrillation process comprises the following steps:

[0071] (1) Weigh 2.45 mg of the dual-emission metal ruthenium complex probe 13 and dissolve it in 10 mL of tris buffer solution (300 mM NaCl / 100 mM Tris, pH = 7.5) to obtain a probe solution with a concentration of 140 μM for use;

[0072] (2) Weigh 1.0 mg of purchased amyloid protein Aβ 40 , dissolved in 300 μL 25 mM NaOH solution, ultrasonicated for two minutes, and then filtered with a 0.2 μm centrifugal filter tube. The filtrate was diluted to 1 mL with a buffer solution (300 mM NaCl / 100 mM Tris, pH = 7.5). The protein solution was then measured spectrophotometrically at 290 nm (ε = 2132 M -1 cm -1 ) was measured by measuring the absorbance of the characteristic absorption peak at 140 μM, and the concentration of the protein solution was calculated to be 140 μM. The prepared protein solution was placed in a thermostatic mixer for incubation at 37°C and 700 rpm.

[0073] (3) Every 15 minutes or 20 minutes, 100 μL of the probe solution of step (1) and the protein solution of step (2) were respectively aspirated and mixed, and diluted to 1 mL with distilled water. The emission intensity ratio of the metal ruthenium complex ratiometric luminescence probe was monitored in real time using a fluorescence spectrometer, wherein the parameters of the fluorescence spectrometer were set as follows: the excitation and emission slit widths were both 10 nm; the delay time was 1 s; the detection voltage was 700 V; the excitation wavelength was 340 nm; the gain was 1; and the sensitivity was 4.0 s.

[0074] The results show that: Figure 4 As shown, when Aβ 40 When the incubation time reaches 150 minutes, the ratio of the luminous intensity of the two emission peaks in the emission spectrum increases by 5 times (I 640 / I 440 ), indicating that the dual-emission metal ruthenium complex probe can bind to Aβ 40 The fiber structure undergoes hydrophobic and hydrogen-building effects.

[0075] Example 5

[0076] The application of the dual-emission metal ruthenium complex probe 13 in real-time detection of the β-amyloid protein fibrillation process comprises the following steps:

[0077] (1) Weigh 2.45 mg of the dual-emission metal ruthenium complex probe 13 and dissolve it in 10 mL of tris buffer solution (300 mM NaCl / 100 mM Tris, pH = 7.5) to obtain a probe solution with a concentration of 140 μM for use;

[0078] (2) Weigh 1.0 mg of purchased amyloid protein Aβ 42 , dissolved in 300 μL 25 mM NaOH solution, ultrasonicated for two minutes, and then filtered with a 0.2 μm centrifugal filter tube. The filtrate was diluted to 1 mL with a buffer solution (300 mM NaCl / 100 mM Tris, pH = 7.5). The protein solution was then measured spectrophotometrically at 290 nm (ε = 2132 M -1 cm -1 ) was measured by measuring the absorbance of the characteristic absorption peak at 140 μM, and the concentration of the protein solution was calculated to be 140 μM. The prepared protein solution was placed in a thermostatic mixer for incubation at 37°C and 700 rpm.

[0079] (3) Every 15 minutes or 20 minutes, 100 μL of the probe solution of step (1) and the protein solution of step (2) were respectively aspirated and mixed, and diluted to 1 mL with distilled water. The changes in the ratio of the dual emission intensity of the metal ruthenium complex probe were monitored in real time using a fluorescence spectrometer, wherein the parameters of the fluorescence spectrometer were set as follows: the excitation and emission slit widths were both 10 nm; the delay time was 1 s; the detection voltage was 700 V; the excitation wavelength was 340 nm; the gain was 1; and the sensitivity was 4.0 s.

[0080] The results show that: Figure 5 As shown, when Aβ 42 When the incubation time reached 150 minutes, the ratio of the luminous intensity of the two emission peaks in the emission spectrum increased by 2.8 times (I 640 / I 440 ), which also shows that the dual-emission metal ruthenium complex probe can bind to Aβ 42 The fiber structure undergoes hydrophobic and hydrogen-building effects.

[0081] 4. Ratio Imaging Detection of β-Amyloid Protein Fibers

[0082] Example 6

[0083] The application of the dual-emission ruthenium complex probe 13 in the ratiometric imaging detection of β-amyloid protein fibers comprises the following steps:

[0084] (1) Weigh 2.45 mg of the dual-emission metal ruthenium complex probe 13 and dissolve it in 10 mL of tris buffer solution (300 mM NaCl / 100 mM Tris, pH = 7.5) to obtain a probe solution with a concentration of 140 μM for use;

[0085] (2) Weigh 1.0 mg of purchased amyloid protein Aβ 40 , dissolved in 300 μL 25 mM NaOH solution, ultrasonicated for two minutes, and then filtered with a 0.2 μm centrifugal filter tube. The filtrate was diluted to 1 mL with a buffer solution (300 mM NaCl / 100 mM Tris, pH = 7.5). The protein solution was then measured spectrophotometrically at 290 nm (ε = 2132 M -1 cm -1 ) was measured by measuring the absorbance of the characteristic absorption peak at 140 μM, and the concentration of the protein solution was calculated to be 140 μM. The prepared protein solution was placed in a thermostatic mixer and incubated for 150 min at a temperature of 37° C. and a rotation speed of 700 rpm.

[0086] (3) 100 μL of the probe solution from step (1) and the protein fiber solution from step (2) were respectively taken and mixed, and diluted to 1 mL with distilled water. The ratio imaging of the metal ruthenium complex probe was detected using an excitation confocal scanning microscope, wherein the parameters of the excitation confocal scanning microscope were set as follows: excitation wavelengths of 405 and 488 nm; signal detection ranges of 425-525 nm and 600-700 nm, and a detection voltage of 600 V.

[0087] The results show that: Figure 6 As shown, when the excitation wavelength is 405nm and the detection range is 425-525nm, or when the excitation wavelength is 488nm and the detection range is 600-700nm, Aβ can be detected. 40 The imaging signal of the fibers was then mapped using FV10-ASW software. 40 Ratio imaging of fibers.

[0088] Example 7

[0089] The application of the dual-emission ruthenium complex probe 13 in the ratiometric imaging detection of β-amyloid protein fibers comprises the following steps:

[0090] (1) Weigh 2.45 mg of the dual-emission metal ruthenium complex probe 13 and dissolve it in 10 mL of tris buffer solution (300 mM NaCl / 100 mM Tris, pH = 7.5) to obtain a probe solution with a concentration of 140 μM for use;

[0091] (2) Weigh 1.0 mg of purchased amyloid protein Aβ 42, dissolved in 300 μL 25 mM NaOH solution, ultrasonicated for two minutes, and then filtered with a 0.2 μm centrifugal filter tube. The filtrate was diluted to 1 mL with a buffer solution (300 mM NaCl / 100 mM Tris, pH = 7.5). The protein solution was then measured spectrophotometrically at 290 nm (ε = 2132 M -1 cm -1 ) was measured by measuring the absorbance of the characteristic absorption peak at 140 μM, and the concentration of the protein solution was calculated to be 140 μM. The prepared protein solution was placed in a thermostatic mixer and incubated for 150 min at a temperature of 37° C. and a rotation speed of 700 rpm.

[0092] (3) 100 μL of the probe solution from step (1) and the protein fiber solution from step (2) were respectively taken and mixed, and diluted to 1 mL with distilled water. The ratio imaging of the metal ruthenium complex probe was detected using an excitation confocal scanning microscope, wherein the parameters of the excitation confocal scanning microscope were set as follows: excitation wavelengths of 405 and 488 nm; signal detection ranges of 425-525 nm and 600-700 nm, and a detection voltage of 600 V.

[0093] The results show that: Figure 7 As shown, Aβ can be detected when the excitation wavelength is 405nm and the detection range is 425-525nm, or when the excitation wavelength is 488nm and the detection range is 600-700nm. 42 The imaging signal of the fibers was then mapped using FV10-ASW software. 42 Ratio imaging of fibers. Ratio imaging of fibers.

[0094] As demonstrated in the aforementioned ratiometric detection examples of β-amyloid fibrils, the ruthenium complex not only enables real-time ratiometric luminescence detection of β-amyloid fibril formation, but also enables ratiometric imaging analysis of β-amyloid fibrils. This demonstrates that the ruthenium complex of the present invention is an ideal ratiometric luminescence probe for real-time monitoring of amyloid fibril formation and holds great promise for early detection of Alzheimer's disease.

Claims

1. A dual-emission ruthenium complex probe, characterized in that: Its general structural formula is shown in I, wherein R1 is a hydrogen atom and R2 is a methoxy group; or R1 is a methoxy group and R2 is a hydrogen atom; Selected from bidentate polypyridyl ligands as well as One of the following; Selected from One of them.

2. The method for preparing a dual-emission ruthenium complex probe according to claim 1, wherein: The steps include: (1) Weigh compound A, p-cymene dichloride ruthenium dimer, and bidentate polypyridine ligand compound B in a molar ratio of 1:2-2.2:2-2.

2. and bidentate polypyridine ligand compound C in, Selected from bidentate polypyridine ligands as well as One of the following; Selected from One of the following; (2) Under a nitrogen atmosphere, a solvent and a bidentate polypyridine ligand compound B are added to compound A, the reaction temperature is controlled at 80°C to 90°C for metal coordination reaction, and the heating reaction is carried out for 3 to 7 hours. The volume ratio of the solvent to the amount of compound A is 8 to 16 mL: 0.1 mmol; (3) Then, the bidentate polypyridine ligand compound C is added to the reaction system of step (2), and the reaction temperature is controlled at 130°C to 150°C to carry out the metal coordination reaction, and the reaction is refluxed for 10 to 15 hours, cooled, filtered, and washed to obtain compound D. (4) Weigh compound D and bidentate ligand compound E in a molar ratio of 1:1.1-1.

5. wherein R1 is a hydrogen atom and R2 is a methoxy group; or R1 is a methoxy group and R2 is a hydrogen atom; (5) Adding bidentate ligand compound E to compound D, and then adding a solvent to carry out a metal coordination reaction to obtain the dual-emission metal ruthenium complex probe, wherein the ratio of the volume of the solvent to the amount of the compound D is 8-12 mL:0.1 mmol.

3. The preparation method according to claim 2, wherein: In step (2), the solvent is one of 1,4-dioxane, acetonitrile and N,N-dimethylformamide.

4. The preparation method according to claim 2, wherein: In step (5), the solvent is one of tert-butyl alcohol, ethylene glycol and ethylene glycol dimethyl ether.

5. Use of the dual-emission ruthenium complex probe according to claim 1 in preparing a ratiometric imaging detection reagent for β-amyloid protein fibers.

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