Fluorescent / photoacoustic bimodal probe responding to Cu (I) as well as preparation method and application of fluorescent / photoacoustic bimodal probe

Abstract

The invention belongs to the technical field of biochemistry, and relates to a fluorescence / photoacoustic bimodal probe responding to Cu (I) and a preparation method and application thereof. The fluorescence / photoacoustic bimodal probe responding to Cu (I) has high selectivity and sensitivity, has efficient tumor targeting ability, can be used for dynamic tracing of Cu (I) in tumor, can dynamically image Cu (I) in tumor by combining with a three-dimensional photoacoustic / living body fluorescence imaging technology and the probe HCyCu1, provides a new detection means for detection of Cu (I), and has wide application prospects. And a potential tool is provided for tumor diagnosis. According to the preparation method of the bimodal probe, the adopted raw materials are easy to obtain, the reaction conditions are mild and easy to control, the reaction cost is saved, and the yield of a target product is guaranteed. The structural formula of the probe is as follows: # imgabs0 #.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and specifically relates to a fluorescence / photoacoustic dual-modal probe responsive to Cu(I), a preparation method thereof, and an application thereof. Background Art

[0002] As an essential trace element for the human body, copper plays an important role in a variety of physiological and pathological processes through its redox properties. Among them, the close association between the imbalance of Cu(I) homeostasis and cancer has received close attention in recent years with the revelation of related molecular mechanisms such as copper hyperplasia and cuproptosis. The dual role of copper metabolism disorders in tumorigenesis and cancer treatment has become one of the hotspots in cancer treatment research. Therefore, the dynamic tracing of Cu(I) in living tumor tissues is of great significance for revealing the pathological functions of Cu(I) in tumorigenesis and development, as well as related research such as early diagnosis and treatment intervention of tumors.

[0003] In recent years, due to its advantages such as non-invasiveness, high sensitivity, and real-time visualization, fluorescence imaging has become an important technology for Cu(I) dynamic imaging. A variety of fluorescence probes with excellent performance have been developed, and accurate imaging of Cu(I) has been achieved in cells and shallow tissues, providing important underlying information for the mechanism analysis of Cu(I)-related physiological and pathological functions. It should be particularly noted that the Chang research group developed a Cu(I) probe, FCP-1, with ratio response ability, revealing the connection among the intracellular labile Cu(I) pool, glutathione metabolism, and carcinogenic transformation, providing an accurate detection tool for exploring the association between the intracellular labile Cu(I) level and the occurrence and development of cancer. However, limited by factors such as poor deep tissue penetration ability of fluorescence signals and interference from tissue autofluorescence, it is difficult for fluorescence probes to achieve high-resolution imaging of Cu(I) in living tumors. Therefore, how to achieve high-resolution imaging of Cu(I) in living tumor tissues has always been one of the challenging topics in the design of molecular probes.

[0004] Photoacoustic imaging (PA), as a non-invasive imaging modality, has received extensive attention in the field of biomedical imaging due to its advantage of high-resolution imaging of deep tissues. So far, small-molecule photoacoustic probes with different performances have been developed to achieve the detection and imaging of various life-related species in deep tissues of living organisms. Recently, small-molecule photoacoustic probes have demonstrated great potential in the high-resolution dynamic imaging of Cu(I) / Cu(II) in vivo. For example, the Chan group developed the first Cu(I) photoacoustic probe and achieved in-situ imaging of the Cu(I) level in a Wilson's disease model mouse. Our research group designed a ratiometric photoacoustic probe with the ability to cross the blood-brain barrier and, combined with three-dimensional photoacoustic imaging technology, achieved high-resolution imaging of Cu(II) in the brain of a Parkinson's disease model mouse and the analysis of its spatial distribution in the brain. However, it should be noted that compared with fluorescence imaging, photoacoustic probes still face the problems of long signal acquisition time and low sensitivity. The design of Cu(I) photoacoustic probes still has the following challenges: (1) the real-time response ability of Cu(I); (2) Cu(I) ratiometric imaging; (3) it is difficult to accurately and dynamically trace Cu(I). Summary of the Invention

[0005] The object of the present invention is to solve the above problems existing in the prior art and provide a fluorescence / photoacoustic dual-modal probe responsive to Cu(I), its preparation method and application. The probe of the present invention simultaneously has tumor targeting ability and real-time response to Cu(I); combined with three-dimensional photoacoustic / in-vivo fluorescence imaging technology, the probe has the application potential of dynamically tracing Cu(I) in living tumors, providing a reliable research tool for clarifying the regulation and intervention mechanism of Cu(I) on tumors and the diagnosis and treatment of related diseases.

[0006] To achieve the above object, in the first aspect of the present invention, a fluorescence / photoacoustic dual-modal probe responsive to Cu(I) is provided, and the structural formula of the probe is: 。

[0007] In the second aspect of the present invention, a preparation method of the fluorescence / photoacoustic dual-modal probe responsive to Cu(I) is provided, including the following steps: (1) Adding compound (I), compound (II), and anhydrous sodium acetate to a first solvent, and performing a first reaction to obtain compound (III); (2) Adding compound (III), N-tert-butoxycarbonyl-1,2-ethylenediamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to a second solvent, and performing a second reaction to obtain compound (IV); (3) Add compound (IV), trifluoroacetic acid, compound (V) and triethylamine to a third solvent and conduct a third reaction to obtain the said probe compound; The reaction process is as follows: .

[0008] The third aspect of the present invention provides the application of the said fluorescence / photoacoustic dual-modal probe responsive to Cu(I) in the preparation of an in vivo tumor Cu(I) imaging agent.

[0009] The present invention has the following beneficial effects: 1. The dual-modal probe provided by the present invention has high selectivity and sensitivity; after reacting with Cu(I), the absorption wavelength of the formed complex blue-shifts by about 10 nm, showing an obvious ratio-type response characteristic; at the same time, the fluorescence is enhanced by about 15 times; it has good stability under physiological pH conditions, which is helpful for real-time in-situ imaging of Cu(I); the probe itself has a strong photoacoustic signal, and at the same time, ratio-type photoacoustic imaging can significantly reduce the interference of the photoacoustic signals of in vivo biomolecules (such as hemoglobin, melanin, etc.), which is beneficial for in vivo imaging.

[0010] 2. For the preparation method of the dual-modal probe provided by the present invention, the raw materials used are easy to obtain, the reaction conditions are mild and easy to control, saving the reaction cost and ensuring the yield of the target product.

[0011] 3. The dual-modal probe HCy Cu 1 has high-efficient tumor targeting ability and can be used for dynamic tracing of Cu(I) in tumors. Combining three-dimensional photoacoustic / in vivo fluorescence imaging technology, the probe HCy Cu 1 can dynamically image the dynamics of Cu(I) in tumors, providing a new detection means for the detection of Cu(I) and a potential tool for the diagnosis of tumors.

[0012] Other features and advantages of the present invention will be described in detail in the subsequent specific implementation part. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] By describing the exemplary embodiments of the present invention in more detail in combination with the accompanying drawings, the above-mentioned and other objects, features and advantages of the present invention will become more obvious.

[0014] Figure 1 Shows the NMR spectrum of HCy Cu 1 in the present invention.

[0015] Figure 2 Shows the spectrograms of HCy Cu 1 before and after reacting with Cu(I) in Mops buffer salt solution, where Figure 2 a in shows the ultraviolet absorption spectrogram,Figure 2 Figure b shows the fluorescence spectrum diagram.

[0016] Figure 3 The fluorescence imaging diagram and fluorescence intensity diagram of the present invention are shown, where Figure 3 Figure a shows HCy Cu 1. HCy Cu 2. ATTM + HCy Cu Group 1 and the in vivo fluorescence imaging diagram responsive to Cu(I) Figure 3 Figure b shows HCy Cu 1. HCy Cu 2. ATTM + HCy Cu The change diagram of the fluorescence intensity of Group 1 over time.

[0017] Figure 4 The two-dimensional photoacoustic imaging diagram and photoacoustic signal intensity diagram of the present invention are shown, where Figure 4 Figure a shows HCy Cu 1 and HCy Cu The two-dimensional photoacoustic imaging of Group 1 and Group 2 in tumor model mice Figure 4 Figure b shows HCy Cu 1 and HCy Cu The change diagram of the photoacoustic signal intensity of Group 1 and Group 2 over time.

[0018] Figure 5 HCy is shown Cu The three-dimensional photoacoustic imaging diagram and photoacoustic signal intensity diagram of 1 in tumor model mice, where Figure 5 Figure a shows HCy Cu The imaging effect diagram of 1 changing over time Figure 5 Figure b shows HCy Cu The change diagram of the photoacoustic signal intensity of 1 over time; Figure 5 Figure c shows HCy Cu The photoacoustic imaging effect diagram of 1 in tumor model mice with different tumor volume sizes Figure 5 Figure d shows HCy Cu The change diagram of the photoacoustic signal intensity of 1 in tumor model mice with different tumor volume sizes. Detailed implementation manners

[0019] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0020] The first aspect of the present invention provides a fluorescence / photoacoustic dual-modal probe responsive to Cu(I), and the structural formula of the probe is: .

[0021] The second aspect of the present invention provides a method for preparing the above-mentioned fluorescence / photoacoustic dual-mode probe responsive to Cu(I), comprising the following steps: (1) Adding compound (I), compound (II), and anhydrous sodium acetate to a first solvent for a first reaction to obtain compound (III); (2) Adding compound (III), N-tert-butoxycarbonyl-1,2-ethylenediamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to a second solvent for a second reaction to obtain compound (IV); (3) Adding compound (IV), trifluoroacetic acid, compound (V), and triethylamine to a third solvent for a third reaction to obtain compound HCy Cu 1; The reaction process is as follows: .

[0022] According to the present invention, preferably, the first solvent, the second solvent, and the third solvent are each independently acetic anhydride and / or dichloromethane.

[0023] According to the present invention, preferably, in step (1), the molar ratio of the amounts of compound (I) and compound (II) used is 1:1.1 to 1.5; based on 1 mol of compound (I), the volume of the first solvent is 500 - 1000 mL, and the mass of sodium acetate is 300 - 500 g.

[0024] According to the present invention, preferably, in step (1), the conditions of the first reaction include: the reaction temperature is 10 - 40°C, and the reaction time is 0.5 - 50 min.

[0025] According to the present invention, preferably, in step (2), based on 1 mol of compound (III), the addition amount of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate is 300 - 500 g, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 100 - 120 g, and the addition amount of N-tert-butoxycarbonyl-1,2-ethylenediamine is 160 - 200 g.

[0026] According to the present invention, preferably, in step (2), the conditions of the second reaction include: the reaction temperature is 10 - 40°C, and the reaction time is 10 - 24 h.

[0027] According to the present invention, preferably, in step (3), the molar ratio of the compound (IV), the compound (V), trifluoroacetic acid and triethylamine is 1: 2.0-2.4: 20-40: 1.0-1.2; The conditions of the third reaction include: the reaction temperature is -10 to 25 °C, and the reaction time is 1 to 10 h.

[0028] According to the present invention, preferably, in step (3), the third reaction comprises the following steps: (a) Dissolve the compound (IV) in a third solvent, lower the reaction temperature to -10 to 5 °C, add trifluoroacetic acid, raise the reaction temperature to 10 to 25 °C for reaction, and spin-dry the reaction solution after the reaction to obtain a reaction product; (b) Dissolve the reaction product in a third solvent again, lower the reaction temperature to -10 to 5 °C, add the compound (V) and triethylamine, raise the reaction temperature to 10 to 25 °C for reaction, and spin-dry the reaction solution after the reaction to obtain the probe.

[0029] The third aspect of the present invention provides the application of the fluorescent / photoacoustic dual-modal probe responsive to Cu(I) in the preparation of an in vivo tumor Cu(I) imaging agent.

[0030] For those not specified in the embodiments regarding specific experimental steps or conditions, the operations or conditions of the conventional experimental steps described in the literature in the art can be followed. For reagents or instruments without indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0031] The device used for two-dimensional photoacoustic imaging in the embodiments is the Vevo F2 LAZR-X photoacoustic imaging platform, the device used for in vivo fluorescence imaging is the IVIS Lumina K series III instrument (PerkinElmer), and the device used for three-dimensional photoacoustic imaging is the LOIS-3D, Tomo Wave Laboratories, US.

[0032] Example 1

[0033] Compound III / HCy CuSynthesis method of 2: Under nitrogen protection, compound I (520 mg, 1 mmol) and compound II (358 mg, 1.1 mmol) dissolved in 10 mL of glacial acetic acid were added to a 50 mL Schlenk tube, and then anhydrous sodium acetate (162 mg, 2 mmol) was added. The reaction mixture was stirred at room temperature for 0.5 h. Extracted with dichloromethane, the pH value was adjusted to neutral with sodium bicarbonate, washed 3 times with saturated brine, and then the organic phase was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 443 mg of a black solid (the eluent was dichloromethane and methanol, dichloromethane: methanol = 40:1, v / v ; Yield: 51%).

[0034] Synthesis method of compound IV: Under nitrogen protection, compound III (400 mg, 0.48 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 184 mg, 0.48 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 48 mg, 0.24 mmol) were dissolved in 20 mL of dry dichloromethane (DCM) and added to a 50 mL Schlenk tube, and stirred at room temperature for 30 minutes. Then N-tert-butoxycarbonyl-1,2-ethylenediamine (92 mg, 0.56 mmol) was added, and the reaction mixture was stirred for 1 h. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 360 mg of a black solid (the eluent was dichloromethane and methanol, dichloromethane: methanol = 50:1, v / v ; Yield: 72%).

[0035] Compound HCy Cu Synthesis method of 1: Under nitrogen protection, compound IV (200 mg, 0.27 mmol) was dissolved in 10 mL of dry dichloromethane (DCM), added to a 25 mL two-necked flask, cooled to 0 o °C, trifluoroacetic acid (TFA, 2 mL, 1.1 mmol) was added dropwise, and the reaction was terminated after 1 h. The solvent was removed under reduced pressure, 10 mL of toluene was added azeotropically to remove trifluoroacetic acid (TFA), and then the residue was dissolved in 10 mL of dry dichloromethane (DCM), added to a 50 mL Schlenk tube, and triethylamine (200 μL, 0.54 mmol) was added dropwise, and the reaction mixture was stirred for 1 h. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 168 mg of a black solid (the eluent was dichloromethane and methanol, dichloromethane: methanol = 50:1, v / v ; Yield: 62%).

[0036] Compound HCy Cu The 1H NMR spectrum is as follows Figure 1 As can be seen from the figure 1 H NMR (600 MHz, CDCl3) δ / ppm 8.60 (d, J = 14.2 Hz, 1H), 7.42 (dd, J = 14.1, 7.6 Hz, 2H), 7.35 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 7.27 – 7.29 (m, 2H), 7.23 (d, J = 6.8 Hz, 1H), 7.12 (m, 2H), 7.02 (d, J = 7.7 Hz, 1H), 6.98 (t, J = 5.5 Hz, 1H), 6.75 (d, J = 8.8 Hz, 1H), 6.57 (s, 1H), 6.33 (d, J = 14.4 Hz, 1H), 5.91 (s, 1H), 4.46 (t, J = 7.1 Hz, 2H), 3.74 (t, J = 7.4 Hz, 4H), 3.39 (d, J = 20.2 Hz, 4H), 3.13 (m, J = 14.1 Hz, 1H), 2.82 (t, J = 7.3 Hz, 6H), 2.73 (t, J = 13.1 Hz, 8H), 2.62 (t, J = 7.0 Hz, 4H), 2.10 (s, 6H), 1.90 (m, 6H), 1.77 (s, 6H), 1.19 (d, J = 6.9 Hz, 6H). It can verify the structure of HCy Cu 1

[0037] Take the probe HCy Cu 1 and dissolve it in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The test solution is ethanol / Mops buffer salt solution (10 / 90, 10 mM, pH 7.0), and the test concentration of the probe is 10 μM. The detection results of the absorption spectrum are as shown in Figure 2 (a) in. The absorption peaks of the probe HCy Cu 1 are around 715 nm and 680 nm. After the probe HCy Cu 1 reacts with Cu(I), the absorption intensities at 715 nm and 680 nm gradually decrease, and a new absorption peak appears at 699 nm and 665 nm, and the absorption intensity gradually increases. The ultraviolet absorption spectrum shows that the probe HCy Cu1 is characterized by an obvious ratio-type response. At the same time, as shown in (b) of Figure 2 , after adding Cu(I) to the probe solution, the fluorescence intensity of the probe at 725 nm gradually increases with the increase of the Cu(I) concentration. When the content of Cu(I) reaches 1 equivalent, the fluorescence of the probe reaches saturation, and the fluorescence intensity of HCy Cu 1 increases by about 15 times.

[0038] Example 2

[0039] Probe HCy Cu 1 fluorescence imaging in response to Cu(I) in in vivo fluorescence imaging experiments.

[0040] To further evaluate the performance of probes HCy Cu 1 and HCy Cu 2 in in vivo fluorescence imaging, these probes were administered to 4T1 tumor-bearing mice by tail vein injection. In the control group receiving normal saline, no significant fluorescence signal was observed at the tumor site. As shown in Figure 3 a, after injecting the probe, the accumulation of HCy Cu 1 and HCy Cu 2 in the tumor region was monitored. As shown in Figure 3 b, the fluorescence at the tumor site in the HCy Cu 1 group gradually increased and reached the maximum value 2 hours after injection. Relatively speaking, the fluorescence signal in the HCy Cu 2 group decreased significantly over time and almost disappeared by 5 hours. These observations indicate that HCy Cu 1 and HCy Cu 2 have superior tumor targeting and accumulation capabilities. To avoid interference from other biological factors, ATTM was pre-injected into the tumor site 30 minutes before the tail vein injection of HCy Cu 1. Fluorescence imaging showed that the tumor fluorescence intensity in the ATTM-treated group was significantly lower than that in the untreated HCy Cu 1 group at all time points, confirming that the observed fluorescence signal mainly originated from the elevated Cu(I) level in the tumor. These results indicate that HCy Cu 1 can effectively achieve tumor targeting and in vivo imaging of Cu(I) dynamics.

[0041] Example 3

[0042] Probe HCy Cu 1 photoacoustic imaging of mouse tumors.

[0043] By tail vein injection, the Mops buffer salt solution of HCy Cu 1 and HCy Cu 2 (concentration 100 M, 150 L) Inject it into the 4T1 tumor-bearing model mice. After 0.5 h, perform photoacoustic imaging on the mice. Through dual-channel photoacoustic imaging analysis, obtain ratio-type photoacoustic imaging pictures, such as Figure 4 shown in a. In the HCy Cu 2 group without a targeting group, since HCy Cu 2 has no targeting property, the photoacoustic intensity remains almost unchanged; while in the HCy Cu 1 group, the probe has high targeting ability and the photoacoustic signal is significantly enhanced. As Figure 4 shown in b, the photoacoustic signal intensity in the HCy Cu 1 group increases with the extension of the injection time and reaches the maximum at 2 h; while the photoacoustic signal intensity in the HCy Cu 2 group hardly changes.

[0044] Then, use three-dimensional photoacoustic imaging technology to further study Cu(I) in the tumor. As Figure 5 shown in a, at the tumor site, the PA 670 / 730 ratio signal increases with the extension of the injection time, which is consistent with the two-dimensional photoacoustic imaging effect. As Figure 5 shown in b, the photoacoustic signal intensity increases with the extension of the injection time and reaches the maximum at 2 h. Subsequently, the imaging effect of the probe HCy Cu 1 in model mice with different tumor volume sizes was studied. As Figure 5 shown in c, as the tumor volume increases, the imaging effect becomes more and more obvious; as Figure 5 shown in d, the photoacoustic signal intensity is the largest in the 200 mm 3 tumor model mice.

[0045] Based on the above experimental results, it can be proved that the probe HCy Cu 1 has high tumor targeting ability. Combining photoacoustic imaging can be used for dynamic tracing of Cu(I) in living tumors and has great application prospects in the field of biomedicine.

[0046] The above has described the embodiments of the present invention. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A fluorescence / photoacoustic dual-modal probe responsive to Cu(I), characterized in that, The structural formula of the probe is as follows: 。 2. The preparation method of the fluorescence / photoacoustic dual-modal probe responsive to Cu(I) according to claim 1, characterized in that, It includes the following steps: (1) Add compound (I), compound (II), and anhydrous sodium acetate to the first solvent, and carry out the first reaction to obtain compound (III); (2) Add compound (III), N-tert-butoxycarbonyl-1,2-ethylenediamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the second solvent, and carry out the second reaction to obtain compound (IV); (3) Add compound (IV), trifluoroacetic acid, compound (V), and triethylamine to the third solvent, and carry out the third reaction to obtain the probe; The reaction process is as follows: 。 3. The preparation method according to claim 2, wherein The first solvent, the second solvent, and the third solvent are each independently acetic anhydride and / or dichloromethane.

4. The preparation method according to claim 2, wherein, In step (1), the molar ratio of the amounts of compound (I) and compound (II) used is 1:1.1 - 1.5; based on 1 mol of compound (I), the volume of the first solvent is 500 - 1000 mL, and the mass of sodium acetate is 300 - 500 g.

5. The preparation method according to claim 2, wherein, In step (1), the conditions of the first reaction include: the reaction temperature is 10 - 40°C, and the reaction time is 0.5 - 50 min.

6. The preparation method according to claim 2, wherein, In step (2), based on 1 mol of compound (III), the addition amount of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate is 300 - 500 g, the addition amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 100 - 120 g, and the addition amount of N-tert-butoxycarbonyl-1,2-ethylenediamine is 160 - 200 g.

7. The preparation method according to claim 2, wherein, In step (2), the conditions of the second reaction include: the reaction temperature is 10 - 40°C, and the reaction time is 10 - 24 h.

8. The preparation method according to claim 2, wherein In step (3), the molar ratio of the amounts of compound (IV), compound (V), trifluoroacetic acid, and triethylamine used is 1:2.0 - 2.4:20 - 40:1.0 - 1.2; The conditions of the third reaction include: the reaction temperature is -10 - 25°C, and the reaction time is 1 - 10 h.

9. The preparation method according to claim 2, wherein, In step (3), the third reaction includes the following steps: (a) Dissolve compound (IV) in the third solvent, lower the reaction temperature to -10 - 5°C, add trifluoroacetic acid, raise the reaction temperature to 10 - 25°C for reaction, and spin-dry the reaction solution after the reaction ends to obtain a reaction product; (b) Dissolve the reaction product in the third solvent again, lower the reaction temperature to -10 - 5°C, add compound (V) and triethylamine, raise the reaction temperature to 10 - 25°C for reaction, and spin-dry the reaction solution after the reaction ends to obtain the probe.

10. Use of the fluorescence / photoacoustic dual-modal probe responsive to Cu(I) described in claim 1 in the preparation of an in vivo tumor Cu(I) imaging agent.

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