A near-infrared fluorescent probe and a preparation method thereof and dual-response detection of Cu 2+ and use in Abeta aggregates

Abstract

This invention belongs to the field of analytical detection, and relates to small molecule fluorescent probes, specifically a near-infrared fluorescent probe, its preparation method, and a dual-response detection method for Cu. 2+ Applications in Aβ aggregates. The probe is modified on the fluorophore of a rhodamine derivative to redshift its emission wavelength to the near-infrared region. The fluorescent probe of this invention is named ( E )-2-amino-6'-(diethylamino)-4'-(4-(piperidin-1-yl)benzylmethyl)-1',2',3',4'-tetrahydrospiro[isoindoline-1,9'-oxanthracene]-3-one, its outstanding feature is: this fluorescent probe can achieve the detection of Cu in Alzheimer's disease (AD). 2+ Simultaneous detection of Aβ aggregates is simple to prepare, and its near-infrared fluorescence emission (765nm) can effectively avoid background interference, exhibiting high sensitivity and good selectivity.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, and relates to small molecule fluorescent probes, specifically a near-infrared fluorescent probe, its preparation method, and a dual-response detection method for Cu. 2+ Applications in Aβ aggregates. Background Technology

[0002] Alzheimer's disease (AD) is a common dementia that severely impacts the quality of life for people worldwide, accounting for approximately two-thirds of all dementia cases. While AD is prevalent in the elderly, its age of onset has been gradually decreasing in recent years. As an insidious neurodegenerative disease, its main characteristic is cognitive impairment, progressing to memory loss and even life-threatening conditions.

[0003] Currently, the clinical diagnosis of Alzheimer's disease requires two features of the brain: extraneuronal senile plaque deposition and intraneuronal neurofibrillary tangles. Due to the complexity of the pathogenesis of AD, specific therapeutic drugs cannot yet be developed, and its asymptomatic period can last 5-10 years, meaning that patients may already have a very severe condition when clinically diagnosed with AD. Therefore, developing efficient tools capable of diagnosing during the latent stage is of great significance for timely prevention and intervention of AD. Careful selection of biomarkers for AD disease progression can promote further understanding of the biochemical processes and underlying pathology of disease development, thereby optimizing the design of preventive treatment trials.

[0004] Excessive aggregation of amyloid peptide (Aβ) to form amyloid plaques is one of the classic biomarkers of Alzheimer's disease (AD). 42 Compared to Aβ 40 The presence of two additional hydrophobic amino acids makes it more prone to aggregation and thus more toxic. However, drug development based on amyloid protein has not yet been successful, indicating that other influencing factors should be considered in research on the pathogenesis of Alzheimer's disease. Metal ion imbalance also plays a crucial role in the development of AD. Studies have shown that trace amounts of metal ions can maintain the homeostasis of the neuronal microenvironment; the erroneous enrichment of metal ions in Alzheimer's patients not only affects the activity of metalloenzymes but also leads to the accumulation and deposition of Aβ plaques. 2+ As one of the representative metal ions in the human body, it has been found to deposit abnormally in Aβ plaques. It not only synergistically accelerates the accumulation of Aβ, but also generates reactive oxygen species through the Fenton reaction, thereby leading to oxidative damage to neurons.

[0005] To date, numerous near-infrared fluorescent probes have been used to detect Aβ plaques or Cu in Alzheimer's disease. 2+According to reports, patent CN116041244A discloses a curcumin-based near-infrared fluorescent probe for Aβ aggregates targeting Aβ aggregates, and patent CN110467582A discloses a near-infrared fluorescent probe for copper ion detection, but it can also be used to simultaneously detect Aβ patches and Cu. 2+ There are few reports on near-infrared fluorescent probes for Cu; therefore, further research on Cu using small molecule fluorescent probe technology is needed. 2+ Understanding the link between Aβ plaques and Alzheimer's disease is crucial for improving the efficiency of early diagnosis of Alzheimer's disease. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a near-infrared fluorescent probe, its preparation method, and a dual-response detection method for Cu. 2+ Its application in Aβ aggregates, with its near-infrared emission advantage, can effectively reduce interference from biological background signals and is beneficial to Cu. 2+ Aβ aggregates exhibit highly sensitive luminescence.

[0007] The technical solution of this invention is implemented as follows:

[0008] A method for detecting Cu 2+ A near-infrared fluorescent probe that is dual-responsive to Aβ aggregates, the probe being ( E )-2-amino-6'-(diethylamino)-4'-(4-(piperidin-1-yl)benzylmethyl)-1',2',3',4'-tetrahydrospiro[isoindoline-1,9'-oxanthracene]-3-one, with the molecular formula C 36 H 40 N4O2, the structural formula is shown below:

[0009] .

[0010] The synthesis of the near-infrared fluorescent probe in this invention mainly includes the following steps:

[0011] (1) Cyclohexanone was added dropwise to concentrated sulfuric acid under ice bath conditions, followed by 2-(4-diethylamino-hydroxybenzoyl)benzoic acid. After stirring evenly, the mixture was heated and stirred under nitrogen protection. After the reaction was complete, it was cooled to room temperature. The reactants were poured into an ice-water mixture, and perchloric acid was slowly added dropwise. The mixture was stirred until a precipitate was formed. After standing and filtering, the precipitate was washed and dried to obtain a red solid, namely compound 1.

[0012] (2) Compound 1 and 4-piperidinebenzaldehyde were dissolved in anhydrous ethanol solution. After adding piperidine, the mixture was heated under reflux until complete. The mixture was cooled to room temperature, the solvent was removed under reduced pressure, and the mixture was purified to obtain compound LDMD.

[0013] (3) The dichloromethane solution of phosphorus oxychloride was added dropwise to the mixture of compound LDMD and dichloromethane. After overnight at room temperature, the solvent was removed under reduced pressure. Acetonitrile was added, followed by the addition of 80% hydrazine hydrate. The mixture was then refluxed until complete. The solvent was removed and the final product, the fluorescent probe LDMD-N, was obtained.

[0014] In step (1) above, the molar ratio of cyclohexanone to 2-(4-diethylamino-hydroxybenzoyl)benzoic acid is 167:135-145.

[0015] The temperature for the above heating and stirring reaction is 85-90℃, and the time is 6-7 h.

[0016] The structural formula of compound 1 is as follows: .

[0017] In step (2) above, the molar ratio of compound 1 to 4-piperidinebenzaldehyde is 18:15-25.

[0018] The structural formula of the above compound LDMD is: .

[0019] In step (3) above, the molar ratio of compound LDMD to hydrazine hydrate is 1:7-9.

[0020] The aforementioned near-infrared fluorescent probes are used for dual-response detection of Cu in the biological environment for non-disease diagnostic purposes. 2+ Applications in Aβ aggregates.

[0021] Preferably, the above-mentioned biological environment includes an in vitro solution environment and an in vivo cellular environment.

[0022] The present invention has the following beneficial effects:

[0023] 1. This invention relates to a near-infrared dual-response fluorescent probe designed based on rhodamine derivative fluorophores. The probe provided by this invention can simultaneously detect Cu in Alzheimer's disease. 2+ The presence of Aβ aggregates is of great significance for the early diagnosis of Alzheimer's disease.

[0024] 2. The fluorescent probe preparation method provided by this invention is relatively simple, and the probe has relatively stable chemical properties, which has the potential for large-scale application.

[0025] 3. The fluorescent probe provided by this invention has a fluorescence emission wavelength of 765 nm after response, which is in the near-infrared emission region. This can effectively reduce the interference of background signals in biological organisms and greatly improve the accuracy of detection results.

[0026] 4. The near-infrared fluorescent probe provided by this invention not only exhibits activity against Cu in vitro in solution. 2+It exhibits good fluorescence response to Aβ aggregates and can also be applied in vivo in cells, zebrafish, and mouse models. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is the synthesis route for the dual-response near-infrared fluorescent probe LDMD-N of this invention.

[0029] Figure 2 Different concentrations of Cu were added to the dual-response near-infrared fluorescent probe LDMD-N (10 μM) of this invention. 2+ UV absorption spectrum, fluorescence emission spectrum and linear relationship graph (0-60 μM).

[0030] Figure 3 Cu was added to the dual-response near-infrared fluorescent probe LDMD-N (10 μM) of this invention. 2+ Fluorescence spectrum of fluorescence over time after reaching 30 μM.

[0031] Figure 4 Cu was added to the dual-response near-infrared fluorescent probe LDMD-N of this invention. 2+ Fluorescence emission spectra and linear relationships of the compound LDMD (2 μM) after the response with different concentrations of Aβ aggregates (0-20 μM).

[0032] Figure 5 This invention relates to the binding of the dual-response near-infrared fluorescent probe LDMD-N (10 μM) with other potential interfering substances (30 μM) and its effect on Cu in the presence of the interfering substances. 2+ The graph shows the fluorescence intensity changes in response, with the vertical axis representing the fluorescence intensity at 765 nm.

[0033] Figure 6 The present invention uses the dual-response near-infrared fluorescent probe LDMD-N (10 μM) and Cu-treated... 2+ Fluorescence imaging of cells after incubation with (20 μM) pretreated cells.

[0034] Figure 7 The fluorescent probe LDMD-N of this invention 1 H NMR spectrum.

[0035] Figure 8This is the high-resolution mass spectrum of the fluorescent probe LDMD-N of this invention. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] The synthesis route of the near-infrared fluorescent probe in this embodiment is as follows: Figure 1 As shown, the synthesis steps are as follows:

[0039] (1) In an ice bath, cyclohexanone (1.5 mL, 16.7 mmol) was added dropwise to 20 mL of concentrated sulfuric acid, followed by 2-(4-diethylamino-hydroxybenzoyl)benzoic acid (1.7 g, 13.5 mmol). After stirring for 10 min, the mixture was heated and stirred at 90 °C for 6 h under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature and poured into a 200 mL ice-water mixture. Then, 3 mL of HClO4 was added dropwise. After stirring, a precipitate was formed. After standing for 2 h, the mixture was filtered. The precipitate was washed three times with cold distilled water and dried at room temperature to obtain red solid compound 1.

[0040] (2) Compound 1 (680 mg, 1.8 mmol) and 4-piperidinebenzaldehyde (480 mg, 1.56 mmol) were dissolved in 15 mL of anhydrous ethanol solution. After adding the catalyst amount of piperidine, the mixture was heated under reflux for 4 h, cooled to room temperature, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (eluent: DCM:CH3OH = 20:1) to obtain compound LDMD.

[0041] (3) 1.2 mL of phosphorus oxychloride was dissolved in dichloromethane, and then added dropwise to compound LDMD (280 mg, 0.59 mmol) dissolved in dichloromethane. The reaction was carried out overnight at room temperature, and the solvent was removed under reduced pressure. Acetonitrile was added as the reaction solvent, and 80% hydrazine hydrate (0.18 mL, 3.89 mmol) was added dropwise. The mixture was then refluxed for 2 h. After removing the solvent, the final product, the fluorescent probe LDMD-N, was purified by column chromatography (eluent: DCM:CH3OH = 50:1). The fluorescent probe LDMD-N was a pale yellow solid with a yield of 30%, and was obtained by... 1 The product was characterized by H NMR spectrum and high-resolution mass spectrometry, and the results showed that it was indeed the target fluorescent probe LDMD-N. 1 H NMR spectrum Figure 7 As shown, high-resolution mass spectrometry is as follows Figure 8 As shown.

[0042] Example 2

[0043] The synthesis route of the near-infrared fluorescent probe in this embodiment is as follows: Figure 1 As shown, the synthesis steps are as follows:

[0044] (1) In an ice bath, cyclohexanone (1.5 mL, 16.7 mmol) was added dropwise to 20 mL of concentrated sulfuric acid, followed by 2-(4-diethylamino-hydroxybenzoyl)benzoic acid (1.74 g, 13.8 mmol). After stirring for 10 min, the mixture was heated and stirred at 90 °C for 6 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature and poured into a 200 mL ice-water mixture. Then, 3 mL of HClO4 was added dropwise. After stirring, a precipitate was formed. After standing for 2 h, the mixture was filtered. The precipitate was washed three times with cold distilled water and dried at room temperature to obtain red solid compound 1.

[0045] (2) Compound 1 (680 mg, 1.8 mmol) and 4-piperidinebenzaldehyde (584.6 mg, 1.9 mmol) were dissolved in 15 mL of anhydrous ethanol solution. After adding the catalyst amount of piperidine, the mixture was heated under reflux for 4 h, cooled to room temperature, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (eluent: DCM:CH3OH = 20:1) to obtain compound LDMD.

[0046] (3) 1.2 mL of phosphorus oxychloride was dissolved in dichloromethane, and then added dropwise to compound LDMD (280 mg, 0.59 mmol) dissolved in dichloromethane. The reaction was carried out overnight at room temperature, and the solvent was removed under reduced pressure. Acetonitrile was added as the reaction solvent, and 80% hydrazine hydrate (0.2 mL, 4.43 mmol) was added dropwise. The mixture was then refluxed for 2 h. After removing the solvent, the final product, the fluorescent probe LDMD-N, was purified by column chromatography (eluent: DCM:CH3OH = 50:1). The fluorescent probe LDMD-N was a pale yellow solid with a yield of 30%, and was obtained by... 1 The product was characterized by H NMR spectrum and high-resolution mass spectrometry, and the results showed that it was indeed the target fluorescent probe LDMD-N. 1 H NMR spectrum Figure 7 As shown, high-resolution mass spectrometry is as follows Figure 8 As shown.

[0047] Example 3

[0048] The synthesis route of the near-infrared fluorescent probe in this embodiment is as follows: Figure 1 As shown, the synthesis steps are as follows:

[0049] (1) In an ice bath, cyclohexanone (1.5 mL, 16.7 mmol) was added dropwise to 20 mL of concentrated sulfuric acid, followed by 2-(4-diethylamino-hydroxybenzoyl)benzoic acid (1.76 g, 14 mmol). After stirring for 10 min, the mixture was heated and stirred at 87 °C for 7 h under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature and poured into a 200 mL ice-water mixture. Then, 3 mL of HClO4 was added dropwise. After stirring, a precipitate was formed. After standing for 2 h, the mixture was filtered. The precipitate was washed three times with cold distilled water and dried at room temperature to obtain red solid compound 1.

[0050] (2) Compound 1 (680 mg, 1.8 mmol) and 4-piperidinebenzaldehyde (461.5 mg, 1.5 mmol) were dissolved in 15 mL of anhydrous ethanol solution. After adding the catalyst amount of piperidine, the mixture was heated under reflux for 4 h, cooled to room temperature, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (eluent: DCM:CH3OH = 20:1) to obtain compound LDMD.

[0051] (3) 1.2 mL of phosphorus oxychloride was dissolved in dichloromethane, and then added dropwise to compound LDMD (280 mg, 0.59 mmol) dissolved in dichloromethane. The reaction was carried out overnight at room temperature, and the solvent was removed under reduced pressure. Acetonitrile was added as the reaction solvent, and 80% hydrazine hydrate (0.18 mL, 3.89 mmol) was added dropwise. The mixture was then refluxed for 2 h. After removing the solvent, the final product, the fluorescent probe LDMD-N, was purified by column chromatography (eluent: DCM:CH3OH = 50:1). The fluorescent probe LDMD-N was a pale yellow solid with a yield of 30%, and was obtained by... 1 The product was characterized by H NMR spectrum and high-resolution mass spectrometry, and the results showed that it was indeed the target fluorescent probe LDMD-N. 1 H NMR spectrum Figure 7 As shown, high-resolution mass spectrometry is as follows Figure 8 As shown.

[0052] Example 4

[0053] The synthesis route of the near-infrared fluorescent probe in this embodiment is as follows: Figure 1 As shown, the synthesis steps are as follows:

[0054] (1) In an ice bath, cyclohexanone (1.5 mL, 16.7 mmol) was added dropwise to 20 mL of concentrated sulfuric acid, followed by 2-(4-diethylamino-hydroxybenzoyl)benzoic acid (1.82 g, 14.5 mmol). After stirring for 10 min, the mixture was heated and stirred at 85 °C for 6.5 h under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature and poured into a 200 mL ice-water mixture. Then, 3 mL of HClO4 was added dropwise. After stirring, a precipitate was formed. After standing for 2 h, the mixture was filtered. The precipitate was washed three times with cold distilled water and dried at room temperature to obtain red solid compound 1.

[0055] (2) Compound 1 (680 mg, 1.8 mmol) and 4-piperidinebenzaldehyde (769.2 mg, 2.5 mmol) were dissolved in 15 mL of anhydrous ethanol solution. After adding the catalyst amount of piperidine, the mixture was heated under reflux for 6 h, cooled to room temperature, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (eluent: DCM:CH3OH = 20:1) to obtain compound LDMD.

[0056] (3) 1.2 mL of phosphorus oxychloride was dissolved in dichloromethane, and then added dropwise to compound LDMD (237.3 mg, 0.5 mmol) dissolved in dichloromethane. The reaction was carried out overnight at room temperature, and the solvent was removed under reduced pressure. Acetonitrile was added as the reaction solvent, and 80% hydrazine hydrate (0.62 mL, 3.5 mmol) was added dropwise. The mixture was then refluxed for 2 h. After removing the solvent, the final product, the fluorescent probe LDMD-N, was purified by column chromatography (eluent: DCM:CH3OH = 50:1). The fluorescent probe LDMD-N was a pale yellow solid with a yield of 30%, and was obtained by... 1 The product was characterized by H NMR spectrum and high-resolution mass spectrometry, and the results showed that it was indeed the target fluorescent probe LDMD-N. 1 H NMR spectrum Figure 7 As shown, high-resolution mass spectrometry is as follows Figure 8 As shown.

[0057] Example 5

[0058] The synthesis route of the near-infrared fluorescent probe in this embodiment is as follows: Figure 1 As shown, the synthesis steps are as follows:

[0059] (1) In an ice bath, cyclohexanone (1.5 mL, 16.7 mmol) was added dropwise to 20 mL of concentrated sulfuric acid, followed by 2-(4-diethylamino-hydroxybenzoyl)benzoic acid (1.82 g, 14.5 mmol). After stirring for 10 min, the mixture was heated and stirred at 85 °C for 6.5 h under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature and poured into a 200 mL ice-water mixture. Then, 3 mL of HClO4 was added dropwise. After stirring, a precipitate was formed. After standing for 2 h, the mixture was filtered. The precipitate was washed three times with cold distilled water and dried at room temperature to obtain red solid compound 1.

[0060] (2) Compound 1 (680 mg, 1.8 mmol) and 4-piperidinebenzaldehyde (615.4 mg, 2 mmol) were dissolved in 15 mL of anhydrous ethanol solution. After adding the catalyst amount of piperidine, the mixture was heated under reflux for 5 h, cooled to room temperature, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (eluent: DCM:CH3OH = 20:1) to obtain compound LDMD.

[0061] (3) 1.2 mL of phosphorus oxychloride was dissolved in dichloromethane, and then added dropwise to compound LDMD (280 mg, 0.59 mmol) dissolved in dichloromethane. The reaction was carried out overnight at room temperature, and the solvent was removed under reduced pressure. Acetonitrile was added as the reaction solvent, and 80% hydrazine hydrate (0.21 mL, 4.5 mmol) was added dropwise. The mixture was then refluxed for 2 h. After removing the solvent, the final product, the fluorescent probe LDMD-N, was purified by column chromatography (eluent: DCM:CH3OH = 50:1). The fluorescent probe LDMD-N was a pale yellow solid with a yield of 30%, and was obtained by... 1 The product was characterized by H NMR spectrum and high-resolution mass spectrometry, and the results showed that it was indeed the target fluorescent probe LDMD-N. 1 H NMR spectrum Figure 7 As shown, high-resolution mass spectrometry is as follows Figure 8 As shown.

[0062] Application Example 1

[0063] Near-infrared fluorescent probe LDMD-N for Cu 2+ The fluorescence response sensing method is as follows:

[0064] First, the near-infrared probe and Cu were prepared using dimethyl sulfoxide and PBS buffer (10 mM, pH=7.4). 2+ The stock solution was prepared and stored at 4 °C. The probe stock solution was diluted to 10 μM using a mixture of PBS buffer (10 mM, pH=7.4) and acetonitrile (2:1), and different concentrations of Cu were added. 2+(0 μM-60 μM), the total volume of the test system was 3 mL, the test temperature was 25℃, the incubation time was 30 min, and then the response UV absorption spectrum was measured using a quartz dish. The fluorescence spectrum was also measured using 610 nm as the excitation wavelength. Figure 2 As shown. Figure 2 The results show that the probe is effective against Cu 2+ It exhibits good sensitivity, and its fluorescence intensity at 765 nm is comparable to that of Cu. 2+ The concentrations showed a good linear relationship (R0). 2 =0.9871) The linear equation is Y=62.16X+71.27.

[0065] Application Example 2

[0066] Sensitivity experiment:

[0067] Near-infrared fluorescent probe LDMD-N with Cu 2+ The fluorescence response of the responsive compound LDMD (2 μM) to different concentrations of Aβ aggregates (0-20 μM) was investigated. The compound concentration was 2 μM, and different concentrations of Aβ aggregates (0 μM-20 μM) were added. The final test volume was 300 μL. After reacting at room temperature for 30 min, the fluorescence spectrum was measured at an excitation wavelength of 610 nm. Figure 4 As shown. Figure 4 The compound also showed good sensitivity to Aβ aggregates, with a good linear relationship between the fluorescence intensity at 750 nm and the concentration of Aβ aggregates (R0). 2 =0.9844) The linear equation is Y=304.7X+299.3.

[0068] Application Example 3

[0069] Detection limit experiment:

[0070] The detection limit (LOD) of probe LY36 is calculated using the following equation:

[0071] LOD = 3σ / k

[0072] Where σ is the standard deviation of the intercept calculated by regression analysis at a 95% confidence level, and k is the slope of the calibration curve.

[0073] pass Figure 2 and Figure 4 The sensitivity test results showed that the detection limit of LDMD-N for copper ions was 0.0543 µM, and the detection limit of LDMD for Aβ aggregates was 0.0421 µM.

[0074] Application Example 4

[0075] Selective experiments:

[0076] The histogram of fluorescence intensity changes of the dual-response near-infrared fluorescent probe LDMD-N (10 μM) after binding with other potential interfering substances (30 μM), and the addition of Cu in the presence of other potential interfering substances. 2+ The subsequent fluorescence intensity change histogram, as shown in the figure. Figure 5 As shown, with an excitation wavelength of 610 nm, the vertical axis of the figure represents the fluorescence intensity at 765 nm. The interfering substance is Ag. + Al 3+ ; Ca 2+ Cd 2+ Co 2+ ; Cr 3+ Fe 3+ Hg 2+ ; K + ; Mg 2+ Na + ; NH 4 + Ni 2+ Pb 2+ Zn 2+ H2O2;ONOO - . Figure 5 The fluorescent probe LDMD-N was shown to affect Cu. 2+ It has good selectivity.

[0077] Implementation Results Example

[0078] Cell imaging experiments were performed using neuroblastoma cells (SH-SY5Y). The experiment was divided into two groups: the control group was directly incubated with the probe LDMD-N (5 μM) for 60 min, while the experimental group was first incubated with Cu... 2+ (20 μM) was incubated with cells for 60 min. After washing, the pretreated cells were co-incubated with the probe LDMD-N (5 μM) for 60 min. Fluorescence images of both groups were obtained under a Nikon AXR confocal microscope. Figure 6 As shown, after exogenous Cu 2+ The fluorescence in the pretreated cells was significantly enhanced, indicating that the fluorescent probe LDMD-N can detect Cu in cells. 2+ .

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe with the molecular formula C 36 H 40 N4O2 has the following structural formula: 。 2. The method for preparing the near-infrared fluorescent probe according to claim 1, characterized in that, The steps are as follows: (1) Cyclohexanone was added dropwise to concentrated sulfuric acid under ice bath conditions, followed by 2-(4-diethylamino-hydroxybenzoyl)benzoic acid. After stirring until homogeneous, the mixture was heated and stirred under nitrogen protection. After the reaction was complete, it was cooled to room temperature, and the reactants were poured into an ice-water mixture. Perchloric acid was then slowly added dropwise while stirring until a precipitate formed. After standing and filtering, the precipitate was washed and dried to obtain a red solid, namely compound 1. The structural formula of compound 1 is: ; (2) Compound 1 and 4-piperidinebenzaldehyde were dissolved in anhydrous ethanol solution. After adding piperidine, the mixture was heated under reflux until complete. The mixture was cooled to room temperature, the solvent was removed under reduced pressure, and the solution was purified to obtain compound LDMD. The structural formula of compound LDMD is as follows: ； (3) A solution of phosphorus oxychloride in dichloromethane was added dropwise to a mixture of compound LDMD and dichloromethane. After incubation at room temperature overnight, the solvent was removed under reduced pressure. Acetonitrile was added, followed by the addition of 80% hydrazine hydrate. The mixture was then refluxed until complete. The solvent was removed, and the final product, the fluorescent probe LDMD-N, was obtained. The structural formula of the fluorescent probe LDMD-N is: 。 3. The method for preparing the near-infrared fluorescent probe according to claim 2, characterized in that: In step (1), the molar ratio of cyclohexanone to 2-(4-diethylamino-hydroxybenzoyl)benzoic acid is 167:135-145.

4. The method for preparing the near-infrared fluorescent probe according to claim 3, characterized in that: The heating and stirring reaction is carried out at a temperature of 85-90℃ for 6-7 hours.

5. The method for preparing the near-infrared fluorescent probe according to claim 4, characterized in that: In step (2), the molar ratio of compound 1 to 4-piperidinebenzaldehyde is 18:15-25.

6. The method for preparing the near-infrared fluorescent probe according to claim 5, characterized in that: In step (3), the molar ratio of compound LDMD to hydrazine hydrate is 1:7-9.

7. The near-infrared fluorescent probe of claim 1 for dual-response detection of Cu in the biological environment for non-disease diagnosis purposes. 2+ Applications in Aβ aggregates.

8. The application according to claim 7, characterized in that: The biological environment includes an in vitro solution environment and an in vivo cellular environment; the concentration of the near-infrared fluorescent probe used is 5-10 μM.

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