Lipid droplet detection kit based on double-response water-soluble fluorescent dye and application of lipid droplet detection kit

Abstract

The invention relates to the technical field of nano material imaging diagnosis, in particular to a lipid droplet detection kit based on double-response water-soluble fluorescent dye and application of the lipid droplet detection kit. The invention provides the lipid droplet detection kit, the lipid droplet detection kit comprises a component A, the component A is a composite fluorescent dye of an anthocyanin derivative fluorescent dye and a metal frame material, the dual response of polarity and RNS can be realized, the lipid droplet imaging effect is good, the dyeing step is simple, the water solubility is better, the lipid droplet is easy to rinse and clean, the experimental result is reliable, and the lipid droplet detection kit has great commercial value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanomaterial imaging diagnosis, and particularly relates to a lipid droplet detection kit based on a double-response water-soluble fluorescent dye and application thereof. BACKGROUND

[0002] The global incidence of metabolic diseases such as non-alcoholic fatty liver (NAFLD) is increasing year by year, and early diagnosis is urgently needed.

[0003] Abnormal accumulation of lipid droplets (LDs) is an early marker for diagnosing NAFLD. Existing methods for imaging and detecting lipid droplets include using commercial oil red O staining methods, but these methods have problems such as complicated procedures, poor water solubility, and low specificity, resulting in low diagnostic efficiency. Although fluorescent dyes such as Nile Red and BODIPY have been reported for use in staining adipocytes, they also have problems such as poor water solubility, high background signal, and low specificity. Therefore, there is an urgent need for a lipid droplet detection method that is easy to operate, has good water solubility, and high specificity.

[0004] Metallic framework materials have highly ordered pore structures, large specific surface areas, and excellent chemical and thermal stabilities, and are used in the fields of catalysis, drug carriers, fluorescent sensors for detecting ions and organic matter, etc. However, there is no related report on the combination of metallic framework materials with fluorescent dyes for lipid droplet detection. SUMMARY

[0005] Therefore, the present application provides a lipid droplet detection kit based on a double-response water-soluble fluorescent dye and application thereof to solve the problems of poor water solubility, low specificity, and complicated procedures in existing lipid droplet detection technologies. By loading a polar-responsive cyanine derivative fluorescent dye into an active nitrogen (RNS)-responsive blue fluorescent metal organic framework material, the polar and RNS double responses of the composite fluorescent dye are achieved, and the detection specificity and water solubility are improved.

[0006] The technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides a lipid droplet detection kit, which comprises components A, B, C, and D, wherein the component A is a composite fluorescent dye of a cyanine derivative fluorescent dye and a metal framework material.

[0008] Preferably, the preparation method of the cyanine derivative fluorescent dye and the metal framework material (UiON) comprises the following steps: dissolving the metal framework material and the cyanine derivative fluorescent dye in DMF, ultrasonicating, stirring, centrifuging, and reserving.

[0009] On the basis of the above technical scheme, preferably, the metal framework material has ONOO-response, and the cyanine derivative fluorescent dye has polarity response.

[0010] On the basis of the above technical scheme, preferably, the metal framework material is any one of UIO-66-(OH)2, UIO-68-B, UIO-66-NH2 and UIO-66-SCH3, and the cyanine derivative fluorescent dye is coumarin-barbituric acid complex (YB).

[0011] On the basis of the above technical scheme, further preferably, the metal framework material is UIO-66-(OH)2. ONOO-response causes framework cracking, and cells in an oxidative stress state will produce high levels of RNS (such as ONOO-). These RNS molecules will diffuse and attack the organic ligands of the framework material, thereby changing the intensity of the blue fluorescence signal.

[0012] The coumarin-based probe can sensitively detect the polarity change of LDs through an intramolecular charge transfer (ICT) mechanism, and the fluorescence intensity thereof is enhanced with the decrease of the environmental polarity. The YB dye is enriched in the low-polarity environment of LDs due to its hydrophobicity, thereby emitting strong red fluorescence.

[0013] On the basis of the above technical scheme, preferably, the metal framework material has a content of 40-60 mg, and the fluorescent dye has a content of 4-6 mg.

[0014] On the basis of the above technical scheme, further preferably, 50 mg of UIO-66-(OH)2 and 5 mg of the cyanine derivative fluorescent dye YB are weighed and dissolved in 10 mL of DMF, ultrasonic treatment is performed to make the dispersion uniform, stirring is performed at 800 rpm at room temperature for 24 hours, centrifugation is performed at 10,000 rpm for 25 minutes, and the prepared product is ready for use.

[0015] On the basis of the above technical scheme, preferably, the lipid droplet detection kit further comprises components B, C and D; the component A is a solution or a freeze-dried powder, the component B is a phosphate buffer, the component C is a fixing solution, and the component D is a mounting medium.

[0016] On the basis of the above technical scheme, preferably, the phosphate buffer has a pH of 7.4 and is packaged in a sterile ampoule; the fixing solution is a ready-to-use 4% paraformaldehyde fixing solution; and the mounting medium is a mounting medium without DAPI.

[0017] The lipid droplet detection kit further comprises component E: a standardized operation instruction, which comprises a cell / tissue staining procedure, microscope parameter settings and result interpretation standards.

[0018] Secondly, the application of the lipid droplet detection kit described above in lipid droplet imaging is provided.

[0019] Based on the above technical solutions, preferably, the sample types used in the application include cells and tissues.

[0020] Based on the above technical solutions, preferably, when used for staining cell lipid droplets, component A is a solution with a concentration of 0.6 to 1.2 mg / mL; when used for staining tissue lipid droplets, component A is a lyophilized powder with a concentration of 40 to 80 mg / mL; more preferably, component A is a solution with a concentration of 0.806 mg / mL.

[0021] Based on the above technical solutions, a further preferred embodiment of the staining steps of the lipid droplet detection kit for cell staining is as follows: incubate cells with component A for 30 min, then discard the culture medium, rinse twice with component B, add component C for 10 min for fixation, rinse twice with component B, mount with component D, and then observe the lipid fluorescence staining results under a fluorescence microscope; for tissue staining, the staining steps of the lipid droplet detection kit include: taking frozen tissue sections, adding component A (the concentration of component A is adjusted according to the thickness of the tissue section), staining for 12 min, and then washing three times with distilled water for 5 min each time.

[0022] Based on the above technical solutions, preferably, the fluorescence emission spectrum of the above dual-response dye is tested, and the emission wavelength of the dye is around 600 nm.

[0023] Based on the above technical solutions, the preferred lipid droplet detection kit, as described above, can also be used for lipid metabolism research, compatible with cell and tissue samples; for rapid screening of fatty liver biopsy samples, and can be integrated into automated diagnostic platforms when combined with standard microscopy parameters; and for lipotoxicity assessment in drug development, especially for metabolic drugs. Intelligent dual-channel validation can reduce false positives and improve data quality.

[0024] The lipid droplet detection kit of the present invention has the following advantages over the prior art:

[0025] (1) A water-soluble fluorescent dye with both polarity and RNS dual response and its staining scheme for adipocytes are provided, avoiding the problems of cumbersome steps, poor water solubility and easy precipitation of existing commercial Oil Red O staining. Based on this water-soluble fluorescent dye kit, it shows good lipid droplet imaging effect, simple staining steps, good water solubility that it is easy to rinse clean, and reliable experimental results, which have significant advantages and great commercial value;

[0026] (2) By loading polar dyes onto RNS-responsive metal framework materials, the water solubility of the dyes and the overall specificity of the kit were improved.

[0027] (3) The kit of this invention proposes a "ready-to-use all-aqueous phase" technical solution: through a pre-packaged system, the key components such as dyes and fixatives are pre-optimized and then dispensed, so that users can use it immediately after opening the bottle; dual morphology adaptation, the cell solution maintains active staining ability, and the lyophilized powder for tissues can be transported at room temperature; intelligent dual-channel verification, through blue / red dual-color fluorescence, i.e., polar / reactive nitrogen co-localization technology, automatically eliminates false positive signals, further improving specificity. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The images show Oil Red O staining and UIO-YB fluorescence of cells at different induction times according to the present invention.

[0030] Figure 2 This is a stained image of an animal pathological section according to the present invention;

[0031] Figure 3 This is a colocation diagram of the UIO-YB of the present invention and the commercial dye BODIPY. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Information on the reagents used in this invention is shown in Table 1.

[0034] Table 1 Reagent Information Table

[0035]

[0036]

[0037] Example 1: Preparation of a dual-response water-soluble lipid droplet detection kit

[0038] 1. Preparation of the metal framework material: 0.3 mmol of HfCl4 and 0.3 mmol of 2-hydroxyterephthalic acid were dissolved in 10 mL of DMF and formic acid solution, with a volume ratio of DMF to formic acid of 9:1. The mixture was placed in a 50 mL autoclave lined with Teflon and heated at 130 °C for 36 hours. The white precipitate obtained after filtration was repeatedly washed with acetone, dried in air, and then dried in an oven at 80 °C to obtain the metal framework material UIO-66-(OH)2.

[0039] 2. Preparation of dual-responsive anthocyanin derivative fluorescent dyes for metal framework materials (UiON): Weigh 50 mg of UIO-66-(OH)2 and 5 mg of anthocyanin derivative fluorescent dye YB and dissolve them in 10 mL of DMF. Sonicate the mixture to ensure uniform dispersion. Stir at 800 rpm for 24 hours at room temperature, centrifuge (10000 rpm, 20-30 minutes), and then vacuum dry for later use. This is the lyophilized component A. Solution-type component A does not require vacuum drying. The preparation method of YB dye is described in the literature "Solvatochromic Two-Photon Fluorescent Probe Enables In Situ Lipid Droplet Multidynamics Tracking for Nonalcoholic Fatty Liver and Inflammation Diagnoses".

[0040] Component B: Phosphate buffered saline (PBS, pH 7.4), packaged in sterile ampoules;

[0041] Component C: Ready-to-use 4% paraformaldehyde fixative;

[0042] Component D: Mounting solution (excluding DAPI).

[0043] Example 2: Preparation of a dual-response water-soluble lipid droplet detection kit

[0044] The preparation method of this embodiment is basically the same as that of Example 1, except that 2-hydroxyterephthalic acid is replaced with an equal amount of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzoic acid to obtain the metal framework material UIO-66-B.

[0045] The prepared UIO-68-B was loaded with YB dye to prepare component A.

[0046] Example 3: Preparation of a dual-response water-soluble lipid droplet detection kit

[0047] The preparation method of this embodiment is basically the same as that of Example 1, except that 2-hydroxyterephthalic acid is replaced with an equal amount of 2-(3,4-diaminobenzoyl)terephthalic acid to obtain the metal framework material UIO-66-NH2.

[0048] The prepared UIO-66-NH2 was loaded with YB dye to prepare component A.

[0049] Example 4: Preparation of a dual-response water-soluble lipid droplet detection kit

[0050] The preparation method of this embodiment is basically the same as that of Example 1, except that 2-hydroxyterephthalic acid is replaced with an equal amount of 2-(benzylthio)terephthalic acid to obtain the metal framework material UIO-66-SCH3.

[0051] The prepared UIO-66-SCH3 was loaded with YB dye to prepare component A.

[0052] Example 5: Preparation of a dual-response water-soluble lipid droplet detection kit

[0053] The preparation method of this embodiment is basically the same as that of Example 1, except that: when preparing UiON, 40mg UIO-66-(OH)2 and 4mg anthocyanin derivative fluorescent dye YB are weighed.

[0054] Example 6: Preparation of a dual-response water-soluble lipid droplet detection kit

[0055] The preparation method of this embodiment is basically the same as that of Example 1, except that: when preparing UiON, 60mg UIO-66-(OH)2 and 6mg anthocyanin derivative fluorescent dye YB are weighed.

[0056] Example 7:

[0057] The staining effect of the kit prepared in Example 1 on lipid droplets in cells.

[0058] The procedure was as follows: HepG2 cells were stimulated with trifluoroacetic acid (FA) solution for several cycles (0, 2, 4, 6, and 8 hours), then the culture medium was removed, the cells were washed with PBS, incubated with 100 μM UIO-YB for 20 minutes, and then washed again with PBS. Subsequently, fluorescence microscopy was used to capture images. UIO blue fluorescence was obtained by λex: 326 nm / λem: 447 nm, and YB red fluorescence was obtained by λex: 537 nm / λem: 598 nm. Simultaneously, HepG2 cells were stained with Oil Red O for comparison, followed by microscopic imaging. The consistency between Oil Red O-labeled lipid droplets and UIO-YB-labeled lipid droplets was analyzed.

[0059] The results are as followsFigure 1 As shown, compared with commercial Oil Red O, the kit of the present invention provides clearer lipid droplet imaging, stronger signal, and avoids lipid droplet displacement problems.

[0060] Example 8:

[0061] The staining effect of the kit prepared in Example 2 on lipid droplets in tissue.

[0062] The procedure was as follows: Eight-week-old male C57BL / 6 mice were purchased from Charles River (Beijing, China). Mice were fed a high-fat diet (HFD) containing 60% calories and fat at different time points (0, 6, 12, and 18 weeks) to induce a liver model. Liver tissue was collected to prepare paraffin sections and frozen sections. The tissue sections were then stained with hematoxylin and eosin (HE) and Oil Red O, respectively, to observe the liver tissue structure and lipid accumulation in different groups of mice. Simultaneously, liver sections from different groups of mice were stained with UIO-YB. The procedure was as follows: frozen tissue sections were washed with water, stained with 200 μM UIO-YB for 20 minutes, washed again with PBS, and observed under a fluorescence microscope (UIO blue fluorescence: λex: 326 nm / λem: 447 nm; YB red fluorescence: λex: 537 nm / λem: 598 nm).

[0063] The results are as follows Figure 2 As shown, the results clearly display the distribution of lipid droplets, exhibiting high specificity and low background noise, making it suitable for the early diagnosis of fatty liver. This provides a solid basis for the clinical translation of this kit.

[0064] according to Figure 1 , 2 As shown, with the accumulation of lipids in cells, the polarity of lipid droplets decreases, while the red fluorescence of polar responses increases; simultaneously, with the increase of RNS content, the blue fluorescence of RNS responses increases. In cell experiments, co-localization results with lipid droplets show that this dye and its corresponding staining scheme have strong staining specificity, good staining effect, and simple and easy-to-operate staining steps. The above results indicate that the simple, easy-to-operate, and highly specific water-soluble lipid droplet dye and staining scheme proposed in this invention exhibit good lipid droplet staining effect.

[0065] Example 9:

[0066] The kit prepared in Example 3 above is isolocalized with the commercial dye BODIPY.

[0067] The specific steps were as follows: HepaG2 cells stimulated with fatty acids were labeled with the lipid droplet dye Bodipy (green channel). Subsequently, the cells were co-incubated with 100 μM UIO-YB (red channel) for 20 minutes, and the slides were sealed with mounting medium containing Hoechst (blue channel). Finally, the cells were observed using a fluorescence microscope.

[0068] The results are as follows Figure 3 As shown, UIO-YB is highly co-located with the commercial dye BODIPY, confirming its accuracy and reliability. Users can seamlessly replace existing methods, reducing barriers to market promotion.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lipid droplet detection kit, characterized by: The complex fluorescent dye comprises component A, which is a cyanine derivative fluorescent dye-metal framework material.

2. The lipid droplet detection kit of claim 1, wherein: The preparation method of the cyanine derivative fluorescent dye-metal framework material comprises the following steps: dissolving the metal framework material and the cyanine derivative fluorescent dye in DMF, ultrasonicating, stirring, centrifuging, and reserving.

3. The lipid droplet detection kit of claim 2, wherein: The metal framework material has ONOO-response, and the cyanine derivative fluorescent dye has polarity response.

4. The lipid droplet detection kit of claim 3, wherein: The metal framework material is any one of UIO-66-(OH)2, UIO-68-B, UIO-66-NH2, and UIO-66-SCH3, and the cyanine derivative fluorescent dye is a coumarin-barbituric acid complex.

5. The lipid droplet detection kit of claim 4, wherein: The content of the metal framework material is 40-60 mg, and the content of the cyanine derivative fluorescent dye is 4-6 mg.

6. The lipid droplet detection kit of claim 1, wherein: The lipid droplet detection kit further comprises components B, C, and D; the component B is a phosphate buffer, the component C is a fixing solution, and the component D is a mounting solution.

7. Application of the lipid droplet detection kit of claim 1 in lipid droplet imaging.

8. Use according to claim 7, wherein: The sample types of the application include cells and tissues.

9. Use according to claim 8, wherein: When the sample type is cells, the component A is a solution with a concentration of 0.6-1.2 mg / mL; when the sample type is tissues, the component A is a lyophilized powder with a concentration of 40-80 mg / mL.

10. The use according to claim 7, characterized in that: The fields of the application include lipid metabolism research, biopsy sample screening, and lipid toxicity evaluation in drug development.

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