Fluorescence ratio probe as well as development method and application thereof

Abstract

The invention belongs to the field of fluorescent probes, and provides a fluorescent ratio probe as well as a development method and application thereof. The fluorescence ratio probe is 4-(4-dimethylaminostyryl) quinoline, QD for short, and is used for imaging and detecting beta-amyloid protein aggregate (Abetas for short) and monitoring the pH change of the environment where the Abetas is located. The invention further discloses a development method of the probe. The invention further discloses application of the probe in preparation of a product for diagnosing the disease progress of the Alzheimer's disease. The product comprises the probe. The invention provides a bifunctional fluorescence ratio probe QD capable of lightening Abetas and monitoring a surrounding pH microenvironment, and the bifunctional fluorescence ratio probe QD has the characteristics of high affinity, good repeatability and simplicity and convenience in operation. QD and Abetas are combined with two-color fluorescence, and the fluorescence ratio changes along with the pH change of the environment where Abetas is located, so that in-vivo and in-vitro Abetas can be marked and the pH microenvironment around Abetas can be displayed by using QD.

Description

Technical Field

[0001] The present disclosure relates to the field of fluorescent probes, and in particular to a fluorescent ratio probe and a development method and application thereof. Background Art

[0002] Alzheimer's disease (AD) has an insidious onset and is one of the most common dementias. AD is highly correlated with age, and the number of patients has increased dramatically with the global aging population. Abnormal accumulation of Aβ aggregates (Aβs) is an early event in AD. Although the relevance of plaques formed by Aβs in the staging of AD disease is controversial, Aβs is an indirect reservoir of other toxic entities, and plaque deposition formed by Aβs is one of the two major pathological characteristics of AD, and is a key target for early diagnosis and confirmation of AD. Therefore, the development of probes targeting Aβs has always been one of the hot topics in the research field.

[0003] In recent years, most of the development of Aβs probes, whether classical fluorescent probes or near-infrared fluorescent probes, are based on the modification of the parent skeleton of existing probes that can identify Aβs. Commonly used skeletons include benzothiazoles, diphenylethylene derivatives, curcumin derivatives, BODIPY (Boron dipyrromethane), oxazine derivatives, cyanine, olefin near-infrared fluorescent probes, etc. The method based on the modification of the parent skeleton can ensure a high affinity with Aβs. On this basis, the structure is modified by chemical synthesis to achieve efficient regulation of the optical properties, specificity, lipid solubility and other properties of the compound. For example, previous studies can increase the molecular conjugated structure and construct a D-π-A / D-π-A-π-D conjugated system, which is beneficial to the red shift of the emission spectrum; and by replacing the charged group, the electroneutrality of the molecule is increased, and the blood-brain barrier permeability is improved. However, the above methods have certain limitations. First, new parent structures cannot be discovered; second, the organic synthesis process is complicated and costly, and cannot be synthesized in batches for large-scale screening; more importantly, this method cannot synthesize intelligent light-up probes to reduce the fluorescence background. Therefore, a new strategy is urgently needed for the development of Aβs probes, which can screen smart-turn-on fluorescent probes with high affinity in a high-throughput and faster manner.

[0004] Most Aβs probes can only indicate the location and quantity of Aβs. However, AD is a multifactorial disease, and various pathological changes in the patient's brain provide important basis for early diagnosis, such as Aβ plaque deposition, neuroinflammation, oxidative stress, lipid metabolism, autophagy disorders, etc. These pathologies do not exist independently, but influence each other and are highly dependent. In order to explore the relationship between the various pathologies, two single-response probes are used to simultaneously detect possible intersections in distribution and spectral position, so the development of dual-function probes has important application value in exploring the relationship between pathologies. At present, simultaneous detection of Aβ-related markers mainly focuses on oxidative stress and lipid metabolism-related markers. Among them, dual-function probes for Aβs and ROS include those for Aβs and H2O2, Aβs and ·OH, Aβs and ONOO - Simultaneous detection of Aβs. The dual-function probes of Aβs and lipid metabolism include those for the simultaneous detection of Aβs and lipid droplets. Although lysosomal autophagy disorders in AD are closely related to Aβ, and lysosomal autophagy disorders cause damage to intracellular lysosomes, leading to the leakage of lysosomes and protons. However, due to the lack of dual-function probes of Aβs and pH, there are currently no studies on the effects of Aβs on the surrounding pH microenvironment. Therefore, there is an urgent need to develop a dual-function probe that can light up Aβs and monitor the surrounding pH microenvironment, to help study the effects of Aβs-induced lysosomal autophagy disorders on the pH microenvironment and the relationship between the effects. Summary of the invention

[0005] The present disclosure provides a fluorescence ratio probe and a development method and application thereof, so as to at least solve the above technical problems existing in the prior art.

[0006] According to the first aspect of the present disclosure, a fluorescence ratio probe is provided, which is 4-(4-dimethylaminophenylvinyl)quinoline, referred to as QD; the fluorescence ratio probe is used for imaging and detection of Aβs, and monitoring changes in pH of the environment in which the Aβs are located.

[0007] Specifically, Aβs is an insoluble β-amyloid protein aggregate, which is the main component of Aβ plaques in the brain tissue of AD transgenic mice. As the acidity of the environment in which Aβs are located in the brain increases, the red fluorescence of QD binding to Aβs increases; as the alkalinity of the environment in which Aβs are located increases, the green fluorescence of QD binding to Aβs increases, that is, as the pH of the environment in which Aβs are located changes from acidic to neutral and alkaline, the fluorescence color changes from red to orange and then to green. Therefore, the pH change of the environment in which Aβs is located can be monitored based on the change in fluorescence color after the fluorescence ratio probe binds to Aβs.

[0008] When the pH of the environment in which Aβs is located changes from acidic to neutral and alkaline, the fluorescence intensity at 530nm increases and the fluorescence intensity at 645nm decreases. The principle is that QD can be ionized and protonated under acidic conditions. After obtaining protons, it is converted into QD (H + ), the electron gain increases the degree of electron delocalization and the spectrum red shifts. pK a It is an important indicator of the ionization ability of a compound. The pK a The value is 5.0. That is, when pH is less than 5.0, QD is in the protonated form QD (H + ) when pH>5.0, it mainly exists in the molecular form of QD, so the pH-sensitive property of QD can be used as a fluorescence ratio probe of Aβs.

[0009] In one embodiment, the binding fluorescence ratio of the fluorescence ratio probe to the Aβs increases as the pH of the environment in which the Aβs are located increases.

[0010] Specifically, as the pH of the environment in which Aβs is located increases, the green fluorescence at 530nm increases and the red fluorescence at 645nm decreases, resulting in a gradual increase in the combined fluorescence ratio. The combined fluorescence ratio refers to the ratio of green fluorescence at 530nm to red fluorescence at 645nm, i.e. Green 530nm / Red 645nm, referred to as G / R.

[0011] In one embodiment, at pK a =5, the fluorescence ratio of the fluorescence ratio probe to the Aβs increases to a peak value; at pK a When ≥6, the increase in the binding fluorescence ratio of the fluorescence ratio probe to the Aβs tends to be gentle.

[0012] According to a second aspect of the present disclosure, a method for developing the above-mentioned fluorescence ratio probe is provided, comprising the following steps:

[0013] S1: extract the smiles information of N,N-dimethylphenyl in the existing Aβs probe, and screen the compounds having the smiles information in the screening library;

[0014] S2: Further screening compounds with Binding energy ≥ -7.5 kcal / mol from the compounds with the smiles information obtained in step S1 to screen out compounds with affinity for Aβs;

[0015] S3: further screening out compounds that bind to the Aβs and have a fluorescence on response from the compounds screened in step S2;

[0016] S4: The compound screened in step S3 is verified by fluorescence screening with the Aβs at the protein level, thereby obtaining the fluorescence ratio probe.

[0017] In one embodiment, the existing Aβs probe in step S1 includes at least one of thioflavin derivatives, stilbene derivatives, curcumin derivatives, BODIPY, and DANIR derivatives.

[0018] Specifically, most existing Aβs probes have a common structure of N,N-dimethylphenyl, and some previous studies have also speculated that this structure is relevant to the recognition of Aβs. Therefore, step S1 extracts compounds with N,N-dimethylphenyl structure from the screening library, and then extracts smiles information of N,N-dimethylphenyl in some existing Aβs probes (smiles is a linear symbol used to input and represent molecular reactions, and is an ASCII code).

[0019] In one embodiment, the smiles information of the N,N-dimethylphenyl group includes two types, namely CN(C)c1ccc(*)cc1 and C(*)c1ccc(N(C)C)cc1.

[0020] In one embodiment, the screening library has a total of 130,000 compounds, including 12,800 compounds in ZINC for sale and 200 commonly used dyes.

[0021] In one embodiment, step S2 first uses openbable to convert the SDF form of the compound with the smiles information obtained by step S1 into a PDB form for further batch molecular docking, and then screens out compounds with Binding energy ≥ -7.5 kcal / mol.

[0022] Specifically, in order to perform Auto vina molecular docking simulation between the compounds with the smiles information screened in step S1 and the protein model of Aβs (PDB: 2BEG), step S2 ranks the compounds according to affinity.

[0023] Specifically, summarizing the existing Aβs fluorescence-on probes, most of them are related to the twisted intramolecular charge transfer (TICT) effect. Fluorescence-on compounds need to have rotatable single bonds or double bonds, as well as coplanar rigid conjugated structures. When the entire molecule is not in a coplanar state, the molecule is in a fluorescence-off state due to the TICT effect; after binding to Aβs, the folded structure of Aβs inhibits the rotation of the chemical bonds, so that the rigid conjugated structure of the compound is in a coplanar state, inhibiting the TICT effect of the compound, and making the molecule in a fluorescence-on state. Based on this, step S3 further screens out compounds that have a fluorescence-on response when bound to Aβs.

[0024] In one embodiment, step S4 performs fluorescence screening verification at the protein level to verify whether the compound recognizes the Aβs at the protein level.

[0025] According to a third aspect of the present disclosure, there is provided a use of the above-mentioned fluorescence ratio probe in the preparation of a product for diagnosing the disease progression of Alzheimer's disease, wherein the product comprises the fluorescence ratio probe.

[0026] Specifically, the fluorescence ratio probe can identify Aβ plaques in tissues, and the binding fluorescence ratio of the fluorescence ratio probe to identify Aβ plaques changes with the pH microenvironment around the Aβ plaques. Based on this, the disease progression of AD can be displayed by the fluorescence ratio of the fluorescence ratio probe to Aβ plaques being affected by pH.

[0027] According to an implementable embodiment of the present disclosure, at least the following beneficial effects are achieved:

[0028] The present disclosure provides a dual-function fluorescence ratio probe OQ that can light up Aβs and monitor the surrounding pH microenvironment. QD and Aβs are combined with dual-color fluorescence. The fluorescence ratio of QD and Aβs changes with the pH of the environment in which Aβs is located. It has the characteristics of high affinity, good repeatability, and simple operation. It can realize the labeling of Aβs in vivo and in vitro and display the pH microenvironment around Aβs.

[0029] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, in which:

[0031] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.

[0032] Figure 1 The types and structural schematic diagrams of the existing Aβs probes summarized in Example 1 of the present disclosure are shown;

[0033] Figure 2 A schematic diagram of the TICT effect of the Aβs fluorescence turn-on probe in Example 1 of the present disclosure is shown;

[0034] Figure 3 The transmission electron microscope morphology of Aβs in Example 2 of the present disclosure is shown;

[0035] Figure 4 The fluorescence spectra and binding affinity results of THT or QD binding to Aβs in Example 2 of the present disclosure are shown; wherein, a-fluorescence spectra of THT binding to Aβs, b-binding affinity graph of THT binding to Aβs, c-fluorescence spectra of QD binding to Aβs, d-binding affinity graph of QD binding to Aβs;

[0036] Figure 5 The kinetic fitting diagram of the fluorescence binding between QD and Aβ peptide monomer fibrillation at different times in Example 2 of the present disclosure is shown;

[0037] Figure 6 The photo graphs of the fluorescence changes of QD binding to Aβs at different pH in Example 2 of the present disclosure are shown;

[0038] Figure 7 The graphs showing the changes in the excitation spectrum and emission spectrum of the QD combined with Aβs at different pH values ​​in Example 2 of the present disclosure are shown; wherein a-the graph showing the changes in the excitation spectrum of the QD combined with Aβs at different pH values, and b-the graph showing the changes in the emission spectrum of the QD combined with Aβs at different pH values;

[0039] Figure 8 The theoretical pK of QD in Example 2 of the present disclosure is shown. a Schematic diagram of pK values ​​and fluorescence determination a The theoretical pK value of a-QD is shown in Figure 2. a Schematic diagram of pK values, b-fluorescence determination a Value result graph;

[0040] Fig. 9 The result graph showing the change of the fluorescence ratio Green 530nm / Red 645nm of the binding of QD and Aβs with pH in Example 2 of the present disclosure is shown;

[0041] Fig.10The diagram shows the recognition effect of QD on Aβ plaques in brain tissue slices of 5×FAD model mice in Example 3 of the present disclosure; wherein, A-immunofluorescence imaging of the Aβ-specific recognition antibody 2C6-Cy5 under the excitation wavelength of 647nm channel, B-immunofluorescence imaging of QD under the excitation wavelength of 570nm channel, C-immunofluorescence imaging after the above two channels are merged;

[0042] Fig.11 The graph shows the change of the fluorescence ratio G / R of the binding of QD to Aβ plaques in brain slice tissue when the pH is 2, 7, and 10 in Example 3 of the present disclosure;

[0043] Fig.12 The graph shows the change in the fluorescence ratio G / R of the binding of QD to Aβ plaques in brain tissue sections of 5×FAD model mice at 4, 8, and 13 months of age in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0044] In order to make the purpose, features, and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0045] Example 1

[0046] This example develops a fluorescence ratio probe, which is used for imaging and detecting Aβs, and monitoring the pH change of the environment in which Aβs are located. The specific development process is as follows:

[0047] (1) The chemical structures of some existing Aβs probes were summarized, including thioflavin derivatives, stilbene derivatives, curcumin derivatives, BODIPY, DANIR derivatives, etc. Figure 1 As shown in the figure, the common structure of most probes is N,N-dimethylphenyl. Some previous studies also speculated that this structure is related to the recognition of Aβs. Therefore, compounds with N,N-dimethylphenyl structure were extracted from the screening library. Therefore, the smiles information of N,N-dimethylphenyl in some existing Aβs probe structures was extracted, including two forms CN(C)c1ccc(*)cc1 and C(*)c1ccc(N(C)C)cc1. Next, the compounds with this smiles were screened in the library. The screening library used a total of 130,000 compounds, including 12,800 compounds in ZINC for sale and 200 commonly used dyes. This part of the screening obtained 566 compounds.

[0048] (2) The screened compounds should have a strong affinity for Aβs. In order to perform Auto Vina molecular docking simulation on the compounds screened in step (1) and the protein model of Aβs (PDB: 2BEG), the compounds were ranked according to affinity. First, the SDF form of the compounds obtained in step (1) was converted into PDB form using openbable for further batch molecular docking, and compounds with Binding energy ≥ -7.5 kcal / mol were screened. A total of 26 compounds were obtained in this part.

[0049] (3) The screened compounds should have a fluorescence turn-on response when bound to Aβs. To summarize the existing Aβs fluorescence turn-on probes, most are related to the twisted intramolecular charge transfer (TICT) effect, such as Figure 2 As shown. In simple terms, a fluorescently turned-on compound needs to have a rotatable single bond or double bond, and a coplanar rigid conjugated structure. When the entire molecule is not in a coplanar state, the molecule is in a fluorescence-off state due to the TICT effect. After binding to Aβs, the folded structure of Aβs inhibits the rotation of the chemical bonds, making the rigid conjugated structure of the compound in a coplanar state, inhibiting the TICT effect of the compound, and making the molecule in a fluorescence-on state. A total of 3 compounds were obtained in this part of the screening.

[0050] (4) The compounds screened in step (3) are subjected to fluorescence screening verification with Aβs at the protein level (i.e., verifying whether the screened compounds recognize Aβs at the protein level), and finally obtaining the fluorescence ratio probe 4-(4-dimethylaminophenylvinyl)quinoline, referred to as QD.

[0051] Example 2

[0052] This example verifies the recognition and binding of the QDs prepared in Example 1 to Aβs at the protein level.

[0053] 1. QD recognizes Aβs at the protein level and detects the kinetic process of Aβ peptide monomer fibrillation.

[0054] Aβs was formed by fibrillation of Aβ peptide monomers in a 37°C constant temperature incubator for 72 hours, with a final concentration of 20μM. Aβs was diluted with ultrapure water at a dilution ratio of 1:100, and 10μL was dropped on a copper mesh (300 mesh). After natural drying, it was stained with 1% (W / V) phosphotungstic acid for 30s, and finally washed twice with ultrapure water, dried overnight, and tested on a transmission electron microscope to observe the morphology of Aβs. The results are shown in Figure 3 shown. Figure 3 It was shown that the Aβs obtained by the above fibrillization method had a typical filamentous fiber structure.

[0055] Aβs were added to a black opaque 96-well plate (in which the Aβs gold standard dye THT or QD was pre-added), and the fluorescence and binding affinity of Aβs and THT or QD were tested at a constant temperature of 37°C using a multifunctional microplate reader. The results are shown in Figure 2. Figure 4 shown. Figure 4 It was shown that Aβs and THT had fluorescence when the excitation wavelength was 440nm (see Figure 4 a), where the binding affinity of THT to Aβs is 1193 nM (see Figure 4 b); After QD binds to Aβs, the fluorescence emission is enhanced 5 times at an excitation wavelength of 430 nm (see Figure 4 c), and its binding affinity to Aβs reached 33.91 nM (see Figure 4 d), which is two orders of magnitude higher than that of the traditional dye THT.

[0056] The Aβ peptide monomer was dissolved with 10mM NaOH, centrifuged at 4°C, 12000rpm for 0.5h to remove the insoluble components, and then diluted with PBS to a final concentration of 20μM, and the final concentration of QD was 50μM. The total test system was 200μL, and the system was placed in a black opaque 96-well plate and fibrillated in a 37°C constant temperature incubator. The fluorescence spectrum was measured at a constant temperature of 37°C using a multifunctional microplate reader every 1h. The excitation wavelengths of QD are 430nm and 570nm, respectively, and the excitation wavelength of the traditional dye THT is 440nm. At a constant temperature of 37°C on a multifunctional microplate reader, the fluorescence intensity change was measured every 1h, and the measurement results are as follows. Figure 5 shown. Figure 5 It was shown that QD can identify the fibrillation dynamics of Aβ peptide monomers.

[0057] 2. The fluorescence ratio of QD binding to Aβs at the protein level changes with pH.

[0058] By adjusting the pH of the test system, it was shown that the binding fluorescence of QD to Aβs has pH-sensitive properties, and the fluorescence ratio changes with pH. The total test system was 200 μL, the final concentration of Aβs was 20 μM, and the final concentration of QD was 50 μM. The pH of the system was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10 by HCl and NaOH, and then the changes in the fluorescence spectrum were measured on a multifunctional microplate reader. The fluorescence changes of QD binding to Aβs at different pH were irradiated with ultraviolet light, and the results are as follows Figure 6 shown. Figure 6 As the acidity increases, the red fluorescence of QDs combined with Aβs increases; as the alkalinity increases, the green fluorescence of QDs combined with Aβs increases. Therefore, as the pH of the system changes from acidic to neutral and alkaline, the fluorescence color changes from red to orange and then to green. The changes in the excitation and emission spectra of QDs combined with Aβs at different pH values ​​are shown in Figure 2. Figure 7 As shown, Figure 7 It shows that when the pH changes from acidic to neutral and alkaline, the excitation light intensity at 430nm increases and the excitation light intensity at 570nm decreases. It can be seen that the fluorescence spectrum results are consistent with Figure 6 The results are corresponding. The fluorescence wavelength also changes accordingly, the fluorescence intensity at 530nm increases, and the fluorescence intensity at 645nm decreases. The principle is that QD can be ionized and protonated under acidic conditions. After obtaining protons, it is converted into QD (H + ), after gaining electrons, the degree of electron delocalization increases and the spectrum red shifts.

[0059] pK a It is an important indicator of the ionization ability of a compound. There is no pK value for QD in the existing data. a Values, such as Figure 8 As shown in a, the pK values ​​of the QD components quinoline and dimethylaminophenyl are a The values ​​are 4.9, respectively, and the pK of QD is estimated. a The value is around 4.9. On this basis, the pK of QD was determined by fluorescence method. a The value is 5.0, such as Figure 8 That is to say, when pH is less than 5.0, QD in the solution is in the protonated form QD (H + ) is the main component; when pH>5.0, the solution is mainly in the form of molecular QDs. The pH-sensitive property of QDs can be used as a fluorescence ratio probe for Aβs. In addition, the changes in the fluorescence of QD-Aβs binding at different pH values ​​were investigated at the protein level. The results are as follows: Fig. 9 shown. Fig. 9 It shows that with the increase of pH, the green fluorescence at 530nm increases and the red fluorescence at 645nm decreases, resulting in a gradual increase in the G / R fluorescence ratio. a =5, the G / R fluorescence ratio increases most significantly. a When the value was ≥6, the increase of G / R fluorescence ratio tended to be gentle.

[0060] Example 3

[0061] This example tests the recognition and binding of the QDs prepared in Example 1 to Aβ plaques at the tissue level.

[0062] 1. QD identifies Aβ plaques at the tissue level.

[0063] 8-month-old 5×FAD mice were selected. After anesthesia, they were perfused with PBS and 4% PFA in turn. They were fixed with 4% PFA for 24 hours and then sliced ​​on a vibrating microtome with a thickness of 30 μm. The slices were permeabilized with 3% BSA + 0.3% triton-100 for 2 hours at room temperature, and stained with 1% BSA + 0.1% triton-100 diluted Aβ direct-labeled antibody 2C6-Cy5 overnight. Finally, 20 μM QD was used for staining for 15 minutes. After sealing, immunofluorescence imaging was performed on FV3000, where the excitation wavelength of QD was 570 nm and the excitation wavelength of 2C6-Cy5 was 647 nm. The results are shown in Figure 2. Fig.10 shown. Fig.10 The recognition effect of QD on Aβ plaques in brain tissue sections of 5×FAD model mice was demonstrated. Using the antibody 2C6-Cy5 that specifically recognizes Aβ as a reference, the two merged well, proving that QD can illuminate Aβ plaques in brain tissue very well.

[0064] 2. The binding fluorescence ratio of QDs to recognize Aβ plaques at the tissue level changes with pH.

[0065] The pH values ​​of the QD staining solution and the 50% glycerol mounting medium were adjusted to 2, 7, and 10 by HCl / NaOH solution to ensure that the pH of the brain slice tissue environment was fixed. The 20 μM QD staining solution was used for staining at room temperature for 15 min. After sealing, laser confocal microscopy imaging was performed, in which the excitation wavelengths of QD were 430 nm and 570 nm, respectively. The results were processed on FIJI, and the fluorescence ratio was G / R. The results are shown in Fig.11 shown. Fig.11 The results showed that the tissue level had similar results to the protein level. As the pH increased, the G / R fluorescence ratio of QDs and Aβ plaques increased.

[0066] 3. The changes in the binding fluorescence ratio of QD to recognize Aβ plaques show the changes in the pH microenvironment around Aβ plaques in the brain tissue of 5×FAD model mice of different ages.

[0067] 5×FAD model mice of different ages were selected, including 4 months, 8 months, and 13 months. After anesthesia, the mice were perfused with PBS and 4% PFA in turn, fixed with 4% PFA for 24 hours, and then sliced ​​on a vibrating microtome with a thickness of 30 μm. 20 μM QD was used for staining at room temperature for 15 minutes, and laser confocal microscopy was performed after sealing. The excitation wavelengths of QD were 430 nm and 570 nm, respectively. The results were processed on FIJI, and the fluorescence ratio was G / R. The results are shown in Fig.12 shown. Fig.12The results show that the fluorescence ratio G / R of QD binding to Aβ plaques decreases as the disease progresses, proving that the pH around extracellular Aβ plaques decreases, and this phenomenon basically exists in the entire brain slice. This finding shows that the progression of AD disease can be displayed by the influence of pH on the fluorescence ratio of QD binding to Aβ plaques.

[0068] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution of this disclosure can be achieved, and this document is not limited here.

[0069] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0070] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A fluorescence ratio probe, characterized in that The fluorescence ratio probe is 4-(4-dimethylaminophenylvinyl)quinoline, referred to as QD; the fluorescence ratio probe is used for imaging and detecting Aβs, and monitoring the pH change of the environment in which the Aβs are located.

2. The fluorescence ratio probe according to claim 1, characterized in that: The binding fluorescence ratio of the fluorescence ratio probe to the Aβs increases as the pH of the environment in which the Aβs are located increases.

3. The fluorescence ratio probe according to claim 2, characterized in that: In pK a =5, the fluorescence ratio of the fluorescence ratio probe to the Aβs increases to a peak value; at pK a When ≥6, the increase in the binding fluorescence ratio of the fluorescence ratio probe to the Aβs tends to be gentle.

4. The method for developing a fluorescence ratio probe according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: extract the smiles information of N,N-dimethylphenyl in the existing Aβs probe, and screen the compounds having the smiles information in the screening library; S2: Further screening compounds with Binding energy ≥ -7.5 kcal / mol from the compounds with the smiles information obtained in step S1 to screen out compounds with affinity for Aβs; S3: further screening out compounds that bind to the Aβs and have a fluorescence on response from the compounds screened in step S2; S4: The compound screened in step S3 is verified by fluorescence screening with the Aβs at the protein level, thereby obtaining the fluorescence ratio probe.

5. The development method according to claim 4, characterized in that: The existing Aβs probe in step S1 includes at least one of thioflavin derivatives, stilbene derivatives, curcumin derivatives, BODIPY, and DANIR derivatives.

6. The development method according to claim 4, characterized in that: The smiles information of the N,N-dimethylphenyl includes two types, namely CN(C)c1ccc(*)cc1 and C(*)c1ccc(N(C)C)cc1.

7. The development method according to claim 4, characterized in that: The screening library contains a total of 130,000 compounds, including 12,800 compounds in ZINC for sale and 200 commonly used dyes.

8. The development method according to claim 4, characterized in that: Step S2 first uses openbable to convert the SDF form of the compound with the smiles information obtained by step S1 into a PDB form for further batch molecular docking, and then screens out compounds with a Binding energy ≥ -7.5 kcal / mol.

9. The development method according to claim 4, characterized in that: Step S4 is to perform fluorescence screening verification at the protein level to verify whether the compound recognizes the Aβs at the protein level.

10. Use of the fluorescent ratio probe according to any one of claims 1 to 3 in the preparation of a product for diagnosing the disease progression of Alzheimer's disease, characterized in that: The product includes the fluorescent ratio probe.

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