A fluorescent ratiometric probe and its development method and application

Abstract

The present disclosure belongs to the field of fluorescent probes, and provides a fluorescent ratio probe and a development method and application thereof. The fluorescent ratio probe is 4-(4-dimethylaminostyryl) quinoline, referred to as QD, which is used for imaging and detection of beta-amyloid aggregates (referred to as Aβs) and monitoring of pH changes of the environment where the Aβs are located. The development method of the probe is also disclosed, and the application of the probe in preparation of a product for diagnosing the disease progression of Alzheimer's disease, which comprises the probe. The present disclosure provides a bifunctional fluorescent ratio probe QD which can light up Aβs and monitor the surrounding pH microenvironment, and has the characteristics of high affinity, good repeatability and simple operation. The QD has dual-color fluorescence when combined with Aβs, and the fluorescence ratio changes with the pH of the environment where the Aβs are located, so that the QD can be used to label Aβs in vivo and in vitro and display the pH microenvironment around the Aβs.

Description

Technical Field

[0001] This disclosure relates to the field of fluorescent probes, and more particularly to a fluorescent ratio probe, its development method, and its application. Background Technology

[0002] Alzheimer's disease (AD) has an insidious onset and is one of the most common forms of dementia. AD is highly age-related, and the number of patients is surging with the global aging population. Abnormal accumulation of Aβ aggregates (Aβs) is an early event in AD. Although the association of Aβ-formed plaques with AD disease staging is controversial, Aβs are an indirect reservoir of other toxic entities, and Aβ-formed plaque deposition is one of the two major pathological features of AD, making it a key target for early diagnosis and confirmation. Therefore, the development of probes targeting Aβs has been a hot research topic.

[0003] In recent years, the development of Aβs probes, whether classical or near-infrared fluorescent probes, has largely relied on modifying existing Aβs-recognizing probes as the parent skeleton. Commonly used skeletons include benzothiazoles, stilbene derivatives, curcumin derivatives, boron dipyrromethane, oxazine derivatives, cyanine, and alkene-based near-infrared fluorescent probes. Using methods based on parent skeleton modification ensures high affinity for Aβs. Furthermore, the structure can be directionally modified through chemical synthesis to efficiently control the optical properties, specificity, and lipophilicity of compounds. For example, previous studies have shown that increasing molecular conjugation to construct D-π-A / D-π-A-π-D conjugated systems can improve the redshift of emission spectra; and replacing charged groups increases the molecule's electroneutrality, improving blood-brain barrier permeability. However, these methods have limitations. First, they cannot discover new parent structures; second, the organic synthesis process is complex and costly, preventing large-scale synthesis for screening; and more importantly, these methods cannot directionally synthesize intelligent lighting probes to reduce fluorescence background. Therefore, a new strategy is urgently needed for the development of Aβs probes, which can screen intelligently activated 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, Alzheimer's disease (AD) is multifactorial, and multiple pathological changes in the patient's brain provide important evidence for early diagnosis, such as Aβ plaque deposition, neuroinflammation, oxidative stress, lipid metabolism, and autophagy disorders. These pathologies do not exist independently but are highly interdependent and mutually influential. To explore the connections between these pathologies, simultaneous detection using two single-response probes may result in overlaps in distribution and spectral location. Therefore, the development of bifunctional probes has significant application value in exploring the connections between pathologies. Currently, simultaneous detection of Aβ-related markers mainly focuses on biomarkers related to oxidative stress and lipid metabolism. Among these, bifunctional probes for Aβs and ROS include those for Aβs with H2O2, Aβs with ·OH, and Aβs with ONOO. - Simultaneous detection. Bifunctional probes for Aβs and lipid metabolism include those for the simultaneous detection of Aβs and lipid droplets. Although lysosomal autophagy impairment in AD is closely related to Aβs, and lysosomal autophagy impairment leads to damage to intracellular lysosomes, resulting in the leakage of lysosomes and protons, the impact of Aβs on the surrounding pH microenvironment is currently lacking due to the absence of bifunctional probes for Aβs and pH. Therefore, there is an urgent need to develop a bifunctional probe that can illuminate Aβs and monitor the surrounding pH microenvironment to aid in research on the influence and relationship between Aβs-induced lysosomal autophagy impairment and the pH microenvironment. Summary of the Invention

[0005] This disclosure provides a fluorescent ratio probe, its development method, and its application, to at least solve the above-mentioned technical problems existing in the prior art.

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

[0007] Specifically, Aβs are insoluble β-amyloid protein aggregates and are the main components of Aβ plaques in the brain tissue of AD transgenic mice. Increased acidity in the brain environment surrounding Aβs leads to increased red fluorescence when QD binds to Aβs; increased alkalinity leads to increased green fluorescence when QD binds to Aβs. That is, as the pH of the Aβs environment changes from acidic to neutral and then to alkaline, the fluorescence color changes from red to orange and then to green. Therefore, the change in fluorescence color after the fluorescent ratio probe binds to Aβs can be used to monitor pH changes in the Aβs environment.

[0008] As the pH of the environment in which Aβs resides changes from acidic to neutral and then to alkaline, the fluorescence intensity at 530 nm increases, while the fluorescence intensity at 645 nm decreases. The principle behind this is that QD can be ionized and, under acidic conditions, undergoes protonation, gaining a proton and transforming into QD(H). + The gain of electrons increases the degree of electron delocalization, resulting in a redshift in the spectrum. pK a QD is an important indicator of a compound's ionization ability, specifically its pK value. a The value is 5.0. That is, when pH < 5.0, QD exists in the protonated form QD(H). + It exists mainly in the form of QD; at pH > 5.0, it exists mainly in the form of molecules, so the pH-sensitive property of QD can be used as a fluorescent ratio probe for Aβs.

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

[0010] Specifically, as the pH of the environment in which Aβs are located increases, the green fluorescence at 530 nm increases, while the red fluorescence at 645 nm decreases, leading to a gradual increase in the binding fluorescence ratio. The binding fluorescence ratio refers to the ratio of green fluorescence at 530 nm to red fluorescence at 645 nm, i.e., Green 530nm / Red 645nm, abbreviated as G / R.

[0011] In one possible implementation, in pK a When pK = 5, the fluorescence ratio of the fluorescent ratio probe to Aβs increases to a peak; at pK a When the value is ≥6, the increase in the fluorescence ratio of the fluorescent ratio probe to the Aβs tends to level off.

[0012] According to a second aspect of this disclosure, a method for developing the aforementioned fluorescent ratio probe is provided, comprising the following steps:

[0013] S1: Extract the smiles information of N,N-dimethylphenyl from the existing Aβs probe, and screen for compounds with the smiles information in the screening library;

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

[0015] S3: Further screen the compounds selected in step S2 to identify compounds that exhibit a fluorescence-on response when binding to the Aβs;

[0016] S4: Verify the compounds screened in step S3 with the Aβs at the protein level through fluorescence screening to obtain the fluorescence ratio probe.

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

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

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

[0020] In one embodiment, the screening library contains 130,000 compounds, including 12,800 from ZINC for sale and 200 commonly used dyes.

[0021] In one possible implementation, step S2 first uses OpenBable to convert the SDF form of the compounds with the smiles information obtained in step S1 into 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 simulations between the compounds with the smiles information obtained in step S1 and the protein model of Aβs (PDB: 2BEG), step S2 ranks the compounds according to their affinity.

[0023] Specifically, most existing Aβs fluorescence-on probes are related to the twisted intramolecular charge transfer (TICT) effect. Fluorescence-once compounds require rotatable single or double bonds and a rigid conjugated structure that can be coplanar. When the molecule is not coplanar, it is in a fluorescence-off state due to the TICT effect; however, after binding to Aβs, the folded structure of Aβs inhibits bond rotation, making the rigid conjugated structure of the compound coplanar, suppressing the TICT effect, and thus putting the molecule in a fluorescence-on state. Based on this, step S3 further screens for compounds that exhibit a fluorescence-on response when bound to Aβs.

[0024] In one embodiment, step S4 involves verifying fluorescence screening at the protein level to confirm whether the compound recognizes the Aβs at the protein level.

[0025] According to a third aspect of this disclosure, the use of the above-described fluorescent ratio probe in the preparation of a product for diagnosing the disease progression of Alzheimer's disease, the product comprising the fluorescent ratio probe.

[0026] Specifically, the aforementioned fluorescent ratio probe can identify Aβ plaques in tissues, and the binding fluorescence ratio of the fluorescent ratio probe to Aβ plaques changes with variations in the pH microenvironment surrounding the Aβ plaques. Based on this, the disease progression of Alzheimer's disease can be demonstrated by observing the pH-dependent effect of the binding fluorescence ratio of the fluorescent ratio probe to Aβ plaques.

[0027] According to one possible implementation of this disclosure, at least the following beneficial effects are achieved:

[0028] This disclosure provides a bifunctional fluorescent ratiometric probe (OQ) that can illuminate Aβs and monitor the surrounding pH microenvironment. The QD binds to Aβs with dual-color fluorescence. The binding fluorescence ratio of QD to Aβs varies with the pH of the Aβs environment. It features high affinity, good reproducibility, and simple operation, enabling the labeling of Aβs in vivo and in vitro and the visualization of the pH microenvironment surrounding Aβs.

[0029] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description

[0030] The above and other objects, features, and advantages of this disclosure will become readily apparent from the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings. Several embodiments of this disclosure are illustrated in the drawings by way of example and not limitation, in which:

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

[0032] Figure 1 This illustration shows a schematic diagram of the types and structures of existing Aβs probes summarized in Embodiment 1 of this disclosure;

[0033] Figure 2 A schematic diagram of the TICT effect of the Aβs fluorescent activation probe in Embodiment 1 of this disclosure is shown;

[0034] Figure 3 The transmission electron microscope (TEM) image of Aβs in Embodiment 2 of this disclosure is shown;

[0035] Figure 4 The following diagrams show the fluorescence spectra and binding affinity results of THT or QD binding to Aβs in Embodiment 2 of this disclosure; wherein, a-THT binding to Aβs fluorescence spectrum, b-THT binding to Aβs binding affinity, c-QD binding to Aβs fluorescence spectrum, and d-QD binding to Aβs binding affinity.

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

[0037] Figure 6 The following are photographs showing the fluorescence changes of QD binding to Aβs at different pH values ​​in Example 2 of this disclosure;

[0038] Figure 7 The diagram shows the changes in excitation and emission spectra of QD binding with Aβs at different pH levels in Embodiment 2 of this disclosure; wherein, a - changes in excitation spectra of QD binding with Aβs at different pH levels, and b - changes in emission spectra of QD binding with Aβs at different pH levels.

[0039] Figure 8 The theoretical pK of QD in Embodiment 2 of this disclosure is shown. a Schematic diagram of pK values ​​and pK measured by fluorescence method a Value results diagram; where, the theoretical pK of a-QD is shown. a Schematic diagram of pK values, measured by b-fluorescence method. a Value results graph;

[0040] Figure 9 The graph shows the results of the binding fluorescence ratio of QD to Aβs (Green 530nm / Red 645nm) as a function of pH in Example 2 of this disclosure;

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

[0042] Figure 11 The graph shows the changes in the fluorescence ratio G / R of QD binding to Aβ plaques in brain slices at pH 2, 7, and 10 in Example 3 of this disclosure.

[0043] Figure 12 The diagram shows the changes in the G / R ratio of QD to Aβ plaque binding in brain tissue sections of 4-month-old, 8-month-old, and 13-month-old 5×FAD model mice in Example 3 of this disclosure. Detailed Implementation

[0044] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0045] Example 1

[0046] This embodiment develops a fluorescent ratiometric probe for imaging and detection of Aβs, as well as for monitoring pH changes in the environment in which Aβs exist. The specific development process is as follows:

[0047] (1) A summary of the existing chemical structures of Aβs probes, including thiosulfinate derivatives, stilbene derivatives, curcumin derivatives, BODIPY, DANIR derivatives, etc., is shown below. Figure 1 As shown, most probes share the structure N,N-dimethylphenyl, and previous studies have speculated that this structure is related to the recognition of Aβs. Therefore, compounds with the N,N-dimethylphenyl structure were extracted from the screening library. This resulted in the extraction of smiles for N,N-dimethylphenyl in some existing Aβs probe structures, including two forms: CN(C)c1ccc(*)cc1 and C(*)c1ccc(N(C)C)cc1. Next, compounds with these smiles were screened from the library, which contained 130,000 compounds, including 12,800 and 200 commonly used dyes from ZINC for sale. This screening yielded 566 compounds.

[0048] (2) The screened compounds should have a strong affinity for Aβs. In order to perform Auto vina molecular docking simulations between the compounds screened in step (1) and the protein model of Aβs (PDB: 2BEG), the compounds were ranked according to their affinity. First, the SDF form of the compounds obtained in step (1) was converted to 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 exhibit a fluorescent on-start response when bound to Aβs. In summary, most existing Aβs fluorescent on-start probes are related to the distorted intramolecular charge transfer (TICT) effect, such as... Figure 2 As shown. In simple terms, fluorescently activated compounds need to have rotatable single or double bonds and a rigid conjugated structure that can be coplanar. When the entire molecule is not coplanar, it is in a fluorescent-off state due to the TICT effect. However, after binding to Aβs, the folding structure of Aβs inhibits the rotation of chemical bonds, making the rigid conjugated structure of the compound coplanar, suppressing the TICT effect, and thus putting the molecule in a fluorescent-on state. This screening yielded three compounds.

[0050] (4) The compounds screened in step (3) are compared with Aβs at the protein level for fluorescence screening verification (i.e., verifying whether the screened compounds recognize Aβs at the protein level), and finally the fluorescent ratio probe 4-(4-dimethylaminostyryl)quinoline, abbreviated as QD, is obtained.

[0051] Example 2

[0052] This embodiment verifies the recognition and binding of Aβs by the QD prepared in Example 1 at the protein level.

[0053] 1. QD identifies Aβs at the protein level and detects the kinetics of Aβ peptide monomer fibrillation.

[0054] Aβs were synthesized from Aβ peptide monomers by fibrillation in a 37℃ incubator for 72 hours, with a final concentration of 20 μM. Aβs were diluted with ultrapure water at a ratio of 1:100. 10 μL of the diluted solution was placed on a 300-mesh copper grid, allowed to air dry, stained with 1% (w / v) phosphotungstic acid for 30 seconds, washed twice with ultrapure water, and dried overnight. The morphology of Aβs was then observed under a transmission electron microscope. The results are as follows: Figure 3 As shown. Figure 3 The results show that Aβs obtained using the above-mentioned fiberization method have a typical filamentous fiber structure.

[0055] Aβs was added to black opaque 96-well plates (the wells were pre-filled with the gold standard dye THT or QD for Aβs). The fluorescence and binding affinity of Aβs to THT or QD were measured using a multi-functional microplate reader at 37°C. The results are as follows: Figure 4 As shown. Figure 4 The results showed that Aβs binding to THT exhibited fluorescence at an excitation wavelength of 440 nm (see [link to data]). Figure 4 (a) where the binding affinity of THT to Aβs is 1193 nM (see a). Figure 4 (b); QD, after binding to Aβs, exhibits a 5-fold enhanced fluorescence emission at an excitation wavelength of 430 nm (see [link]). Figure 4 (c), and its binding affinity to Aβs reaches 33.91 nM (see c). Figure 4 The d) is two orders of magnitude higher than that of the traditional dye THT.

[0056] The Aβ peptide monomer was dissolved in 10 mM NaOH and centrifuged at 12000 rpm for 0.5 h at 4 °C to remove insoluble components. It was then diluted with PBS to a final concentration of 20 μM, and the final concentration of QD was 50 μM. The total test volume was 200 μL, which was placed in a black opaque 96-well plate and incubated at 37 °C for fibrillation. Fluorescence spectra were measured every 1 h using a multi-mode microplate reader at 37 °C. The excitation wavelengths of QD were 430 nm and 570 nm, while the excitation wavelength of the traditional dye THT was 440 nm. Fluorescence intensity changes were measured every 1 h using a multi-mode microplate reader at 37 °C. The results are shown below. Figure 5 As shown. Figure 5 The results show that QD can identify the fibrinolysis kinetics of Aβ peptide monomers.

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

[0058] The pH of the test system was adjusted to demonstrate that the fluorescence of QD recognizing Aβs exhibits pH sensitivity, with the fluorescence ratio changing with pH. The total test volume was 200 μL, with a final Aβs concentration of 20 μM and a QD concentration of 50 μM. The pH of the system was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10 using HCl and NaOH, and the changes in fluorescence spectra were then measured using a multi-functional microplate reader. The fluorescence changes of QD binding to Aβs at different pH levels were also observed under UV light irradiation, and the results are shown below. Figure 6 As shown. Figure 6 The results show that, with increasing acidity, the red fluorescence of QD binding to Aβs increases; with increasing alkalinity, the green fluorescence of QD binding to Aβs increases. Therefore, as the pH of this system changes from acidic to neutral and then to alkaline, the fluorescence color changes from red to orange and then to green. The changes in the excitation and emission spectra of QD binding to Aβs at different pH values ​​are shown below. Figure 7 As shown, Figure 7 The results show that during the pH change from acidic to neutral and then alkaline, the excitation light intensity at 430 nm increases, while the excitation light intensity at 570 nm decreases. This is consistent with the fluorescence spectral results. Figure 6 The results correspond. Similarly, the fluorescence wavelength changes accordingly, with the fluorescence intensity increasing at 530 nm and decreasing at 645 nm. The principle is that QD can be ionized, protonated under acidic conditions, and converted into QD(H) after gaining a proton. + When electrons are gained, the degree of electron delocalization increases, resulting in a red shift in the spectrum.

[0059] pK a It is an important indicator of a compound's ionization ability, but the existing data do not include the pK of QD. a Value, such as Figure 8 As shown in a, the components of QD are quinoline and dimethylaminophenyl pK a The values ​​are 4.9, guessing QD's pK a The value was around 4.9. Based on this, the pK of QD was determined using a fluorescence method. a The value is 5.0, such as Figure 8 As shown in b. That is, at pH < 5.0, QD in the solution exists in the protonated form QD(H). + QD exists predominantly in the solution at pH > 5.0; it exists mainly in molecular form. The pH-sensitive nature of QD can be used as a fluorescent ratio probe for Aβs. Furthermore, the changes in Green 530nm / Red 645nm (G / R) fluorescence of QD binding to Aβs at different pH levels were investigated at the protein level. The results are as follows: Figure 9 As shown. Figure 9 The results showed that with increasing pH, green fluorescence at 530 nm increased, while red fluorescence at 645 nm decreased, leading to a gradual increase in the G / R fluorescence ratio, particularly at pK. a The increase in the G / R fluorescence ratio is most pronounced near pK = 5. a When the ratio is ≥6, the increase in the G / R fluorescence ratio tends to level off.

[0060] Example 3

[0061] This embodiment tested the recognition and binding of the QD prepared in Example 1 to Aβ plaques at the tissue level.

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

[0063] Eight-month-old 5×FAD mice were anesthetized and then subjected to cardiac perfusion with PBS and 4% PFA. After fixation with 4% PFA for 24 hours, sections were prepared on a vibratory microtome to a thickness of 30 μm. The sections were blocked for 2 hours at room temperature with 3% BSA + 0.3% Triton-100 permeabilized membrane. The sections were then stained overnight with a 1% BSA + 0.1% Triton-100 diluted Aβ direct-label antibody 2C6-Cy5. Finally, the sections were stained with 20 μM QD for 15 minutes. After mounting, immunofluorescence imaging was performed on an FV3000 microscope. The excitation wavelength for QD was 570 nm, and for 2C6-Cy5, it was 647 nm. The results are shown below. Figure 10 As shown. Figure 10 The study demonstrated the effectiveness of QD in recognizing Aβ plaques in brain tissue sections from 5×FAD model mice. Using the antibody 2C6-Cy5, which specifically recognizes Aβ, as a reference, the two antibodies merged well, proving that QD can effectively illuminate Aβ plaques in brain tissue.

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

[0065] The pH of the QD staining solution was adjusted to 2, 7, and 10 using HCl / NaOH solution, and the pH of the 50% glycerol mounting medium was also adjusted to 2, 7, and 10 to ensure a constant pH environment for the brain slices. Staining was performed at room temperature using 20 μM of the above QD staining solution for 15 min. After mounting, laser confocal microscopy was performed, with QD excitation wavelengths of 430 nm and 570 nm. The results were processed on FIJI, with a fluorescence ratio of G / R. The results are shown below. Figure 11 As shown. Figure 11 The results showed similar patterns at the tissue and protein levels, with the G / R fluorescence ratio of QD to Aβ plaques increasing with increasing pH.

[0066] 3. Changes in the binding fluorescence ratio of QD to Aβ plaques demonstrate the changes in the pH microenvironment surrounding Aβ plaques in the brain tissue of 5×FAD model mice of different ages.

[0067] Five-fold FAD model mice of different ages (4 months, 8 months, and 13 months) were selected. After anesthesia, the mice underwent cardiac perfusion with PBS and then 4% PFA, followed by fixation with 4% PFA for 24 hours. Sections were then prepared on a vibratory microtome to a thickness of 30 μm. The sections were stained with 20 μM QD for 15 min at room temperature, mounted, and then subjected to laser confocal microscopy. The excitation wavelengths of QD were 430 nm and 570 nm. The results were processed on FIJI with a fluorescence ratio of G / R. The results are shown below. Figure 12 As shown. Figure 12The results showed that the fluorescence ratio (G / R) of QD binding to Aβ plaques decreased with disease progression, demonstrating a decrease in pH around extracellular Aβ plaques, and this phenomenon was prevalent throughout the entire brain slice. This finding suggests that the progression of Alzheimer's disease can be visualized by the pH-dependent influence of 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 rearrange, add, or delete steps. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution of this disclosure can be achieved, and this is not limited herein.

[0069] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means two or more, unless otherwise explicitly specified.

[0070] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. The application of fluorescent ratiometric probes in the preparation of products for diagnosing the disease progression of Alzheimer's disease, characterized in that, The product includes a fluorescent ratio probe, which is 4-(4-dimethylaminostyryl)quinoline.

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