Specific bacterial fluorescent dye as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of chemical synthesis and biology, and particularly relates to a specific bacterial fluorescent dye and a preparation method and application thereof, in particular to a fluorescent dye which can be used for dyeing bacteria but not fungi. Existing fluorescent dyes are limited in capacity of distinguishing bacteria from other microorganisms (such as fungi) and are prone to causing false positive. The stability is poor, and the specific storage requirement is generally required according to a product specification; meanwhile, the emission wavelength of the existing dye belongs to the range of green light or red light. Different from the existing fluorescent dye for staining bacteria, fungal staining is difficult to distinguish and the stability is poor, the invention discloses a specific living bacteria fluorescent dye which is extremely high in stability, the emission wavelength belongs to the blue light category, living cells and dead cells of gram-positive bacteria and gram-negative bacteria can be stained, and the specific living bacteria fluorescent dye has the advantages of high specificity and high specificity. However, RNA and DNA in eukaryotic cells cannot be dyed.

Description

Technical Field

[0001] This invention belongs to the fields of chemical synthesis and biotechnology, specifically relating to a specific bacterial fluorescent dye and its preparation method and application. It is a fluorescent dye that can be used to stain bacteria but not fungi. Background Technology

[0002] As the most widely distributed and abundant group of microorganisms in nature, bacteria play a vital role in medical diagnosis, environmental monitoring, food hygiene, and microbiological research through morphological observation, species identification, and activity analysis. With the development of fluorescent probe technology, fluorescent staining has gradually become the mainstream method in bacterial research due to its advantages such as high sensitivity, strong specificity, and the ability to achieve live-cell imaging. Bacterial fluorescent staining technology achieves visual labeling and analysis of bacteria through the specific binding of dyes to bacterial cellular components, and is one of the core technologies in microbiological research.

[0003] Traditionally used fluorescent dyes capable of penetrating cell membranes to stain bacteria include the following: the Syto series, such as Syto 9, Sytox Green, Sytox Orange, DMAO, and MycoLight Green JJ98 / JJ99 and MycoLight Red JJ94. These dyes all suffer from insufficient specificity: their ability to distinguish bacteria from other microorganisms (such as fungi) is limited, easily leading to false positives. The stability of existing dyes is also relatively poor, and specific storage requirements usually need to be followed according to the product instructions; moreover, the emission wavelengths of existing dyes are all in the green or red light range. Summary of the Invention

[0004] In view of the technical problems existing in the fluorescent dyes used for staining bacteria, the purpose of this invention is to provide a synthetic process and application method for a fluorescent probe with strong ability to distinguish bacteria from other microorganisms (such as fungi).

[0005] The present invention adopts the following technical solution.

[0006] A specific bacterial fluorescent dye, the chemical structural formula of which is as follows:

[0007] As is common knowledge, the chemical structure of the specific bacterial fluorescent dye of this invention contains a coordinating anion, which includes halide ions, sulfonate ions, carboxylate ions, etc., such as chloride ions, iodide ions, bromide ions, etc.

[0008] The preparation method of the above-mentioned specific bacterial fluorescent dye includes the following steps: compound 4 reacts with compound 5 to prepare the specific bacterial fluorescent dye.

[0009] In this invention, 2-(methylthio)benzoxazole and methyl p-toluenesulfonate are reacted to prepare compound 5.

[0010] In this invention, compound 3 is reacted with 3-fluoro-4-methoxybenzoyl chloride to prepare compound 4.

[0011] In this invention, compound 2 is deprotected to obtain compound 3.

[0012] In this invention, compound 1 is reacted with 1-iodopropane to prepare compound 2.

[0013] In this invention, the chemical structural formulas of compounds 1, 2, 3, 4, and 5 are as follows:

[0014] As is common knowledge, the chemical structures of compounds 2, 3, 4 and 5 contain coordination anions, including halide ions, sulfonate ions, carboxylate ions, etc.

[0015] In this invention, the reaction between compound 4 and compound 5 is carried out in the presence of an organic base, which includes an amine compound, such as triethylamine.

[0016] In this invention, the reaction temperature of compound 4 and compound 5 is room temperature, and the reaction time is 5 to 30 hours, preferably 10 to 20 hours.

[0017] In this invention, after compound 4 reacts with compound 5, a halide salt is added to obtain a specific bacterial fluorescent dye; the halide salt is a halogen compound, such as sodium iodide.

[0018] This invention discloses a method for bacterial staining, comprising the following steps: incubating a system containing bacteria and the above-mentioned specific bacterial fluorescent dye, collecting cells, and completing bacterial staining.

[0019] This invention discloses a method for bacterial detection, comprising the following steps: incubating a system containing bacteria and the above-mentioned specific bacterial fluorescent dye, collecting cells, and then observing them using a fluorescence microscope or analyzing them by flow cytometry to complete the bacterial detection.

[0020] In the bacterial staining method or bacterial detection method of the present invention, the specific bacterial fluorescent dye does not stain fungi.

[0021] In the bacterial staining method or bacterial detection method of the present invention, in the system containing bacteria and the above-mentioned specific bacterial fluorescent dye, the dispersion medium includes sodium chloride solution, buffer solution or culture medium; the concentration of the specific bacterial fluorescent dye is 5-50 μM; preferably, in the system containing bacteria and the above-mentioned specific bacterial fluorescent dye, the concentration of the specific bacterial fluorescent dye is 10-40 μM; for example, 15 μM, 20 μM, 25 μM, 30 μM or any concentration within the range.

[0022] In the bacterial staining method or bacterial detection method of the present invention, the incubation is carried out at room temperature or 37°C in the dark for 15 to 60 minutes.

[0023] The bacterial staining method or bacterial detection method of the present invention is a method for non-disease diagnosis and treatment purposes.

[0024] This invention discloses the application of the above-mentioned specific bacterial fluorescent dye in bacterial staining or bacterial detection, or the application of the above-mentioned specific bacterial fluorescent dye in bacterial staining where fungi are not stained or in bacterial detection where fungi cannot be detected; the bacterial staining or bacterial detection is for non-disease diagnosis and treatment purposes.

[0025] This invention discloses the application of the above-mentioned specific bacterial fluorescent dye in the preparation of bacterial staining reagents or bacterial detection reagents; or, the application of the above-mentioned specific bacterial fluorescent dye in the preparation of bacterial staining reagents that do not stain fungi or bacterial detection reagents that cannot detect fungi.

[0026] In this invention, bacteria include live cells and dead cells of Gram-positive and Gram-negative bacteria.

[0027] Unlike existing fluorescent dyes used to stain bacteria, which are difficult to distinguish from fungal staining and have poor stability, this invention discloses a specific fluorescent dye for live bacteria. It has extremely high stability, emits light in the blue light range, and can stain both live and dead cells of Gram-positive and Gram-negative bacteria. However, it cannot stain RNA and DNA in eukaryotic cells. Attached Figure Description

[0028] Figure 1 This is a mass spectrum of a specific live bacterial fluorescent dye.

[0029] Figure 2 The absorption and emission spectra of specific live bacterial fluorescent dyes are shown.

[0030] Figure 3 The specific live bacterial fluorescent dye cannot stain Saccharomyces cerevisiae.

[0031] Figure 4 Saccharomyces cerevisiae stained with Calcofluor White Stain.

[0032] Figure 5 These are specific live bacterial fluorescent dyes and CF 633-labeled wheat germ agglutinin-stained bacteria.

[0033] Figure 6 The bacteria were stained with a newly prepared specific live bacterial fluorescent dye.

[0034] Figure 7 These are bacteria that have been repeatedly frozen and thawed and stored at room temperature without light for 6 months, and are stained with fluorescent dyes to identify specific live bacteria. Detailed Implementation

[0035] Bacterial fluorescent staining technology, through the specific binding of dyes to bacterial cellular components, enables the visual labeling and analysis of bacteria, and is one of the core technologies in microbiology research. Existing dyes suffer from insufficient specificity; these fluorescent dyes have limited ability to distinguish bacteria from other microorganisms (such as fungi), easily leading to false positives; they also have relatively poor stability, and specific storage requirements usually need to be followed according to the product instructions; furthermore, the emission wavelengths of existing dyes are all in the green or red light range. Unlike existing fluorescent dyes for staining bacteria, which are difficult to distinguish from fungal staining and have poor stability, this invention discloses a specific fluorescent dye for live bacteria. It has extremely high stability, an emission wavelength in the blue light range, and can stain both live and dead cells of Gram-positive and Gram-negative bacteria, but it cannot stain RNA and DNA in eukaryotic cells.

[0036] The method for bacterial detection using the specific live bacterial fluorescent dye of the present invention includes the following steps: incubating a system containing bacteria and the above-mentioned specific bacterial fluorescent dye, collecting cells, and then observing them using a fluorescence microscope or analyzing them by flow cytometry to complete the bacterial detection.

[0037] In this invention, bacteria are directly mixed with the aforementioned specific bacterial fluorescent dye in a dispersion medium (or solvent) to obtain the system; alternatively, bacteria can be cultured first, and then mixed with the aforementioned specific bacterial fluorescent dye in a dispersion medium (or solvent) to obtain the system. The specific operation follows conventional techniques, such as culturing bacteria in a growth medium at 37°C overnight; collecting the cells by centrifugation, then resuspending them in a 0.85% (150 mM) sodium chloride solution, and then adding the aforementioned specific bacterial fluorescent dye to obtain the system. The sodium chloride solution can be replaced with Tris buffer, phosphate-buffered saline (PBS), or culture medium.

[0038] The following specific experiments illustrate the technological advancements of this invention. The raw materials used are existing products, and the specific preparation operations and performance tests employ conventional techniques. *Saccharomyces cerevisiae* (Catalog No.: XY-WSW-1545); *Escherichia coli* (Catalog No.: XY-WSW-1969); *Staphylococcus aureus* (Catalog No.: XY-WSW-1802); sourced from Shanghai Xuanya Biotechnology Co., Ltd. The culture medium used for *Staphylococcus aureus* was tryptone soybean broth (TSB), catalog No. CM301; the culture medium used for *Escherichia coli* was LB broth medium, catalog No. 028320.

[0039] Example 1: Synthesis of Specific Live Bacterial Fluorescent Dyes

[0040]

[0041]

[0042] (1) Synthesis of compound 1: 2-amino-4-methylpyridine (10.8 g, 0.1 mol), ditert-butyl dicarbonate (43.6 g, 0.2 mol), and 500 mL of acetone were added to a 1 L three-necked flask and stirred under reflux for 24 h. At this time, TLC detection (n-hexane: ethyl acetate = 3:1) showed that the reaction of the starting material 2-amino-4-methylpyridine was complete. Heating was stopped and the mixture was allowed to cool naturally to room temperature. The entire reaction solution was concentrated and purified directly by silica gel column chromatography. The eluent used was 90% dichloromethane and 10% ethyl acetate. Finally, 8.9 g of oily compound 1 was obtained. (2) Synthesis of compound 2: Compound 1 (8.32 g, 0.04 mol) and 1-iodopropane (17 g, 0.1 mol) were placed in a 500 ml three-necked flask, stirred magnetically and refluxed, and reacted at 110 °C for 3 days. After TLC detection (CH2Cl2:MeOH=2:1), the reactants were basically completely reacted. The mixture was cooled to room temperature and the entire reaction solution was concentrated. Then 200 mL of n-hexane was added, refluxed for 1 h, and then filtered at 4 °C. The filter cake was washed with n-hexane and then dried under reduced pressure to obtain 6.2 g of pale yellow solid compound 2. (3) Synthesis of compound 3: Compound 2 (5.67 g, 0.015 mol) and dichloromethane (50 mL) were added to a 250 mL three-necked flask and stirred at room temperature for 5 min. Then, trifluoroacetic acid (12 mL) was added at 0 °C and stirred at room temperature for 1 h. TLC detection (CH2Cl2:MeOH=1:1) showed that the reactants were basically completely reacted. The entire reaction solution was concentrated and purified directly by silica gel column chromatography. The eluent used was 95% dichloromethane and 5% methanol. Finally, 1.6 g of pale yellow gel-like compound 3 was obtained. (4) Synthesis of compound 4: Compound 3 (1.32 g, 0.005 mol) was dissolved in 20 mL of dichloromethane in a 100 mL three-necked flask. Then, 3-fluoro-4-methoxybenzoyl chloride (0.94 g, 0.005 mol, pre-dissolved in 5 mL of anhydrous DMF) was added dropwise at 0 °C over 10 minutes. The mixture was stirred at room temperature for 12 hours. The resulting mixture was then concentrated, and the residue was poured into a 0.5% hydrochloric acid aqueous solution (100 mL) and extracted with chloroform (2 x 100 mL). The chloroform layers were combined and washed with water (3 x 100 mL). The organic chloroform layer was dried with anhydrous sodium sulfate and then concentrated to obtain 1.6 g of yellow oily compound 4. This oily substance was used directly in the next step (6). (5) Synthesis of compound 5: 2-(methylthio)benzoxazole (6 g, 0.036 mol) and methyl p-toluenesulfonate (8.1 g, 0.043 mmol) were placed in a 100 mL single-necked flask and reacted at 120 °C for 14 h under magnetic stirring. The reaction was detected by TLC (PE / EA=8 / 1) and the result showed that the reaction was complete. Then, 50 mL of ethyl acetate was added at room temperature and stirred twice, each time for 4 h. The mixture was then filtered and the filter cake was dried under vacuum to constant weight to obtain 9.8 g of light yellow solid compound 5.

[0043] (6) Synthesis of compound 6: Compound 4 (676 mg, 0.002 mol), compound 5 (703 mg, 0.002 mol), and 5 mL of DMF were added to a 100 mL single-necked flask and magnetically stirred to dissolve. Then, triethylamine (2 mL) was added, and the resulting mixture was reacted at room temperature for 14 h. TLC (alumina ACN / H2O = 5 / 1) showed that the reaction was basically complete. Then, DMF was dried under vacuum, 20 mL of H2O and 11.6 g of sodium iodide were added, and the mixture was stirred for 24 h and then evaporated to dryness. The resulting mixed solid was purified by column chromatography (alumina, eluent ACN / H2O = 5%). The 90% pure product was collected and evaporated to dryness. The resulting solid was stirred for 12 h with 10 mL of methanol and 100 mL of dichloromethane and then filtered. The filter cake was vacuum dried to a constant weight of 0.83 g, yielding off-white solid compound 6, which is a specific fluorescent dye for live bacteria. Its mass spectrum is shown in [reference needed]. Figure 1 Molecular weight: C 25 H 25 IFN3O3, 561.40; Figure 2 Its absorption and emission spectra.

[0044] Example 2 The experimental protocol for detecting bacteria using the specific live bacterial fluorescent dye of this invention is as follows: 1. The bacteria are suspended in 0.85% (150 mM) sodium chloride solution (or 10 mM Tris buffer, phosphate-buffered saline (PBS), or culture medium at pH 7.5), and the above-mentioned specific live bacterial fluorescent dye is added to achieve a final concentration of 5-50 μM, preferably 10-25 μM. 2. Incubate the sample at room temperature or 37°C in the dark for 30 minutes; 3. Collect cells by centrifugation and resuspend them in fresh buffer solution; 4. For fluorescence microscopy observation: You can take 5 μL of sample and place it on a glass slide, then cover it with an 18 mm coverslip; Alternatively, use a pipette to aspirate 100 μL of sample into the 96-well optical substrate; Use the Pacific Blue or DAPI channels to image cells; 5. To perform flow cytometry analysis: (1) The sample can be diluted with flow cytometry washing buffer (PBS + 1% serum) or similar buffer at a ratio of 1:10; (2) Dilute the sample in the flow cytometer buffer according to the experimental requirements to achieve a suitable flow rate; (3) Detect cells in the DAPI channel.

[0045] Example 3: Cannot be infected with fungi Saccharomyces cerevisiae was cultured in complete yeast medium (product code: BTN130895) at 37°C for 18 hours. After centrifugation, the Saccharomyces cerevisiae cells were resuspended in 10 mM Tris buffer (pH 7.5), divided in half, and one half was further inoculated with the aforementioned specific live bacterial fluorescent dye to achieve a final concentration of 10 μmol. The cells were then incubated at room temperature with shaking in the dark for 20 minutes. Imaging was performed under a fluorescence microscope using a DAPI emission filter. See [link to relevant documentation]. Figure 3 The experimental results verified that the dye of the present invention failed to stain.

[0046] Take another portion of Saccharomyces cerevisiae Tris buffer and add Calcofluor White Stain (18909, Sigma-Aldrich) to achieve a final concentration of 10 μmol. Incubate at room temperature with shaking in the dark for 20 minutes. Image under a fluorescence microscope using a DAPI emission filter; see [link to relevant documentation]. Figure 4The Saccharomyces cerevisiae was stained with Calcofluor White Stain, and the results confirmed that the Saccharomyces cerevisiae was normal.

[0047] Example 4: Bacterial Infection Escherichia coli and Staphylococcus aureus were cultured separately in culture medium at 37°C for 18 hours, followed by centrifugation. The resulting E. coli and Staphylococcus aureus cells were then resuspended in 10 mM Tris buffer at pH 7.5. The aforementioned specific live bacterial fluorescent dye and CF 633-labeled wheat germ lectin (catalog number: 29024) were then added to achieve a final dye concentration of 10 μmol. The mixture was incubated at room temperature with shaking in the dark for 20 minutes. Imaging was performed under a fluorescence microscope using a DAPI emission filter. The experimental results verified that the dyes of this invention can stain bacteria and have universality.

[0048] Escherichia coli and Staphylococcus aureus were stained with the specific live bacterial fluorescent dye (blue) described above and wheat germ lectin (WGA, red) labeled with CF 633. See also Figure 5 Spherical Gram-positive Staphylococcus aureus is stained with WGA, while rod-shaped Gram-negative Escherichia coli is not stained by it; in contrast, the specific live bacterial fluorescent dye product of the present invention stains all cells blue.

[0049] Example 5 Stability SYTO 9, a commonly used nucleic acid fluorescent dye with good industrial application performance (often used for staining live bacteria / fungi, cell viability detection, etc.), is explicitly stated in the LIVE / DEAD series product instructions to be stored in a sealed container at -20°C, protected from light, and to avoid repeated freeze-thaw cycles. This invention solves the problems of existing dyes not being able to be stored at room temperature and requiring repeated freeze-thaw cycles. The DMSO solution (0.1M) of the specific live bacterial fluorescent dye described in this invention, after 50 repeated freeze-thaw cycles, showed no significant difference in staining effect after 6 months of storage at room temperature without the need for light protection, compared to the initial preparation.

[0050] Escherichia coli and Staphylococcus aureus were cultured separately in culture medium at 37°C for 18 hours, followed by centrifugation. The resulting E. coli and Staphylococcus aureus cells were then resuspended in 10 mM Tris buffer at pH 7.5, and the aforementioned specific live bacterial fluorescent dye (freshly prepared or repeatedly frozen and thawed 50 times + stored at room temperature for 6 months without protection from light) was added to achieve a final dye concentration of 15 μmol. The cells were incubated at room temperature with shaking in the dark for 20 minutes. Imaging was performed under a fluorescence microscope using a DAPI emission filter.

[0051] Figure 6 The staining results of the initially prepared dye show that both spherical Gram-positive Staphylococcus aureus and rod-shaped Gram-negative Escherichia coli were stained. Figure 7 After 50 freeze-thaw cycles, without needing to be protected from light, and left at room temperature for 6 months, the dye staining results showed that both spherical Gram-positive Staphylococcus aureus and rod-shaped Gram-negative Escherichia coli were stained.

[0052] The ability of the specific live bacterial fluorescent dye of this invention to distinguish between bacteria and other microorganisms (such as fungi) has been verified. It can greatly reduce false positives caused during staining and has universal applicability to bacteria.

Claims

1. A specific bacterial fluorescent dye, characterized in that, The chemical structural formula of the specific bacterial fluorescent dye is as follows: 。 2. The specific bacterial fluorescent dye according to claim 1, characterized in that, The chemical structure of the specific bacterial fluorescent dye contains a coordinating anion.

3. The method for preparing the specific bacterial fluorescent dye according to claim 1, characterized in that, The process includes the following steps: reacting compound 4 with compound 5 to prepare a specific bacterial fluorescent dye.

4. The method for preparing the specific bacterial fluorescent dye according to claim 3, characterized in that, The reaction between compound 4 and compound 5 was carried out in the presence of an organic base; the reaction temperature between compound 4 and compound 5 was room temperature, and the reaction time was 5 to 30 hours.

5. A method for bacterial staining, characterized in that, The procedure includes the following steps: incubating a system containing bacteria with the specific bacterial fluorescent dye described in claim 1, collecting cells, and completing bacterial staining.

6. A method for detecting bacteria, characterized in that, The procedure includes the following steps: incubating a system containing bacteria with the specific bacterial fluorescent dye described in claim 1, collecting cells, and then observing them using a fluorescence microscope or analyzing them using flow cytometry to complete bacterial detection.

7. The bacterial staining method according to claim 5 or the bacterial detection method according to claim 6, characterized in that, In the system containing bacteria and the above-mentioned specific bacterial fluorescent dye, the concentration of the specific bacterial fluorescent dye is 5–50 μM.

8. A bacterial staining reagent or bacterial detection reagent, characterized in that, The active ingredient of the bacterial staining reagent or bacterial detection reagent includes the specific bacterial fluorescent dye described in claim 1.

9. The application of the specific bacterial fluorescent dye according to claim 1 in bacterial staining or bacterial detection.

10. The use of the specific bacterial fluorescent dye of claim 1 in the preparation of bacterial staining reagents or bacterial detection reagents.

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