Congo red derivative photocatalytic probe as well as preparation method and use method thereof

Abstract

The invention relates to the technical field of application of Congo red derivative photocatalytic probes, in particular to a Congo red derivative photocatalytic probe as well as a preparation method and a use method thereof.The Congo red derivative photocatalytic probe comprises a compound I, the compound I is selected from at least one of the Congo red derivative photocatalytic probes, and the Congo red derivative photocatalytic probe comprises two arenes, arene represents a heteroaromatic ring, and the heteroaromatic ring is selected from one of a benzene ring, furan, thiophene and a naphthalene ring. The Congo red derivative photocatalytic probe provided by the invention can specifically mark amyloid plaques in AD brain tissue slices, the binding affinity is remarkably improved (Kd of Abeta1-40 is equal to 0.07 mu m), the molecular heterogeneity of amyloid deposition and the key regulation and control effect of mitochondrial autophagy-lysosomal axis are disclosed, a new tool is provided for AD pathological mechanism research, and the Congo red derivative photocatalytic probe has a wide application prospect. And cross-brain region (hippocampus / cortex) proteome analysis and pathological mechanism analysis are supported, and the molecular structure and application in neurodegenerative disease research are simulated to be protected.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of application of Congo red derivative photocatalytic probes, and particularly relates to a Congo red derivative photocatalytic probe, a preparation method and a use method thereof. BACKGROUND

[0002] The existing photocatalytic proximity labeling technology mainly relies on Type II photosensitizers (such as singlet oxygen) to realize biological labeling, which is easily disturbed in a complex pathological environment (such as amyloid deposition) and the labeling efficiency is limited. The existing technology modifies the Congo red skeleton through molecular engineering, introduces halogen atoms and heterocyclic structures, and endows it with Type I photosensitizer characteristics, while retaining excellent binding ability to amyloid (Kd of Aβ 1-40 Based on this, the probe P5 can precisely mediate the proximity labeling of amyloid deposition under light irradiation, for the first time realizing micro-cleavage and high-resolution proteomic analysis of amyloid plaques in the brain tissue (hippocampus and cortex) of Alzheimer's disease mice, revealing the core regulatory role of the "mitophagy-lysosome axis" in the pathological process, and providing a new direction for targeted treatment strategies.

[0003] Abnormal aggregation of amyloid is closely related to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Precise detection and analysis of the molecular composition and spatial distribution of amyloid deposition is of great significance for disease mechanism research and development of diagnostic tools. Traditional detection techniques (such as tissue staining) can locate amyloid deposition, but cannot realize in-situ analysis at the molecular level.

[0004] In recent years, molecular imaging probes (such as thioflavin T and Congo red derivatives) have been widely used in in vitro fluorescence detection and PET tracer development by binding to the β-sheet structure of amyloid protein, such as classic amyloid targeting probes. Based on their fluorescence enhancement properties after binding to the β-sheet, they offer advantages such as high signal-to-noise ratio, but lack chemical cross-linking capabilities and cannot capture molecular information at the binding site. Structural modifications (such as introducing fluorescent groups or radioactive isotopes) can improve targeting and imaging dynamics. However, they are still limited by visible light wavelengths and have insufficient tissue penetration. Probes optimized based on the BODIPY or curcumin backbone extend the emission wavelength to the near-infrared region, making them suitable for in vivo imaging. This significantly improves the sensitivity of fluorescence imaging and PET detection. However, existing probes can only provide macroscopic binding information. For example, component analysis techniques analyze peptide composition after enzymatically digesting amyloid deposits, but this requires destroying the sample's spatial structure and cannot distinguish between in-situ bound proteins and contaminants. Furthermore, the molecular composition of the deposits cannot be resolved by enzymatically digesting amyloid deposits to analyze peptide composition. Emerging proximity labeling technologies, such as APEX2, utilize photosensitizers (e.g., methylene blue) to generate reactive oxygen species (ROS) under light, oxidizing neighboring biomolecules and coupling them with tags like biotin to achieve microenvironment analysis. Previous studies have used this strategy for protein-protein interaction networks and subcellular organelle transcriptome localization, but it has not yet been combined with amyloid-targeting probes. Component analysis still relies on liquid chromatography-mass spectrometry (LC-MS / MS), but this technique requires sample separation, has low spatial resolution, and is prone to artifacts. Summary of the Invention

[0005] The purpose of this invention is to solve the problem that it is difficult to prepare Congo red derivative photocatalytic probes in the prior art and that there is a lack of targeted applications. The invention proposes a Congo red derivative photocatalytic probe, its preparation method and application method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The Congo red derivative photocatalytic probe includes a compound I selected from at least one of the Congo red derivative photocatalytic probes, and the Congo red derivative photocatalytic probe includes two arenes, where each arene represents an aromatic heterocycle.

[0008] Preferably, the aromatic heterocycle is selected from one of the following: benzene ring, furan, thiophene, and naphthalene ring.

[0009] A method for preparing a Congo red derivative photocatalytic probe, the method comprising the following steps:

[0010] Step S1, solvent and atmosphere preparation;

[0011] Step S2: Cooling in an ice bath and adding reagents;

[0012] Step S3: Low-temperature stirring activation;

[0013] Step S4, introduction and reaction of aromatic aldehydes;

[0014] Step S5, reaction quenching and extraction;

[0015] Step S6: Purify the target product by column chromatography.

[0016] Preferably, step S1, solvent and atmosphere preparation, refers to dissolving the tetraethyl bisphosphonate compound in THF under an argon atmosphere;

[0017] In step S2, ice bath cooling and reagent addition refers to slowly adding sodium hydride in batches after the ice bath has cooled to 0°C.

[0018] In step S3, the low-temperature stirring activation refers to stirring the resulting mixture at 0°C for 30 minutes.

[0019] In step S4, the aromatic aldehyde introduction and reaction refers to the subsequent addition of aromatic aldehyde II and stirring at room temperature for 16 hours.

[0020] In step S5, after the reaction is completed, the reaction is quenched with ice water and then the crude organic phase product is extracted with ethyl acetate.

[0021] Step S6, column chromatography purification of the target product, refers to purifying the target product I using column chromatography.

[0022] Preferably, the tetraethyl is (2-methoxy-1,4-phenylene)bis(methylene);

[0023] In step S3, the substrate is stirred for 30 minutes during low-temperature stirring activation until the base reacts fully.

[0024] In step S4, aromatic aldehyde II is first added to the activated system during the reaction, then the ice bath is removed, and then the reaction is carried out at room temperature for 16 hours.

[0025] In step S5, the remaining active reagent is quenched with ice water during reaction quenching and extraction.

[0026] The method of using the Congo red derivative photocatalytic probe, wherein the method of use refers to its precise application in the pathological analysis of brain tissue sections from Alzheimer's disease mice.

[0027] Preferably, the Congo red derivative photocatalytic probe has the characteristic of efficiently labeling amyloid deposits through a Type I photosensitization pathway.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1. The Congo red derivative photocatalytic probe provided by this invention can specifically label amyloid plaques in AD brain tissue slices, with significantly enhanced binding affinity (Aβ). 1-40 Kd = 0.07 μm.

[0030] 2. The Congo red derivative photocatalytic probe provided by this invention achieves efficient micro-cutting of amyloid plaques through proximity labeling mediated by Type I reactive oxygen species, avoiding non-specific interference.

[0031] 3. The Congo red derivative photocatalytic probe provided by this invention supports cross-brain region (hippocampus / cortex) proteomic analysis, revealing the molecular heterogeneity of amyloid deposition and the key regulatory role of the mitochondrial autophagy-lysosome axis, providing a new tool for the study of AD pathological mechanisms.

[0032] In summary, the Congo red derivative photocatalytic probe proposed in this invention can perform photocatalytic proximity labeling of amyloid plaques in brain slices from Alzheimer's disease (AD) model mice, supporting cross-brain region (hippocampus / cortex) proteomic analysis and pathological mechanism elucidation, and aims to protect its molecular structure and its application in neurodegenerative disease research. Attached Figure Description

[0033] Figure 1 Here is a schematic diagram of the chemical structure of compound I;

[0034] Figure 2 This is a schematic diagram of the chemical structure of compound II;

[0035] Figure 3 This is a schematic diagram of the synthesis route;

[0036] Figure 4 A schematic diagram for verifying the Type I photosensitization mechanism of the probe molecule;

[0037] Figure 5 This is a schematic diagram illustrating the high binding affinity of probe molecules for amyloid protein.

[0038] Figure 6 Schematic diagram of probe molecules selectively labeling aggregated proteins;

[0039] Figure 7 A schematic diagram illustrating the identification of protein components across brain regions using photocatalytic proximity labeling technology for probe molecules. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0041] Reference Figures 1-7The Congo red derivative photocatalytic probe, as it should be noted, is a molecular probe that enhances detection sensitivity through photocatalysis. Based on the structural modification of Congo red, it specifically binds to amyloid protein deposits in the brains of Alzheimer's disease mice for pathological labeling analysis. This type of probe can perform photocatalytic proximity labeling of amyloid plaques in brain slices from Alzheimer's disease (AD) model mice, supporting cross-brain region (hippocampus / cortex) proteomics analysis and pathological mechanism elucidation, aiming to protect its molecular structure and application in neurodegenerative disease research. It includes a compound I, selected from at least one of the Congo red derivative photocatalytic probes, and the Congo red derivative photocatalytic probe includes two arenes. Arenes represent aromatic heterocycles, which are cyclic compounds containing one or more non-carbon atoms and possess aromaticity. Due to the delocalization of π electrons, aromatic heterocycles are more stable than their non-aromatic counterparts and are relatively inert chemically. The atoms (including heteroatoms) on the ring are usually coplanar to ensure effective overlap of π orbitals. Aromatic heterocycles are the core structure of many natural products, drugs, and materials. Among these are drugs such as antibiotics (penicillin contains a thiazole ring), antihistamines (chlorpheniramine contains a pyridine ring), and anticancer drugs (paclitaxel contains an indole ring); biomolecules such as bases in nucleic acids (purines, pyrimidines), porphyrin rings in hemoglobin (containing pyrrole rings), and corrin rings in vitamin B12; and functional materials such as conductive polymers (polypyrrole) and organic light-emitting materials (containing pyridine or quinoline structures).

[0042] Congo red derivative photocatalytic probes specifically labeled amyloid plaques in AD brain tissue sections, with significantly enhanced binding affinity (Aβ). 1-40 With a Kd of 0.07 μm, this method enables efficient micro-dissection of amyloid plaques through proximity labeling mediated by Type I reactive oxygen species, avoiding non-specific interference. It supports cross-brain region (hippocampus / cortex) proteomic analysis, revealing the molecular heterogeneity of amyloid deposition and the key regulatory role of the mitochondrial autophagy-lysosome axis, providing a new tool for the study of AD pathological mechanisms.

[0043] Aromatic heterocycles are selected from one of the following: benzene ring, furan, thiophene, and naphthalene ring. Aromatic rings refer to cyclic compounds with aromatic properties, including two types:

[0044] First, aromatic hydrocarbons (non-heterocyclic): composed only of C and H, such as benzene rings and naphthalene rings.

[0045] Second, aromatic heterocycles: the ring contains at least one non-carbon heteroatom (such as O, S, N), such as furan and thiophene.

[0046] The core difference between the two lies in whether they contain heteroatoms - heterocycles must contain non-carbon atoms, while benzene rings and naphthalene rings are pure carbon rings and do not belong to heterocycles.

[0047] A method for preparing a Congo red derivative photocatalytic probe, comprising the following steps:

[0048] Step S1, solvent and atmosphere preparation, refers to dissolving the tetraethyl bisphosphonate compound in THF under an argon atmosphere;

[0049] Step S2, ice bath cooling and reagent addition, refers to slowly adding sodium hydride in batches after the ice bath has cooled to 0°C.

[0050] Step S3, low-temperature stirring activation, refers to stirring the obtained mixture at 0°C for 30 min;

[0051] Step S4, Aromatic Aldehyde Introduction and Reaction: Step S4, Aromatic Aldehyde Introduction and Reaction refers to the subsequent addition of aromatic aldehyde II and stirring at room temperature for 16 hours. It should be noted that high-frequency stirring was performed using a nuclear magnetic resonance spectrometer, and the nuclear magnetic resonance spectrometer was Bruker AVANCE III 700MHz.

[0052] Step S5, reaction quenching and extraction, refers to the process after the reaction is completed, quenching with ice water, and then extracting the crude organic phase with ethyl acetate. During this process, a confocal fluorescence microscope (Olympus FV1000 FluoView) is used. TM Confocalmicroscope;

[0053] Step S6, column chromatography purification of the target product. Step S6, column chromatography purification of the target product refers to the purification of target product I by column chromatography. In this process, an OrbitrapExploris480 mass spectrometer is used.

[0054] Further explanation:

[0055] Tetraethyl is (2-methoxy-1,4-phenylene)bis(methylene);

[0056] Step S3 involves low-temperature stirring for 30 minutes until the substrate and base fully react. This is achieved by using the base to remove active hydrogen atoms from the substrate (such as those in carboxylic acids, phenols, and alkynes), or to initiate elimination or nucleophilic substitution reactions. Stirring accelerates molecular diffusion through mechanical force, ensuring uniform mixing of the substrate and base, increasing the collision probability, and shortening the reaction induction period. Furthermore, low-temperature conditions suppress side reactions and stabilize intermediates. Low temperatures reduce the reaction rate, selectively generating the target product. Certain reaction intermediates (such as carbanions and Grignard reagents) are more stable at low temperatures, preventing decomposition or rearrangement.

[0057] Low temperature control can be achieved in the following ways:

[0058] Ice bath (0℃): The most commonly used method, suitable for reaction temperatures between 0 and 5℃.

[0059] Ice-salt bath (-5℃~-20℃): such as NaCl / ice (-18℃) or CaCl2·6H2O / ice (-55℃), the ratio needs to be selected according to the target temperature.

[0060] Cryogenic coolant circulation pump: Precisely controls low temperatures of -40℃ to -80℃, suitable for temperature-sensitive reactions.

[0061] Liquid nitrogen cooling: used for ultra-low temperature reactions (<-80℃), requires a cryogenic bath (such as a liquid nitrogen / ethanol bath).

[0062] Most reactions reach equilibrium or become fully mixed within 30 minutes, but the specific progress needs to be monitored using TLC (thin-layer chromatography) or GC (gas chromatography). It is crucial to use a cryothermometer or thermocouple to monitor the temperature in real time to avoid localized overheating. Solvents that do not solidify at low temperatures should be selected (e.g., THF remains liquid at -77°C, while diethyl ether solidifies at -116°C). When using a cryogenic bath, wear antifreeze gloves and goggles to avoid skin contact. After the reaction is complete, the temperature should be slowly raised to room temperature before quenching (e.g., by adding dilute acid to neutralize the base) to prevent sudden heating that could lead to product decomposition. The structure of reaction intermediates can be tracked using low-temperature NMR (e.g., at -50°C) to verify the reaction mechanism.

[0063] Step S4: Aromatic aldehyde introduction and reaction. First, add aromatic aldehyde II to the activated system, then remove the ice bath, and proceed with the reaction at room temperature for 16 hours. If there is still raw material remaining after 16 hours, appropriate heating (e.g., 40-60℃) or additional catalyst can be added. This step-by-step operation helps to achieve:

[0064] Add aromatic aldehydes at low temperatures: to avoid side reactions between the aldehyde group and excess base or nucleophile.

[0065] Removing the ice bath and raising the temperature provides sufficient energy to continue the reaction at room temperature, promoting the conversion of the intermediate into the target product.

[0066] Long-term room temperature reaction: Most condensation reactions require several hours to tens of hours to reach equilibrium.

[0067] When adding aromatic aldehydes, please note the following:

[0068] Solid aldehyde: After preparing a solution, slowly add it dropwise using a constant pressure dropping funnel (10-30 min);

[0069] Liquid aldehydes: can be added directly dropwise, but the rate needs to be controlled to avoid local overheating.

[0070] To further clarify, the feeding temperature should be maintained in an ice bath (0-5℃) to avoid contact between the aldehyde group and excessive alkali.

[0071] Various abnormal situations can easily occur during this process:

[0072] The first type produces no products.

[0073] Possible causes: insufficient alkali strength, low aldehyde activity, or excessive water content in the solvent.

[0074] Solutions: Use a stronger base (such as NaHMDS), increase the reaction temperature, and dry the solvent again.

[0075] The second type generates a large number of byproducts.

[0076] Possible causes: improper temperature control, excessive alkali, or excessively long reaction time.

[0077] Solutions: Lower the feeding temperature, add the alkali in batches, and shorten the reaction time.

[0078] The third type involves difficulties in product separation.

[0079] Possible reason: It generates isomers with similar polarity.

[0080] Solution: Adjust column chromatography conditions and use preparative HPLC for separation.

[0081] In summary, by controlling the order of addition, temperature, and time, the "aromatic aldehyde introduction-heating reaction" strategy can efficiently construct carbon-carbon bonds and has wide applications in the total synthesis of natural products and the preparation of pharmaceutical intermediates. In practice, conditions need to be optimized according to the substrate structure and reaction type, and the reaction process needs to be monitored using modern analytical techniques.

[0082] Step S5: Quenching of the reaction and extraction with ice water to quench the remaining active reagent.

[0083] It should be noted that the optimal preparation method for the Congo red derivative photocatalytic probe was obtained through one set of examples and four sets of test cases:

[0084] Example 1

[0085] Bisphosphonate (204 mg, 0.5 mM) was dissolved in THF (5 mL) under an inert atmosphere and cooled to 0 °C in an ice bath. Sodium hydride (60 mg, 1.5 mM, 60 wt. dispersed in mineral oil) was added incrementally, and the resulting mixture was stirred at 0 °C for 30 min. A separate THF solution of aldehyde (219 mg, 1.25 mM) was prepared and added to the reaction mixture, followed by stirring at room temperature for 16 h. After the reaction was complete, the mixture was carefully quenched with ice-cold water and extracted three times with ethyl acetate. The combined organic layers were washed with saturated NaHCO3 solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give the target compound (56 mg, yield 25.0%).

[0086] The synthesis route in this embodiment is referenced in the appendix of the instruction manual. Figure 3 P2 was tested using nuclear magnetic resonance spectroscopy, and its structure was characterized and its purity determined using liquid chromatography-high-resolution time-of-flight mass spectrometry (Q-TOF6540). The results are as follows:

[0087] 1 HNMR(400MHz, DMSO-d6)7.88(s,1H),7.40(s,1H),7.39(d,J=16.1Hz,1H),7.23-7.16(m,2H),7.12-7.06(m,2H) ,6.71(d,J=3.4Hz,1H),6.69-6.66(m,2H),6.59(d,J=3.4Hz,1H),3.95(s,3H)ppm.HRMS(m / z)Anal.Calc'dforC 19 H 14 O3Br2(M+Na)+:470.9202,Found(M+Na) + :470.9207.

[0088] Test Example 1

[0089] Verification of P5's Type I photosensitization mechanism

[0090] The probe P5 (1.0 mM) was reacted with superoxide anion radicals (O2) respectively. .- The scavenging agents were DMPO (25.0 mM, methanol solution), BMPO (100.0 mM, aqueous solution), and singlet oxygen. 1 O2) was mixed with TEMP (200.0 mM, aqueous solution) as a scavenger, and the illumination (white light, 25 mW·cm⁻¹) was detected by electron spin resonance (EPR). -2The reactive oxygen species (ROS) signal was measured at 20 min. Vitamin C (5.0 mM) was added to the experimental groups as a quencher of superoxide anions and hydroxyl radicals, and rhodamine was used as a... 1 The positive control for O2 detection. The results showed that P5 light irradiation significantly generated superoxide anions and hydroxyl radicals, but produced almost no singlet oxygen.

[0091] The results are as shown in the attached instruction manual. Figure 4 As shown, P5 generates Type I free radicals (O2) under white light irradiation. .- It produces ·OH, but does not produce singlet oxygen (1O2).

[0092] Test Example 2

[0093] P5 has a high affinity for amyloid protein.

[0094] P5 was adjusted to different concentration gradients (0.0-50.0 μM) with DMSO and then incubated with amyloid protein (25.0 μM) at room temperature for 30 min. Subsequently, the fluorescence spectrum of each sample was collected individually using a 96-well plate reader. Finally, the fluorescence intensity and probe concentration were fitted to determine the dissociation constant (Kd).

[0095] The results are as shown in the attached instruction manual. Figure 5 As shown, P5 has a high binding affinity for amyloid protein.

[0096] Test Example 3

[0097] P5 selectively labels aggregated proteins

[0098] Aggregated tau-K18 and monomeric tau-K18 (150 μM each) were mixed with P5 (5-25 μM concentration gradient), and propargylamine (10 mM) was added as a labeled substrate. After incubation at room temperature for 30 min, the mixture was irradiated with white light (25 mW / cm²). 2 The system was precipitated with acetone at -20℃ (4 volumes, 1 h), centrifuged (13,000 rpm, 10 min), and the precipitate was redissolved with 10% SDS. Click reaction solution (containing 1.7 mM TTBTA, 50 mM CuSO4, 50 mM TCCEP, and 50 μM TMR-N3) was added, and the reaction was carried out in the dark for 1 h. The mixture was then purified again by acetone precipitation / centrifugation. The final sample was analyzed by SDS-PAGE and dot blot analysis.

[0099] The results are as shown in the attached instruction manual. Figure 6 As shown, p5 selectively labels aggregated proteins, and the labeling contrast becomes more pronounced as the probe concentration increases.

[0100] Test Example 4

[0101] P5 photocatalytic proximity labeling technology reveals the identification of protein components across brain regions;

[0102] Brain tissue sections were subjected to antigen retrieval in sodium citrate buffer (10 mM, pH 6.0, 95℃, 20 min) followed by the following sequential steps: endogenous biotin blocking (room temperature, 30 min), P5 probe staining in the dark (150 μM, 30 min), washing three times with PBS, incubation with amino-PEG2-biotin in the dark (10 mM, 10 min), and irradiation with white light (25 mW / cm²). 2 After treating the samples with lysis buffer (6M guanidine hydrochloride, containing 1% protease / phosphatase inhibitor), the solution was diluted to 1M guanidine hydrochloride and incubated with streptavidin-agarose resin (Cytiva, catalog number 17511301) at room temperature for 2 hours. The resin was washed twice with 6M guanidine hydrochloride / 2M NaCl, resuspended in 0.3M guanidine hydrochloride, and then subjected to reduction (10mM MTT, 1 hour) and alkylation (20mM iodoacetamide, protected from light for 30 minutes). The samples were washed twice with ammonium bicarbonate (50mM) and digested with trypsin (1:30, w / w) at 37°C for 12 hours. The enzymatic digests were centrifuged, lyophilized, and loaded onto mass spectrometers for data analysis.

[0103] The experimental results are as shown in the attached instruction manual. Figure 7 As shown, 16 common proteins (such as APP and ApoE) were identified in the hippocampal and cortical amyloid deposition lesions, suggesting abnormalities in the mitochondrial-lysosomal pathway.

[0104] The method of using Congo red derivative photocatalytic probes refers to their precise application in the pathological analysis of brain tissue sections from Alzheimer's disease mice. The core structure of Congo red is bis(azobiphenyl). By modifying sulfonate groups or introducing photosensitive groups, the targeting and photocatalytic activity are enhanced. Hydrophobic groups help to enhance the binding affinity with the Aβ hydrophobic region, and photosensitive groups help to generate singlet oxygen or free radicals after absorbing light of specific wavelengths, inducing local photochemical reactions.

[0105] The application of Congo red derivative photocatalytic probes can be achieved through the following steps:

[0106] Probe solution preparation → Incubation condition optimization → Photocatalytic reaction activation → Multimodal detection

[0107] By optimizing probe design and strictly controlling experimental conditions, Congo red derivative photocatalytic probes can provide highly sensitive and specific detection tools for Alzheimer's disease (AD) pathology research, aiding in the elucidation of disease mechanisms and drug development. In practical applications, multiple validation methods must be combined, and care must be taken to avoid the impact of technical errors on the results. A more detailed operational procedure is as follows:

[0108] 1. Preparation of brain tissue sections (frozen or paraffin sections)

[0109] 2. Dewaxing and hydration (only required for paraffin sections)

[0110] Blocked with 3.5% BSA / PBS (room temperature, 30 min).

[0111] 4. Incubate with probe working solution (1-10 μM, 37℃, 1 h, protected from light)

[0112] 5. Wash with PBS (3 x 5 min)

[0113] 6. Confocal microscopy observation (excitation 635nm, emission 650-750nm)

[0114] 7. Photocatalytic activation (635nm laser, 20mW / cm²) 2 , irradiate for 2 minutes)

[0115] 8. Secondary imaging (comparing signal changes before and after photocatalysis)

[0116] 9. Image analysis and pathological scoring

[0117] Congo red derivative photocatalytic probes are characterized by highly efficient labeling of amyloid deposition via a Type I photosensitization pathway. The core advantage of Type I probes lies in:

[0118] 1. High selectivity and stability

[0119] The free radical reaction occurs only near the probe-Aβ complex, avoiding damage to non-targeted tissues.

[0120] Covalently labeled products are resistant to washing and tissue processing, making them suitable for long-term preservation of pathological specimens.

[0121] 2. Penetration and Deep Imaging

[0122] Near-infrared photosensitizers (such as indocyanine green ICG derivatives) can penetrate brain tissue (to a depth of 2-3 mM).

[0123] By combining photoacoustic imaging technology, the penetration limit of optical microscopes (~100μm) has been broken.

[0124] 3. Potential for multimodal applications

[0125] Fluorescence imaging: Directly detect the fluorescence signal of the probe-Aβ complex.

[0126] Mass spectrometry analysis: Aβ is quantified by characteristic peaks of oxidation products (e.g., m / z 240.08 corresponds to dityrosine).

[0127] Electron microscopy: Combined with metal markers (such as gold nanoparticles) to achieve ultrastructural observation.

[0128] It should be noted that the specific model and specifications to be adopted need to be determined based on the actual specifications of the device. The specific selection and calculation methods use existing technology in this field, so they will not be elaborated here.

[0129] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A Congo red derivative photocatalytic probe, comprising compound I, characterized in that, The compound I is selected from at least one of Congo red derivative photocatalytic probes, and the Congo red derivative photocatalytic probe includes two arenes, where each arene represents an aromatic heterocycle.

2. The Congo red derivative photocatalytic probe according to claim 1, characterized in that, The aromatic heterocycle is selected from one of the following: benzene ring, furan, thiophene, and naphthalene ring.

3. A method for preparing the Congo red derivative photocatalytic probe as described in claim 2, characterized in that, The preparation method includes the following steps: Step S1, solvent and atmosphere preparation; Step S2: Cooling in an ice bath and adding reagents; Step S3: Low-temperature stirring activation; Step S4, introduction and reaction of aromatic aldehydes; Step S5, reaction quenching and extraction; Step S6: Purify the target product by column chromatography.

4. The method for preparing the Congo red derivative photocatalytic probe according to claim 3, characterized in that, Step S1, solvent and atmosphere preparation, refers to dissolving the tetraethyl bisphosphonate compound in THF under an argon atmosphere; In step S2, ice bath cooling and reagent addition refers to slowly adding sodium hydride in batches after the ice bath has cooled to 0°C. In step S3, the low-temperature stirring activation refers to stirring the resulting mixture at 0°C for 30 minutes. In step S4, the aromatic aldehyde introduction and reaction refers to the subsequent addition of aromatic aldehyde II and stirring at room temperature for 16 hours. In step S5, after the reaction is completed, the reaction is quenched with ice water and then the crude organic phase product is extracted with ethyl acetate. Step S6, column chromatography purification of the target product, refers to purifying the target product I using column chromatography.

5. The method for preparing the Congo red derivative photocatalytic probe according to claim 4, characterized in that, The tetraethyl is (2-methoxy-1,4-phenylene)bis(methylene); In step S3, the substrate is stirred for 30 minutes during low-temperature stirring activation until the base reacts fully. In step S4, aromatic aldehyde II is first added to the activated system during the reaction, then the ice bath is removed, and then the reaction is carried out at room temperature for 16 hours. In step S5, the remaining active reagent is quenched with ice water during reaction quenching and extraction.

6. A method of using the Congo red derivative photocatalytic probe as described in claim 5, characterized in that, The method of use refers to its precise application in the pathological analysis of brain tissue sections from Alzheimer's disease mice.

7. The method of using the Congo red derivative photocatalytic probe according to claim 6, characterized in that, The Congo red derivative photocatalytic probe is characterized by its ability to efficiently label amyloid deposits via a Type I photosensitization pathway.

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