Fluorescent probe for identifying bacterial species as well as preparation method and application of fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and application of a fluorescent probe for identifying bacterial species. The structural formula of the fluorescent probe for identifying the bacterial species is as follows: # imgabs0 #, and n is equal to 1-6. In the research, a hemicyanine group cationic cell wall dye (DP) connected with rigid steric hindrance is designed, and the hemicyanine group cationic cell wall dye (DP) not only can specifically distinguish gram-positive bacteria and gram-negative bacteria, but also can distinguish the dead and living states of the gram-negative bacteria. The work provides a reasonable design strategy for a fluorescent probe for identifying bacterial species and opens up a new way for bacterial diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and application of a fluorescent probe for identifying bacterial species. Background Art

[0002] Bacterial infectious diseases have become one of the major challenges faced by the world today, posing a severe threat to human health and social civilization. Numerous research materials show that bacteria not only directly cause diseases but also promote the occurrence of diseases through indirect pathways. For example, bacteria such as Staphylococcus aureus, Bacillus cereus, and Mycobacterium tuberculosis can induce various diseases such as infective endocarditis, meningitis, food poisoning, and tuberculosis (TB). The pathogenicity of bacteria is often closely related to the composition of their cell walls, and the composition and dynamic changes of the cell walls exhibit a high degree of heterogeneity, including differences in the types and ratios of membrane phospholipids and membrane proteins, as well as different surface charges of bacteria. For a long time, the differentiation between Gram-positive bacteria and Gram-negative bacteria has mainly relied on the traditional Gram staining method. However, this method is time-consuming and cumbersome. In contrast, fluorescence imaging technology has shown great potential in the visualization and tracking of biological analytes due to its high detection sensitivity, fast response speed, and multi-color imaging capabilities, and has been developed and applied to the detection of pathogenic bacteria. Currently, a series of fluorescent probes for labeling and identifying bacteria have been designed based on specific components of the bacterial cell wall. However, these fluorescent probes are often complex to synthesize, difficult to accurately distinguish between Gram-positive bacteria and Gram-negative bacteria, and usually unable to monitor bacterial viability through staining. Therefore, in view of the problems existing in the current fluorescent probes for selectively identifying bacteria, there is an urgent need to develop new fluorescent molecular probes for the identification of bacterial species and the monitoring of bacterial viability. Summary of the Invention

[0003] The first object of the present invention is to propose a fluorescent probe for selectively identifying bacterial species and monitoring bacterial viability in view of the above problems existing in the existing fluorescent probes.

[0004] The object of the present invention can be achieved by the following technical solutions:

[0005] A fluorescent probe for identifying bacterial species, the structural formula of the fluorescent probe is as follows:

[0006] Wherein, n = 1 - 6.

[0007] In the above-mentioned fluorescent probe for identifying bacterial species, it is characterized in that the fluorescent probe is a DP fluorescent probe.

[0008] A preparation method for preparing the above fluorescent probe, comprising the following steps:

[0009] a. Synthesis of 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide;

[0010] b. Synthesis of alkyl-substituted (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde derivatives;

[0011] c. Synthesis of the target DP fluorescent probe.

[0012] In the above preparation method of the above fluorescent probe, the specific operation of step a is as follows: Add 1-(3-bromoethyl)-1,4-diazabicyclo[2,2,2]octane-1-ium bromide to 4-methylpyridine in an ethanol solution, then reflux the mixture. After the reaction is completed, white crystals precipitate on the wall of the round-bottom flask. Filter out the precipitate and wash it with EA. The final product is a white powder after drying, and 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide is obtained.

[0013] In the above preparation method of the above fluorescent probe, in step a, the dosage ratio of 1-(3-bromoethyl)-1,4-diazabicyclo[2,2,2]octane-1-ium bromide, 4-methylpyridine, and ethanol is: 5 mmol: 50 mmol: 50 mL.

[0014] In the above preparation method of the above fluorescent probe, the specific operation of step b is as follows: Add (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde to 1-bromohexane in a two-necked flask, heat under reflux. After the reaction is completed, a red solid precipitates at the bottom of the round-bottom flask. Filter out the precipitate and wash it with EA. The final product is a red powder after drying.

[0015] In the above preparation method of the above fluorescent probe, in step b, the dosage ratio of (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde and 1-bromohexane is: 12.5 mmol: 25 mmol.

[0016] In the above preparation method of the above fluorescent probe, the specific operation of step c is as follows: Stir the mixture of 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide, alkyl-substituted (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde derivatives, and pyrrolidine (5 drops) in ethanol overnight, evaporate the solvent under reduced pressure, and purify the residue by alumina chromatography (DCM:MeOH = 50:1) to obtain a red solid.

[0017] In the above-mentioned method for preparing the above-mentioned fluorescent probe, in the step c, the dosage ratio of 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide, alkyl-substituted (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde derivative, pyrrolidine and ethanol is: 9.9 mmol: 8.3 mmol: 5 drops: 30 mL.

[0018] An application of the above-mentioned fluorescent probe in bacterial imaging.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: In this study, we designed a hemicyanine group cationic cell wall dye (DP) linked with a rigid steric hindrance, which can not only specifically distinguish Gram-positive bacteria and Gram-negative bacteria, but also distinguish the living and dead states of Gram-negative bacteria. This work provides a reasonable design strategy for fluorescent probes for identifying bacterial species and opens up a new way for bacterial diagnosis. Brief Description of the Drawings

[0020] Figure 1 A is the ultraviolet-visible absorption spectrum of the fluorescent probe DP in aqueous solution (10.0 μM).

[0021] Figure 1 B is the fluorescence emission spectrum of the fluorescent probe DP in aqueous solution (10.0 μM).

[0022] Figure 1 C is the fluorescence spectrum of DP (10.0 μM) in water before and after adding Cellulose (50 mg / mL).

[0023] Figure 1 D is the fluorescence spectrum of PI (10.0 μM) in water before and after adding Cellulose (50 mg / mL).

[0024] Figure 2 Comparison of laser scanning confocal microscope images of Bacillus subtilis in different living and dead states stained with DP (5.0 μM, 5 minutes), scale bar = 5 μM.

[0025] Figure 3 Comparison of laser scanning confocal microscope images of Escherichia coli in different living and dead states stained with DP (5.0 μM, 5 minutes), scale bar = 5 μM. Detailed Embodiments

[0026] The following are specific examples of the present invention and in combination with the accompanying drawings, the technical solutions of the present invention are further described, but the present invention is not limited to these examples.

[0027] The cell wall plays a crucial role in bacterial characteristic recognition. By precisely identifying the cell wall of bacteria, we can effectively distinguish different bacterial species. We synthesized a Gram-negative bacteria-selective DP fluorescent probe. In the application scenario of bacterial imaging, it can not only clearly identify the differences between Gram-positive and Gram-negative bacteria, but also further achieve the effective discrimination of living and dead Gram-negative bacteria. The purpose of the present invention is to provide a fluorescent probe for selectively identifying bacterial species and monitoring bacterial viability, as well as a preparation method for preparing the DP fluorescent probe. By introducing rigid steric hindrance and multi-charge groups, the fluorescent probe is endowed with the molecular property of non-permeability to the membrane and the fluorescence property activated by the cell wall, enabling it to interact with the cell wall through weak forces such as electrostatics, and realizing the specific imaging of the cell wall. Based on the non-permeability characteristic of the probe to the cytoplasmic membrane, it is also used for selectively identifying Gram-positive bacteria and monitoring the activity of Gram-negative bacteria.

[0028] The present invention provides a fluorescent probe for identifying bacterial species, its preparation method and application. The fluorescent probe is a DP fluorescent probe, and its structural formula is shown as follows:

[0029] Among them, n = 1 - 6.

[0030] The DP fluorescent probe has an alkyl chain, rigid steric hindrance and two positive charges. The two positive charges are used to target the negative charges of the cell wall and ensure the water solubility of the probe molecule. The introduction of the rigid group can effectively prevent the probe molecule from penetrating through the cell wall by steric hindrance effect, so that this series of probe molecules have an extremely long residence time on the cell wall.

[0031] Using the structure with D-A effect and the fluorescence turn-on characteristic of the cell wall as the molecular backbone, the molecule has a relatively high fluorescence turn-on characteristic.

[0032] A lipophilic unit with a shorter length is introduced at the front end of the probe structure to ensure that there is no strong interaction between the probe molecule and the cytoplasmic membrane, and at the same time make the probe molecule unable to target the cytoplasmic membrane.

[0033] The rear end of the probe structure is connected by a rigid group and two positive charges are introduced at the same time to ensure that the probe has good water solubility, and under the combined action of the rigid group and the charge, the probe has a large steric hindrance and charge attraction with the cell wall, effectively preventing the internalization of the probe by the cell wall, so as to achieve ultra-long time cell wall imaging.

[0034] Based on the above series of design principles, we synthesized a fluorescent probe for identifying bacterial species. The preparation method of the above fluorescent probe includes the following steps:

[0035] a. Synthesis of 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide;

[0036] b. Synthesis of alkyl-substituted (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde derivatives;

[0037] c. Synthesis of the target product DP fluorescent probe.

[0038] The above invention content will be further described in detail through specific examples below.

[0039] Example 1:

[0040] The present invention provides a preparation method for a fluorescent probe for identifying bacterial species. The structural formula of the fluorescent probe DP is as follows:

[0041] At this time, n = 1.

[0042] Its synthesis route is as follows:

[0043]

[0044] The preparation method of the DP fluorescent probe specifically includes the following steps:

[0045] a. Synthesis of 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide: Add 1-(3-bromoethyl)-1,4-diazabicyclo[2,2,2]octane-1-ium bromide (1.57 g, 5 mmol) to 4-methylpyridine (4.66 g, 50 mmol) in 50 ml of ethanol solution, and then reflux the mixture for 48 h. After the reaction is completed, white crystals precipitate on the wall of the round-bottom flask. Filter out the precipitate and wash it with EA. The final product is a white powder after drying, with a yield of 80%.

[0046] Product nuclear magnetic resonance data: 1 H NMR(400MHz,DMSO-d6)δ9.03(d,J = 6.7Hz,2H),8.05(d,J = 6.3Hz,2H),4.64(t,J = 7.4Hz,2H),3.34–3.27(m,8H),3.09–3.00(m,7H),2.62(s,3H),2.47–2.37(m,2H). 13 C NMR(101MHz,DMSO)δ159.69,144.43,128.87,60.07,57.31,52.19,45.08,23.87,21.93.

[0047] c. Synthesis of 1-(3-(4-((E)-3-((E)-1,3,3-Trimethylindolin-2-ylidene)prop-1-en-1-yl)pyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octan-1-ium dibromide: A mixture of 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide (3.38 g, 8.3 mmol), (E)-2-(1,3,3-trimethylindolin-2-ylidene)acetaldehyde (2 g, 9.9 mmol) and pyrrolidine (5 drops) in 30 mL of ethanol was stirred overnight. The solvent was removed by evaporation under reduced pressure. The residue was purified by alumina chromatography (DCM:MeOH = 50:1) to give a red solid in 35% yield.

[0048] NMR data of the product: 1 H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 7.0 Hz, 2H), 8.06–7.98 (m, 1H), 7.87 (d, J = 6.7 Hz, 2H), 7.40 (d, J = 6.8 Hz, 1H), 7.22 (d, J = 7.4 Hz, 1H), 7.01 (dd, J = 16.6, 7.7 Hz, 2H), 6.33 (d, J = 14.3 Hz, 1H), 5.84 (d, J = 12.4 Hz, 1H), 4.50 (t, J = 7.2 Hz, 2H), 3.80 (t, J = 5.4 Hz, 2H), 3.40 (s, 8H), 3.02 (s, 6H), 2.44–2.36 (m, 2H), 1.59 (d, J = 6.0 Hz, 8H), 1.27–1.23 (m, 6H), 0.82 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 165.48, 154.01, 143.64, 142.93, 139.99, 127.62, 126.10, 122.43, 122.10, 121.04, 116.61, 108.88, 97.85, 65.47, 52.11, 51.93, 50.92, 48.97, 47.26, 45.12, 44.86, 31.44, 28.68, 26.30, 22.48, 14.30.

[0049] Performance characterization

[0050] The DP fluorescent probe prepared in Example 1 was characterized by UV-visible absorption spectroscopy and fluorescence emission spectroscopy. The test results of the UV-visible absorption spectrum of the DP fluorescent probe in aqueous solution are as Figure 1As shown in a, the aqueous solution of the DP fluorescent probe appears red under sunlight, and the maximum absorption peak of the aqueous solution is located at 535 nm. The test results of its fluorescence emission spectrum are as Figure 1 shown in b. The emission spectrum of the DP fluorescent probe was tested, and the maximum emission peak wavelength of the aqueous solution of the DP fluorescent probe was 590 nm.

[0051] To evaluate the fluorescence response of DP to the cell wall, we used cellulose, the main component of the cell wall, to observe the fluorescence turn-on of DP in aqueous solution. The dye molecules showed very weak fluorescence under ultraviolet irradiation. However, after adding cellulose, their fluorescence intensity increased significantly. As Figure 1 shown in c and d, after adding cellulose, the fluorescence intensity of the traditional commercial dye PI increased by 5.2 times, and that of DP increased by 5.6 times. Therefore, this probe can exhibit a fluorescence turn-on process for cellulose.

[0052] The above characterizations confirmed the fluorescence turn-on behavior of the DP fluorescent probe for cellulose.

[0053] The present invention also provides the use of such a fluorescent probe for identifying bacterial species to identify Gram-positive bacteria and Gram-negative bacteria, and experiments have proven that this phenomenon is due to different cell wall components resulting in different fluorescence intensities of the probe on the cell wall.

[0054] Applications in identifying bacterial species among different bacterial species.

[0055] The following will be described in detail through specific application examples.

[0056] Application Example 1

[0057] Imaging of the DP fluorescent probe in Gram-positive bacteria.

[0058] Specific implementation method: Test strains: Escherichia coli, Bacillus subtilis. The above strains were all purchased from the China General Microbiological Culture Collection Center (CGMCC).

[0059] Strain activation: Streak the E. coli and B. subtilis strains stored at -80 °C on an LB plate medium, place them in an incubator at 37 °C for activation culture. After culturing for 24 h, pick a single colony and transfer it into a triangular flask containing 20 ml of LB culture medium, place it on a shaker at 37 °C and 200 r.min-1 for 12 h, centrifuge at 8000 rpm for 15 min, discard the upper supernatant, and rinse the collected bacterial cells 3 times with sterile water to obtain a bacterial suspension for standby.

[0060] Take 200 μL of each of the above two types of bacteria and mix them with DP (10 μM) dye in 1800 μL of sterile water. Stain for 5 min. Drop 5 μL of the mixed solution on a glass slide. Under the condition of an excitation light wavelength of 561 nm, use a confocal laser scanning microscope with a 100× oil lens to collect images.

[0061] Live bacteria imaging was performed as described above. To image dead bacteria, transfer a single spot on the solid LB medium containing bacteria to a glass slide and bake it over a spirit lamp at high temperature for five minutes. Drop 5 μL of sterile water containing DPD-M (10 μM) dye on the glass slide for 5 min. Under the condition of an excitation of 561 nm, use a confocal laser scanning microscope with a 100× oil lens to collect images.

[0062] As Figure 2 shown, the DP dye has the ability to stain Gram-positive bacteria in both living and dead states. This property is attributed to the uniqueness of the cell wall structure of Gram-positive bacteria, which consists of a single peptidoglycan layer. It should be noted that when this dye is used to treat Bacillus subtilis in rod shape, it shows non-specific staining behavior, meaning that it cannot effectively distinguish Bacillus subtilis according to cell viability (i.e., live bacteria and dead bacteria), thus limiting its application in accurately identifying the life and death states of Gram-positive bacteria.

[0063] Application Example 2

[0064] Imaging of DP fluorescent probe in Gram-negative bacteria.

[0065] The specific implementation method and instrument conditions are the same as those in Application Example 1.

[0066] As Figure 3 shown, the DP dye exhibits a unique staining property, that is, it can only stain Gram-negative bacteria in the dead state. This property deeply reflects the complexity of the cell wall structure of Gram-negative bacteria, which is composed of an outer layer of lipopolysaccharide, outer membrane proteins, and an embedded peptidoglycan layer. Notably, when the DP dye is applied to Escherichia coli, its staining behavior shows a high degree of specificity, and it can accurately identify and distinguish Escherichia coli according to the cell viability state (i.e., distinguish live bacteria and dead bacteria). This discovery not only deepens our understanding of the staining mechanism of the DP dye but also provides a powerful tool for the identification of the life and death states of Gram-negative bacteria.

[0067] Application Example 1-2 fully demonstrates the unique advantages of the DP fluorescent probe in bacterial classification and determination of active status. Specifically, the probe can effectively distinguish Gram-positive bacteria from Gram-negative bacteria based on the differences in the composition of the bacterial cell wall. More advanced, the DP fluorescent probe exhibits specific recognition ability for the living and dead states of Gram-negative bacteria, which makes it have broad application prospects in the field of bacterial diagnosis. By accurately identifying the types and active states of bacteria, the DP fluorescent probe provides strong technical support for the early diagnosis and precise treatment of bacterial diseases.

[0068] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced by the present invention. Any reference signs in the claims should not be construed as limiting the claims concerned.

Claims

1. A fluorescent probe for identifying bacterial species, and the structural formula of the fluorescent probe is shown as follows: Among them, n＝1-6。 2. The fluorescence probe for identifying bacterial species according to claim 1, characterized in that, The fluorescent probe is a DP fluorescent probe.

3. A preparation method of the fluorescent probe according to claim 1 or 2, comprising the following steps: a. Synthesize 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide; b. Synthesize an alkyl-substituted (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde derivative; c. Synthesize the target product, the DP fluorescent probe.

4. The method for preparing the fluorescent probe according to claim 3, wherein The specific operation of step a is as follows: Add 1-(3-bromoethyl)-1,4-diazabicyclo[2,2,2]octane-1-ium bromide to 4-methylpyridine in an ethanol solution, then reflux the mixture. After the reaction is completed, white crystals precipitate on the wall of the round-bottom flask. Filter out the precipitate and wash it with EA. The final product is a white powder after drying, and 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide is obtained.

5. The preparation method of the fluorescent probe according to claim 3, characterized in that, In step a, the dosage ratio of 1-(3-bromoethyl)-1,4-diazabicyclo[2,2,2]octane-1-ium bromide, 4-methylpyridine, and ethanol is: 5 mmol: 50 mmol: 50 mL.

6. The preparation method of the fluorescent probe according to claim 3, characterized in that, The specific operation of step b is as follows: Add (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde to 1-bromohexane in a two-necked flask, heat and reflux. After the reaction is completed, a red solid precipitates at the bottom of the round-bottom flask. Filter out the precipitate and wash it with EA. The final product is a red powder after drying.

7. The preparation method of the fluorescent probe according to claim 3, wherein, In step b, the dosage ratio of (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde and 1-bromohexane is: 12.5 mmol: 25 mmol.

8. The preparation method of the fluorescent probe according to claim 3, characterized in that, The specific operation of step c is as follows: Stir the mixture of 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide, the alkyl-substituted (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde derivative, and pyrrolidine (5 drops) in ethanol overnight, evaporate the solvent under reduced pressure, and purify the residue by alumina chromatography (DCM:MeOH = 50:1) to obtain a red solid.

9. The preparation method of the fluorescent probe according to claim 3, wherein, In step c, the dosage ratio of 1-(3-(4-methylpyridin-1-ium-1-yl)propyl)-1,4-diazabicyclo[2.2.2]octane-1-ium dibromide, the alkyl-substituted (E)-2-(3,3-dimethylindolin-2-ylidene)acetaldehyde derivative, pyrrolidine, and ethanol is: 9.9 mmol: 8.3 mmol: 5 drops: 30 mL.

10. An application of the fluorescent probe according to claims 1-9 above in bacterial imaging.

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