Fluorescent Probe for Detecting Aβ Aggregates, Preparation Method and Application Thereof

Abstract

The present invention discloses a fluorescent probe for detecting Aβ aggregates, its preparation method and applications. Specifically, the structure of the probe is shown in Formula I. The Aβ aggregate probe provided by the present invention has various uses, can be used for the recognition and detection of Aβ aggregates in vitro and in biological tissue sections, is used for β-amyloid protein imaging in biological tissues with high sensitivity, and has wide applications in the early diagnosis of Alzheimer's disease, preclinical research and development of anti-AD drugs, and the revelation of pathological mechanisms.

Description

Technical Field

[0001] The present invention relates to a class of fluorescent probes for detecting Aβ aggregates, their preparation methods and applications. Specifically, it relates to the biological use of detecting Aβ aggregates, especially the application as a detection tool molecule for a pathological marker of Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD), accounting for about 70% of the total number of dementia patients, is a central nervous system degenerative disease highly related to age, mainly featuring progressive cognitive impairment and memory damage. The currently recognized pathological features of AD are extracellular misfolded β-amyloid protein plaques and intracellular tau protein tangles. In recent years, major global biopharmaceutical companies have invested a large amount of manpower and material resources in the research and development of new anti-AD drugs. Due to the complex pathogenic mechanism of AD, the research and development of anti-AD drugs have all ended in failure. Although the basic research on AD has developed rapidly, there is still no specific drug for AD clinically. The existing treatment plans mainly target the cognitive and memory impairments manifested by AD to relieve the symptoms of patients. So far, AD is still a major disease with unmet clinical needs, and there is an urgent need to develop new treatment drugs and diagnostic methods.

[0003] There are many hypothesis theories for the AD mechanism. So far, it can be confirmed that the overproduction and abnormal aggregation of amyloid β (Aβ) aggregates to form senile plaques are the most important pathological markers of Alzheimer's disease, the central link of AD pathology, and the common pathway for the occurrence and development of AD caused by various factors. In the current research on AD diseases and drugs, the pathological changes of Aβ are important biomarkers for the early diagnosis of AD patients and the evaluation of the efficacy of clinical drugs. Most AD patients are diagnosed at an advanced stage. Therefore, improving the detection and diagnosis level of AD is of great significance for the early diagnosis and treatment of AD and the research on its pathogenesis.

[0004] Compared with traditional radioactive probe technologies, fluorescent probes have significant advantages such as rapidity, low cost, high sensitivity, non-invasiveness, innovative visualization, and high spatial resolution. They are an innovative and advantageous way for the visualization and quantitative evaluation of specific molecular targets in modern biomedical research and clinical research. Currently, fluorescent probes for AD are usually used to detect Aβ and tau proteins. Although numerous probe molecules have been reported, there is still a lack of probe molecules with high sensitivity, strong binding force, longer emission wavelength, and excellent physical and chemical properties.

[0005] Therefore, the development of Aβ fluorescent probe molecules with high specificity and affinity has attracted much attention. After combining Aβ fluorescent probes with techniques such as molecular imaging, biological tissue imaging, and in vivo fluorescence imaging, real-time and non-invasive Aβ plaque imaging, tracking, and detection can be achieved. Furthermore, it facilitates the development of diseases and drug research in the preclinical and clinical fields of AD. Summary of the Invention

[0006] The object of the present invention is to provide a class of fluorescent probes targeting Aβ aggregates, a preparation method thereof, and uses thereof in the detection, imaging, etc. of Aβ as a pathological marker in AD-related research.

[0007] In the first aspect of the present invention, a fluorescent probe is provided, and the fluorescent probe is a compound of formula I:

[0008]

[0009] Wherein,

[0010] X is selected from O or NH; and

[0011] R is selected from CN or C1-C6 alkyl OC(=O)-.

[0012] In another preferred embodiment, X is selected from O or NH.

[0013] In another preferred embodiment, R is selected from CN or C1-C3 alkyl OC(=O)-.

[0014] In another preferred embodiment, R is selected from CN or CH3CH2OC(=O)-.

[0015] In another preferred embodiment, the compound of formula I is selected from the following group:

[0016]

[0017] In the second aspect of the present invention, there is provided the use of the fluorescent probe as described in the first aspect of the present invention in the preparation of a fluorescent probe reagent for detecting Aβ aggregates.

[0018] In another preferred embodiment, the fluorescent probe reagent can be used for the research and development of anti-AD drugs and / or the revelation of pathological mechanisms.

[0019] In another preferred embodiment, the detection is Aβ aggregate fluorescence imaging.

[0020] In the third aspect of the present invention, an Aβ aggregate fluorescence detection kit is provided, which includes the fluorescent probe as described in the first aspect of the present invention and an optional instruction manual.

[0021] In the fourth aspect of the present invention, there is provided the use of the fluorescent probe as described in the first aspect of the present invention in the preparation of an Alzheimer's disease diagnostic reagent.

[0022] In another preferred example, the diagnostic reagent is used for diagnosing early Alzheimer's disease.

[0023] In the fifth aspect of the present invention, there is provided a diagnostic kit for Alzheimer's disease, which is characterized in that it comprises the fluorescent probe described in the first aspect of the present invention, and an optional instruction manual.

[0024] In the sixth aspect of the present invention, there is provided a method for in vitro fluorescence detection of Aβ aggregates, comprising the steps of:

[0025] (1) contacting a sample to be tested with a solution of the fluorescent probe described in the first aspect of the present invention to stain the sample; and

[0026] (2) using the stained sample for fluorescence detection.

[0027] In another preferred example, the sample to be tested is a biological tissue section or a body fluid sample, such as a brain tissue section.

[0028] In another preferred example, the fluorescence detection is fluorescence imaging of a biological tissue section.

[0029] In another preferred example, the sample to be tested is from a mammal, such as a human, a rat or a mouse.

[0030] In another preferred example, in step (2), the excitation wavelength of the fluorescence detection is 405 nm ± 10 nm (such as ±5 nm or ±2 nm), and fluorescence is detected in the range of 550–650 nm.

[0031] In the sixth aspect of the present invention, there is provided a method for diagnosing Alzheimer's disease, comprising the steps of:

[0032] (1) contacting a sample to be tested from a test subject with a solution of the fluorescent probe described in the first aspect of the present invention to stain the sample; and

[0033] (2) using the stained sample for fluorescence detection.

[0034] In another preferred example, the sample to be tested is a biological tissue section or a body fluid sample, such as a brain tissue section.

[0035] In another preferred example, the fluorescence detection is fluorescence imaging of a biological tissue section.

[0036] In another preferred example, the test subject is a mammal, such as a human, a rat or a mouse.

[0037] In another preferred example, in step (2), the excitation wavelength of the fluorescence detection is 405 nm ± 10 nm (such as ±5 nm or ±2 nm), and fluorescence is detected in the range of 550–650 nm.

[0038] In another preferred example, the method further includes the step of comparing the detection result of step (2) with the detection result of a positive control or a negative control sample, so as to determine whether the detection object has Alzheimer's disease.

[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the ultraviolet absorption and fluorescence emission spectrogram of the preferred probes KAD0202, KAD0240 and KAD0242 of the present invention;

[0041] Figure 2 is the Aβ aggregate protein affinity determination curve of the preferred probes KAD0202, KAD0240 and KAD0242 of the present invention;

[0042] Figure 3 is the single- and two-photon confocal brain slice imaging result of the preferred probe of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] Through extensive and in-depth research, and through a large number of screenings and tests, the present inventors have provided a class of Aβ aggregate fluorescent probes. The Aβ aggregate probes provided by the present invention have various uses and can be used for the identification and detection of Aβ aggregates in vitro and in biological tissue sections. After recognizing Aβ, the excitation wavelength is in the red light range (550 - 650 nm), close to near-infrared, and the probe itself has two-photon characteristics and can be excited by near-infrared light. This fluorescent probe can be used for β-amyloid protein imaging in biological tissues with high sensitivity and has wide applications in the early diagnosis of Alzheimer's disease, preclinical research and development of anti-AD drugs, and the revelation of pathological mechanisms. On this basis, the present invention has been completed.

[0044] TERMINOLOGY

[0045] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0046] As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0047] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of", or "consisting of".

[0048] As used herein, the term "room temperature" or "ambient temperature" refers to a temperature of 4 - 40 °C, preferably, 25 ± 5 °C.

[0049] Aβ aggregates

[0050] In the present invention, Aβ aggregates refer to amyloid-β protein aggregates (Amyloid β, Aβ). The overproduction and abnormal aggregation of Aβ aggregates to form senile plaques are the most important pathological markers of Alzheimer's disease (AD), the central link of AD pathology, and the common pathway for the occurrence and development of AD caused by various factors. In the early stage of AD, Aβ aggregates can circulate from the brain to body fluids. Therefore, the early diagnosis of AD patients can be achieved by detecting Aβ proteins in body fluids.

[0051] Fluorescent probes and their applications

[0052] The present invention provides an Aβ aggregate fluorescent probe represented by Formula I:

[0053]

[0054] Wherein,

[0055] X is selected from O or NH; and

[0056] R is selected from CN or C1 - C6 alkyl OC(=O)-.

[0057] In another preferred embodiment, X is selected from O or NH.

[0058] In another preferred embodiment, R is selected from CN or C1 - C3 alkyl OC(=O)-.

[0059] In another preferred embodiment, R is selected from CN or CH3CH2OC(=O)-.

[0060] In another preferred embodiment, the compound of Formula I is selected from the following group:

[0061]

[0062] Experimental results show that the fluorescent probe of the present invention can be used for the recognition and detection of Aβ aggregates in vitro and in biological tissue sections. After recognizing Aβ, its excitation wavelength is in the red light range (550 - 650 nm), close to the near-infrared region. The probe itself has two-photon properties and can be excited by near-infrared light. In addition, the fluorescent probe can be used for imaging β-amyloid protein in biological tissues with high sensitivity, and has wide applications in the early diagnosis of Alzheimer's disease, preclinical research and development of anti-AD drugs, and the revelation of pathological mechanisms.

[0063] Further, the present invention provides the use of the fluorescent probe in the preparation of a fluorescent probe reagent for detecting Aβ aggregates. The fluorescent probe reagent can be used for the research and development of anti-AD drugs and / or the revelation of pathological mechanisms.

[0064] In addition, an Aβ aggregate fluorescence detection kit is also provided, which includes the fluorescent probe as described above and an optional instruction manual.

[0065] Further, the present invention also provides the use of the fluorescent probe in the preparation of a diagnostic reagent for Alzheimer's disease. In particular, it is used for diagnosing early Alzheimer's disease.

[0066] In addition, a diagnostic kit for Alzheimer's disease is also provided, which is characterized in that it includes the fluorescent probe described in the first aspect of the present invention and an optional instruction manual.

[0067] Furthermore, the present invention also provides a method for in vitro fluorescence detection of Aβ aggregates, comprising the steps of:

[0068] (1) contacting a sample to be tested with a solution of the fluorescent probe of the present invention to stain the sample; and

[0069] (2) using the stained sample for fluorescence detection.

[0070] The present invention has no specific requirements for the sample to be tested, and it can be various common samples to be detected, such as samples prepared in the laboratory, or biological tissue sections or body fluid samples from organisms, such as brain tissue sections.

[0071] In another preferred example, the fluorescence detection is fluorescence imaging of biological tissue sections.

[0072] In another preferred example, the sample to be tested is from a mammal, such as a human, a rat or a mouse.

[0073] In another preferred example, in step (2), the excitation wavelength of the fluorescence detection is 405 nm ± 10 nm (such as ±5 nm or ±2 nm), and fluorescence is detected in the range of 550 - 650 nm.

[0074] A diagnostic method for Alzheimer's disease is further provided, comprising the steps of:

[0075] (1) contacting a sample to be tested from a test subject with a solution of the fluorescent probe described in the first aspect of the present invention to stain the sample; and

[0076] (2) using the stained sample for fluorescence detection.

[0077] In another preferred example, the sample to be tested is a biological tissue section or a body fluid sample, such as a brain tissue section.

[0078] In another preferred example, the fluorescence detection is fluorescence imaging of a biological tissue section.

[0079] In another preferred example, the detection object is a mammal, such as a human, a rat or a mouse.

[0080] In another preferred example, in step (2), the excitation wavelength of the fluorescence detection is 405 nm ± 10 nm (such as ±5 nm or ±2 nm), and fluorescence is detected in the range of 550–650 nm.

[0081] In another preferred example, the method further includes the step of comparing the detection result of step (2) with the detection result of a positive control or a negative control sample, so as to determine whether the detection object has Alzheimer's disease.

[0082] Preparation method

[0083] The present invention has no specific requirements for the preparation method of the fluorescence probe, and it can be prepared with reference to the examples of the present invention in combination with the commonly used methods in the art.

[0084] Preferably, the preparation method can be selected from the following routes:

[0085]

[0086] Route 1: Using 4-bromo-2-hydroxybenzaldehyde and 4-dimethylaminostyrene as raw materials, in a mixed solvent of DMF and water, with palladium acetate etc. as catalysts, the intermediate KAD0202A is obtained through a Heck reaction, and then a Knoevenagel condensation reaction is carried out with malononitrile to obtain the probe KAD0202.

[0087] Route 2: Using the probe KAD0202 as a raw material, the probe KAD0240 is obtained after hydrolysis with an acidic ethanol solution.

[0088] Route 3: Using the intermediate KAD0202A as a raw material, a Knoevenagel condensation reaction is carried out with diethyl malonate to obtain the probe KAD0242.

[0089] The main advantages of the present invention include:

[0090] The present invention has the following advantages and effects compared with the prior art: (1). The probe of the present invention is based on the traditional coumarin fluorescent mother nucleus, and has the advantages of high fluorescence efficiency and large Stokes shift. (2). The fluorescent probe of the present invention has good selectivity, high affinity and detection sensitivity for Aβ. (3). The fluorescent probe of the present invention can sensitively detect Aβ aggregates in biological tissues and can better image Aβ plaques in tissues. The excitation wavelength of this series of probes is close to the near-infrared, and the probes have two-photon characteristics with strong tissue penetration, and can be used for preclinical small animal in vivo imaging research. (4). The synthesis route of this series of fluorescent probes is short, the operation is simple, and the products are easy to obtain, and it has broad prospects in the early diagnosis and pathological mechanism research of Alzheimer's disease.

[0091] The following is a further elaboration of the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions indicated in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0092] Example 1

[0093] Preparation of probe KAD0202 and probe KAD0240:

[0094]

[0095] (1) Synthesis of intermediate KAD0202A

[0096] 4-Bromo-2-hydroxybenzaldehyde (500 mg, 2.5 mmol) was dissolved in a mixed solvent of an appropriate amount of N,N-dimethylformamide and H2O (60 mL: 10 mL). Under stirring, 4-dimethylaminostyrene (735 mg, 5 mmol) was added, and under N2 replacement protection, triethylamine (505 mg, 5 mmol), triphenylphosphine (132 mg, 0.5 mmol), and palladium acetate (57 mg, 0.25 mmol) were added. The reaction was carried out at 100 °C for 18 h. After quenching the reaction with water, the reaction system was extracted with ethyl acetate. The organic phase was collected and washed three times with an equal amount of saturated NaCl solution. After thoroughly drying the organic phase with anhydrous Na2SO4 crystals, it was distilled under reduced pressure. The obtained crystals were subjected to column chromatography using a petroleum ether:dichloromethane = 1:2 system to obtain 400 mg of dark red solid KAD0202A, with a yield of 63%. 11H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 10.13 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 16.3 Hz, 1H), 7.19 (dd, J = 8.2, 1.5 Hz, 1H), 7.06 (d, J = 1.5 Hz, 1H), 6.97 (d, J = 16.3 Hz, 1H), 6.73 (d, J = 8.8 Hz, 2H), 2.96 (s, 6H). EI-HRMS: m / z calc. for C 17 H 17 NO2: 267.1259, found: 267.1262。

[0097] (2). Synthesis of probe KAD0202:

[0098] Put KAD0202A (200 mg, 0.75 mmol) into a flask, add an appropriate amount of absolute ethanol as the reaction solution, add malononitrile (60 mg, 0.9 mmol) and ammonium acetate (30 mg, 0.375 mmol) under stirring. After reacting at room temperature for 2 h, detect the reaction by TLC, and the ratio of the developing agent is ethyl acetate: petroleum ether = 1:2. The substrate is partially insoluble in absolute ethanol and is a yellow solid precipitate at the bottom. As the reaction proceeds, the reaction solution turns red, and the yellow solid is converted into a red solid and precipitates out. Pour the reaction system into ice water, extract the reaction system with dichloromethane, wash it twice with water, then wash it with saturated NaCl solution, and then dry it thoroughly with Na2SO4 and distill it under reduced pressure and then perform column chromatography. Use a petroleum ether: dichloromethane = 2:1 system for column chromatography, and collect 200 mg of the red product KAD0202 solid, with a yield of 83%. 1 1H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 1.6 Hz, 1H), 8.29 (d, J = 1.6 Hz, 1H), 7.53 - 7.42 (m, 4H), 7.39 (d, J = 16.3 Hz, 1H), 7.31 (s, 1H), 7.03 (d, J = 16.4 Hz, 1H), 6.73 (d, J = 8.6 Hz, 2H), 2.96 (s, 6H). 13 13C NMR (151 MHz, DMSO-d6) δ 154.88, 152.41, 151.08, 146.96, 144.95, 140.71, 133.70, 131.37, 130.10, 129.40, 128.87, 128.37, 124.50, 122.18, 122.10, 116.14, 112.61, 112.54, 40.53. EI-HRMS: m / z calc. for C20 H 17 N3O: 315.1172, found: 315.1175。

[0099] (3). Synthesis of probe KAD0240:

[0100] Place the solid KAD0202 (100 mg, 0.32 mmol) in a 100 ml flask, add an appropriate amount of anhydrous ethanol as the reaction solvent, acidify the system with 1N HCl solution, then set up a reflux heating device and react in an 80 °C oil bath for 3 h. Pour the reaction system into ice water while it is hot, extract the reaction system with dichloromethane, wash it twice with water, then wash it with saturated NaCl solution, and then dry it thoroughly with Na2SO4 and perform column chromatography under reduced pressure. Use the PE:DCM = 2:1 system for column chromatography, and collect 80 mg of the red-black product KAD0240 solid, with a yield of 80%. 1 H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.69 - 7.61 (m, 2H), 7.57 - 7.48 (m, 3H), 7.12 (d, J = 16.3 Hz, 1H), 6.80 - 6.73 (m, 2H), 2.99 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 155.49, 153.23, 146.49, 141.67, 135.06, 130.58, 129.15, 124.34, 123.44, 121.83, 118.92, 116.34, 113.22, 112.57, 99.91. EI-HRMS: m / z calc. for C 20 H 16 N2O2: 316.1212, found: 316.1214。

[0101] Example 2

[0102] Synthesis of probe KAD0242:

[0103]

[0104] KAD0202A (200 mg, 0.75 mmol) was put into a flask, and an appropriate amount of absolute ethanol was added as the reaction solution. Diethyl malonate (145 μL, 0.9 mmol) was added with stirring, and after mixing, a few drops of piperidine were added as the catalyst. The mixture was heated to 80 °C and refluxed overnight. The reaction was monitored by TLC, and the developing solvent ratio was ethyl acetate: petroleum ether = 1:2. The substrate was partially insoluble in absolute ethanol and formed a yellow solid precipitate at the bottom. As the reaction proceeded, the reaction solution turned red, and the yellow solid was converted into a red solid and precipitated out. The reaction system was poured into ice water, and the reaction system was extracted with dichloromethane, washed twice with water, then washed with saturated NaCl solution, and then thoroughly dried with Na2SO4 and subjected to column chromatography under reduced pressure. Column chromatography was carried out using a petroleum ether: dichloromethane = 2:1 system, and 230 mg of the red product KAD0242 solid was collected, with a yield of 84.5%. 1 H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.59 (dd, J = 8.2, 1.6 Hz, 1H), 7.54 (d, J = 1.5 Hz, 1H), 7.52 - 7.43 (m, 3H), 7.09 (d, J = 16.3 Hz, 1H), 6.75 (d, J = 16.3 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 2.97 (s, 6H), 1.31 (t, J = 7.1 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 163.25, 156.77, 155.89, 151.12, 149.01, 145.45, 134.22, 130.90, 128.94, 124.44, 122.87, 122.07, 116.61, 116.59, 115.82, 112.68, 112.53, 61.52, 14.59. EI-HRMS: m / z calc. for C 22 H 21 NO4: 363.1471, found: 363.1476。

[0105] Example 3

[0106] Determination of the ultraviolet absorption and fluorescence emission spectra of the probes KAD0202, KAD0240, and KAD0242

[0107] The specific preparation method of the Aβ aggregates used in the experiment is as follows: An appropriate amount of commercially available Aβ 1-42The monomer (purchased from Shanghai Gil Biochemical, product number 052487) was dissolved in PBS to obtain a stock solution of 100 μM. After incubation at 37 °C for 24 h, the preparation was completed and could be stored at -80 °C for long-term storage. Preparation of the preferred probe stock solution: An appropriate amount of probe solid was taken and dissolved in DMSO to prepare a 10 mM stock solution for standby. It could be stored at -80 °C for long-term. Before use, it was diluted to a 100 μM probe stock solution. Preparation of the experimental solution: 200 μL was taken from the stock solution and added to a 5 mL centrifuge tube, and 1800 μL of 1×PBS (pH = 7.2) buffer was added for dilution to obtain the solution for testing the ultraviolet absorption curve. The final concentration of the probe was 10 μM. 1700 μL of 1×PBS (pH = 7.2) buffer was pre-added to two 5 mL centrifuge tubes respectively, and then 200 μL of the probe preparation solution was added. After mixing evenly, 100 μL of Aβ 1-42 aggregate preparation solution was added to one tube as the experimental group, and 100 μL of 1×PBS (pH = 7.2) buffer was added to the other tube as the blank group. After thorough mixing, the fluorescence emission spectrum was measured. The results are as Figure 1 shown. It can be seen that the fluorescence intensities of the three preferred probes changed significantly before and after binding to Aβ aggregates, and they can be used for the recognition imaging of Aβ aggregates.

[0108] Example 4

[0109] Determination of the protein affinity of the preferred probes KAD0202, KAD0240, and KAD0242

[0110] The preparation methods of Aβ aggregates and probe solutions refer to Example 3, only changing the probe concentration. The final concentrations of the probes were 5, 2, 1, 0.5, 0.25, 0.1, 0.05, and 0 μM in sequence. Under the fluorescence emission conditions measured in Example 3, the fluorescence saturation curves of the probes KAD0202, KAD0240, and KAD0242 for Aβ aggregates were respectively tested. The detection range was 400–800 nm, the detection interval was 2 nm, the scanning speed was 1200 nm / min. After mixing the probe and Aβ evenly to the selected concentration, the fluorescence intensity was measured, and the results were subjected to K d value fitting using GraphPad Prism 8.0 software. The results are as Figure 2 shown. The K d values of the probes KAD0202, KAD0240, and KAD0242 for Aβ aggregates were 272.6 nM, 148.0 nM, and 66.3 nM respectively, indicating that the three preferred probes can all bind to Aβ aggregates relatively firmly.

[0111] Example 5

[0112] Single- and two-photon confocal microscopy imaging experiments of the preferred probes

[0113] Paraffin sections of the brain tissues of normal 10-week-old male mice were stained with probes and used as the blank control group; paraffin sections of the brain tissues of 10-week-old male 3xTg-AD transgenic mice were stained with probes and used as the experimental group. The staining method was dropwise staining. Among them, under the imaging mode of a 20x objective lens of a single-photon confocal microscope, a commercially available gold standard probe, Thioflavine T (ThT), was used as a positive control. The shooting conditions of ThT were excitation at 405 nm and detection of fluorescence near 452 nm; the preferred probe was excited at a wavelength of 405 nm and fluorescence was detected in the range of 550–650 nm; while under the imaging mode of a 0x objective lens of a two-photon confocal microscope, the excitation light wavelength of the two-photon femtosecond laser source was adjusted from 700 nm until 1000 nm to find the optimal excitation light. A commercially available known Aβ two-photon Methoxy-XO4 was used as a positive control probe. Methoxy-XO4 was photographed using the EGFP channel (wavelength 500–550 nm) and an excitation light wavelength of 750 nm; the probe was photographed using an excitation light of 920 nm and the Cy5 channel (wavelength 600–657 nm). Among them, the preferred probes KAD0202 and KAD0242 are as Figure 3 shown in part, it can be seen by single-photon confocal microscopy imaging that the probe can better label Aβ plaques in the brain tissue. The preferred probe KAD0202, as Figure 3 shown, it can be seen by two-photon confocal microscopy imaging that the probe can better label Aβ plaques in the brain tissue. In summary, this series of probes can better image Aβ plaques in the brain tissue on both single- and two-photon confocal systems.

[0114] All documents mentioned in this invention are cited in this application as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the claims appended to this application.

Claims

1. A fluorescent probe, characterized in that, The fluorescent probe is a compound of Formula I: Wherein, X is selected from O or NH; and R is selected from CN or CH3CH2OC(=O)-.

2. The fluorescent probe according to claim 1, wherein X is O.

3. The fluorescent probe according to claim 1, wherein R is CN.

4. The fluorescent probe according to claim 1, wherein The compound of Formula I is selected from the following group:

5. Use of the fluorescent probe according to any one of claims 1-4 in the preparation of a fluorescent probe reagent for detecting Aβ aggregates.

6. A fluorescence detection kit for Aβ aggregates, characterized in that, It comprises the fluorescent probe according to any one of claims 1-4, and an optional instruction manual.

7. Use of the fluorescent probe according to any one of claims 1-4 in the preparation of a diagnostic reagent for Alzheimer's disease.

8. An Alzheimer's disease diagnostic kit, characterized in that, It comprises the fluorescent probe according to any one of claims 1-4, and an optional instruction manual.

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