A wash-free fluorescent probe for specific identification of bacteria and its preparation method and application

Abstract

The present invention provides a wash-free fluorescent probe for specifically identifying bacteria, a preparation method thereof, and an application thereof, and belongs to the field of biochemical technology. The present invention uses a fluoroboron dipyrrole compound (BODIPY) as a fluorophore and connects an alkyl group of a specific length through a covalent bond to obtain the wash-free fluorescent probe. The wash-free fluorescent probe obtained by the present invention can effectively label bacteria, achieve the effect of rapid and naked eye visualization of bacterial concentration detection, and has no toxic effect on bacteria under light-proof conditions. At the same time, the present invention provides a method for specifically identifying bacteria based on the wash-free fluorescent probe, which is simple to operate, easy to observe and detect, does not require a complicated decolorization step, and has strong resistance to biological interference.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and in particular relates to a wash-free fluorescent probe for specifically identifying bacteria, and a preparation method and application thereof. Background Art

[0002] Bacterial infections, particularly foodborne and hospital-acquired infections, pose a significant challenge to healthcare. Therefore, rapid and accurate detection and identification of pathogenic bacteria is crucial for disease prevention, diagnosis, and treatment. Traditional bacterial detection methods, such as culture and biochemical tests, while highly accurate, are cumbersome and time-consuming, making them inadequate for rapid clinical diagnosis.

[0003] In recent years, fluorescent labeling-based bacterial detection technologies have attracted widespread attention due to their advantages, including high sensitivity, ease of operation, and rapid response. However, existing fluorescent dyes have limitations during application, such as poor fluorescence signal stability, susceptibility to environmental factors, and toxicity to bacterial growth and metabolism. Furthermore, the labeling process for some fluorescent dyes requires complex washing steps, which not only increases operational complexity but can also lead to bacterial loss or weakening of the fluorescence signal. Patent applications CN201911388988.2 and CN201911058048.7 describe microbial detection methods based on fluorescent staining. While these methods provide valuable detection tools, the complex washing steps leave room for improvement in operational simplicity and detection efficiency. Furthermore, while antibiotic-based probes can exhibit high specificity in bacterial identification, they may affect bacterial biological functions and induce antibiotic resistance. Therefore, the development of highly specific, wash-free probes without any washing steps or biological interference is highly desirable and of great significance for improving the accuracy and clinical application of bacterial detection. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a wash-free fluorescent probe for specific identification of bacteria, which can achieve the effect of rapid and visually visible detection of bacteria.

[0005] The present invention also aims to provide a method for specifically identifying bacteria, which is simple to operate, convenient for observation and detection, has no complicated decolorization steps, and has strong resistance to biological interference.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a wash-free fluorescent probe for specifically identifying bacteria. The structural formula of the wash-free fluorescent probe is as follows:

[0008]

[0009] Wherein, n is 13 to 21, and X is selected from I - 、Cl - Br - Any one of .

[0010] Preferably, when n is 13, the bacteria are Gram-positive live bacteria, Gram-positive dead bacteria, Gram-negative live bacteria and Gram-negative dead bacteria.

[0011] Preferably, when n is 17, the bacterium is a Gram-positive live bacterium.

[0012] The present invention also provides a method for preparing a wash-free fluorescent probe for specific bacterial identification, comprising the following steps: dissolving compound B and an alkyl halide in acetonitrile, refluxing, and purifying to obtain a wash-free fluorescent probe for specific bacterial identification; the structural formula of compound B is shown below:

[0013]

[0014] Preferably, the alkyl halide is selected from any one of bromotetradecane, iodotetradecane, chlorotetradecane, bromopentadecane, iodopentadecane, chloropentadecane, bromohexadecane, iodohexadecane, chlorohexadecane, bromoheptadecane, iodoheptadecane, chloroheptadecane, bromooctadecane, iodooctadecane, chlorooctadecane, bromononadecane, iodononadecane, chlorononadecane, bromoeicosane, iodoeicosane, chloroeicosane, bromoheunescocane, iodoheunescocane, chloroheunescocane, bromodocosane, iodocosane, and chlorodocosane.

[0015] Preferably, the mass volume ratio of the compound B to the alkyl halide is 95-100 mg:0.1 mL.

[0016] Preferably, the reflux time is 20 to 26 hours; and the purification method is silica gel column chromatography purification.

[0017] Preferably, the synthesis steps of the compound B include: dissolving 4-pyridinecarboxaldehyde and 2,4-dimethylpyrrole in dichloromethane, adding trifluoroacetic acid to catalyze the reaction under nitrogen protection; adding DDQ after the reaction is completed, stirring at room temperature; adding Et3N under ice-water bath conditions, stirring; adding B F3·Et2O dropwise under ice-water bath conditions, returning to room temperature after the addition, stirring; filtering, spin drying, extraction, and purification to obtain compound B.

[0018] The present invention also provides a method for specifically detecting bacteria, wherein the wash-free fluorescent probe is mixed with a sample solution, and the mixture is incubated in the dark before detection.

[0019] Preferably, the detection method includes any one or more of fluorescence intensity detection, laser confocal imaging and ultraviolet light irradiation.

[0020] Beneficial effects of the present invention:

[0021] The present invention specifically develops a wash-free fluorescent probe that specifically identifies bacteria. The probe can effectively label bacteria, enabling rapid and visual detection of bacteria. It is non-toxic to bacteria under light-proof conditions and will not cause bacterial resistance.

[0022] Based on the developed wash-free fluorescent probe, the present invention further provides a method for specific detection of bacteria. The method is simple to operate, convenient for observation and detection, can realize rapid and visual detection of bacterial concentration, does not require complicated decolorization steps, and has strong resistance to biological interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :Compound B 1 H NMR spectrum;

[0024] Figure 2 :Compound B 13 C NMR spectrum;

[0025] Figure 3 : High-resolution mass spectrum of compound B;

[0026] Figure 4 :Compound B-8 1 H NMR spectrum;

[0027] Figure 5 :Compound B-8 13 C NMR spectrum;

[0028] Figure 6 : High-resolution mass spectrum of compound B-8;

[0029] Figure 7 :Compound B-14 1 H NMR spectrum;

[0030] Figure 8 :Compound B-14 13 C NMR spectrum;

[0031] Figure 9 : High-resolution mass spectrum of compound B-14;

[0032] Figure 10 :Compound B-18 1 H NMR spectrum;

[0033] Figure 11 :Compound B-18 13 C NMR spectrum;

[0034] Figure 12: High-resolution mass spectrum of compound B-18;

[0035] Figure 13 :Compound B-22 1 H NMR spectrum;

[0036] Figure 14 :Compound B-22 13 C NMR spectrum;

[0037] Figure 15 : High-resolution mass spectrum of compound B-22;

[0038] Figure 16 : Fluorescence spectra of probe B-8 in response to live and dead Escherichia coli and Staphylococcus aureus;

[0039] Figure 17 : Fluorescence spectra of probe B-14 in response to live and dead Escherichia coli and Staphylococcus aureus;

[0040] Figure 18 : Wash-free fluorescence imaging of live and dead Staphylococcus aureus and Escherichia coli stained with probe B-14 and the commercially available nucleic acid dye Sytox deep red (scale bar: 5 μm);

[0041] Figure 19 : Laser confocal imaging of probe B-14 and four Gram-negative bacteria and four Gram-positive bacteria (scale bar: 10 μm);

[0042] Figure 20 : Fluorescence spectra of probe B-18 in response to live and dead Escherichia coli and Staphylococcus aureus;

[0043] Figure 21 : Wash-free fluorescence imaging of live and dead Staphylococcus aureus and Escherichia coli stained with probe B-18 and the commercially available nucleic acid dye Sytox deep red (scale bar: 5 μm);

[0044] Figure 22 : Laser confocal imaging of probe B-18 and four Gram-negative bacteria and four Gram-positive bacteria (scale bar: 10 μm);

[0045] Figure 23 : Fluorescence spectra of probe B-22 in response to live and dead Escherichia coli and Staphylococcus aureus;

[0046] Figure 24 : Dynamic light scattering particle size distribution of probes B-14 and B-18 after aggregation in 1% EtOH / PBS system;

[0047] Figure 25: Scanning electron microscopy images of probes B-14 and B-18 after aggregation in pure water system;

[0048] Figure 26 :The effects of probes B-14 and B-18 on bacterial growth under light-protected conditions;

[0049] Figure 27 : Fluorescence intensity changes over time after Staphylococcus aureus was co-incubated with probe B-18;

[0050] Figure 28 : Naked eye UV light images after incubation of Staphylococcus aureus with different concentrations and probe B-18;

[0051] Figure 29 : Relationship between brightness value and bacterial concentration after incubation of different concentrations of Staphylococcus aureus with probe B-18. DETAILED DESCRIPTION

[0052] The present invention provides a wash-free fluorescent probe for specifically identifying bacteria. The structural formula of the wash-free fluorescent probe is as follows:

[0053]

[0054] Wherein, n is 13 to 21, and X is selected from I - 、Cl - Br - Any one of .

[0055] The wash-free fluorescent probe described in the present invention uses fluoroboron dipyrrole compound (4,4-difluoro-boradiazaindacene, abbreviated as BODIPY) as a fluorophore, which is modified by covalently linking an alkyl group of a specific length. The n described in the present invention is 13 to 21, more preferably n=13, n=17. When the alkyl chain is too short, the synthesized probe cannot aggregate, resulting in the presence of a fluorescent background signal; when the alkyl chain is too long, the bacterial surface microenvironment cannot disaggregate the probe aggregates. The anion X in the wash-free fluorescent probe described in the present invention is a substitution on the alkyl chain during the synthesis process, and the anion X of the synthesized wash-free fluorescent probe is in a free state and is not connected to the chemical structure of the wash-free fluorescent probe.

[0056] The wash-free fluorescent probe of the present invention has an excitation wavelength range of 450 to 550 nm, an emission wavelength range of 550 to 700 nm, low dark toxicity (almost no dark toxicity when the probe concentration is <80 μM), and has no toxic effect on bacteria under light-proof conditions.

[0057] Preferably, when n in the probe is 13 (denoted as probe B-14), the structural formula is as follows:

[0058]

[0059] The probe B-14 has a molecular mass of 522.3830 amu and is capable of broad-spectrum bacterial labeling, including live Gram-positive bacteria, dead Gram-positive bacteria, live Gram-negative bacteria, and dead Gram-negative bacteria. The Gram-negative bacteria include, but are not limited to, Salmonella enterica, Pseudomonas aeruginosa, Proteus vulgaris, and Escherichia coli, while the Gram-positive bacteria include, but are not limited to, Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus.

[0060] Preferably, when n in the probe is 17 (denoted as probe B-18), the structural formula is as follows:

[0061]

[0062] The molecular mass of the probe B-18 is 578.4462 amu, and it can selectively label Gram-positive live bacteria, enabling simultaneous Gram typing and bacterial activity identification. The Gram-positive live bacteria include, but are not limited to, Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. When the Gram-positive live bacteria is Staphylococcus aureus, detection of Staphylococcus aureus can be completed in as fast as 10 minutes, with a detection limit of 47 CFU / mL. The concentration range of Staphylococcus aureus that can be distinguished by the naked eye is 5×10 5 ~1×10 9 CFU / mL.

[0063] The present invention also provides a method for preparing a wash-free fluorescent probe for specific bacterial identification, comprising the following steps: dissolving compound B and an alkyl halide in acetonitrile, refluxing, and purifying to obtain a wash-free fluorescent probe for specific bacterial identification; the structural formula of compound B is shown below:

[0064]

[0065] The alkyl halide of the present invention is selected from any one of bromotetradecane, iodotetradecane, chlorotetradecane, bromopentadecane, iodopentadecane, chlorpentadecane, bromohexadecane, iodohexadecane, chlorhexadecane, bromoheptadecane, iodoheptadecane, chlorheptadecane, bromooctadecane, iodooctadecane, chloroctadecane, bromononadecane, iodononadecane, chlornonadecane, bromoeicosane, iodoeicosane, chloreicosane, bromoheneicosane, iodoheneicosane, chloreicosane, bromodoceicosane, iododoceicosane, and chloredoceicosane; preferably, the alkyl halide is selected from any one of bromotetradecane, iodotetradecane, chlortetradecane, bromooctadecane, iodooctadecane, and chloroctadecane; more preferably, bromotetradecane or iodooctadecane.

[0066] The mass volume ratio of compound B to alkyl halide of the present invention is 95-100 mg:0.1 mL, preferably 97-98 mg:0.1 mL, and more preferably 97.5 mg:0.1 mL. The reflux time of the present invention is 20-26 h, preferably 22-24 h; the purification method is silica gel column chromatography purification, and the eluent used in the purification is DCM:MeOH=10:1 (V:V). As an optional embodiment, the present invention weighs compound B and alkyl halide separately, dissolves them in acetonitrile, refluxes, and removes the solvent by reduced pressure distillation after the reaction is completed. The crude product is purified by silica gel column chromatography to obtain a deep red solid, which is the wash-free fluorescent probe.

[0067] The synthesis steps of the compound B of the present invention include: dissolving 4-pyridinecarboxaldehyde and 2,4-dimethylpyrrole in dichloromethane, adding trifluoroacetic acid to catalyze the reaction under nitrogen protection; adding DDQ after the reaction, stirring at room temperature; adding Et3N under ice-water bath conditions, stirring; adding BF3·Et2O dropwise under ice-water bath conditions, returning to room temperature after the addition, stirring; and filtering, spin-drying, extracting, and purifying to obtain compound B.

[0068] The ratio of 4-pyridinecarboxaldehyde to 2,4-dimethylpyrrole in the present invention is 1 mmol: 2-2.2 mmol, preferably 1 mmol: 2.1 mmol; the volume ratio of 4-pyridinecarboxaldehyde: 2,4-dimethylpyrrole: dichloromethane: trifluoroacetic acid in the present invention is 0.25-0.35: 1.2-1.3: 95-105: 0.05-0.08, preferably 0.3: 1.22: 100: 0.06; the catalytic reaction in the present invention is stirred at room temperature under light-proof conditions for 4-6 hours, preferably 5 hours; The DDQ of the present invention is dissolved in benzene and then added to the system after the above reaction is completed. The amount of DDQ added is the same as that of 4-pyridinecarboxaldehyde. After adding DDQ, the mixture is stirred at room temperature for 1.5 to 2.5 hours, preferably 2 hours. The volume of Et3N added in the present invention is 3 to 4% of the reaction system, and the mixture is stirred for 12 to 18 minutes, preferably 15 minutes. The volume of BF3·Et2O added in the present invention is 6 to 7% of the reaction system, and the mixture is returned to room temperature after dropwise addition and stirred for 1.5 to 2.5 hours, preferably 2 hours. The present invention preferably uses a Buchner funnel for rapid filtration and rinses the filter cake with dichloromethane. The present invention preferably uses a rotary evaporator to spin dry the liquid to obtain a crude product. The present invention preferably dissolves the crude product in dichloromethane for extraction, preferably 2 to 4 times. The organic phase of the extract is dried over anhydrous Na2SO4, and the obtained organic phase is concentrated under reduced pressure and the product is purified by silica gel column chromatography. The eluent is PE:EA = 5:1 (v:v), and an orange solid is finally obtained, which is compound B.

[0069] The present invention also provides a method for specific detection of bacteria, wherein the aforementioned wash-free fluorescent probe is mixed with a sample solution, incubated in the dark, and then detected. The sample solution may also be a resuspended bacterial solution. The probe concentration is preferably 8 to 12 μM, more preferably 10 μM; the incubation time is preferably 10 to 20 minutes. The concentration of bacteria that can be detected by the present invention is preferably 5×10 5 ~1×10 9 CFU / mL. The detection method includes any one or more of fluorescence intensity detection, laser confocal imaging, and ultraviolet light irradiation. The method for detecting bacteria of the present invention can be used for product safety identification, including food, health products, medicines, feed, etc.

[0070] The technical solutions provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention. In the following examples, unless otherwise specified, conventional methods are used. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0071] Example 1

[0072] 1. Synthesis of compound B:

[0073] A 250 mL round-bottom flask was filled with 100 mL of DCM(AR). 4-Pyridinecarboxaldehyde (0.3 mL, 3 mmol) and 2,4-dimethylpyrrole (1.22 mL, 6.3 mmol) were added to the flask and stirred to dissolve. After stirring, 60 μL of trifluoroacetic acid (60 μL) was added and the reaction was stirred at room temperature for 5 h under nitrogen and in the dark. After TLC monitoring, 685 mg of the highly active oxidant DDQ (3 mmol) was dissolved in 10 mL of benzene and added to the reaction system. Stirring was continued at room temperature for 2 h. After the reaction was complete, 4 mL of Et3N was added in an ice-water bath. After stirring for 15 min, 8 mL of BF3·Et2O was added dropwise with a syringe. After the addition was complete, the mixture was returned to room temperature and stirred for another 2 h. The reaction was monitored by TLC using a handheld UV lamp. After the reaction was complete, the mixture was rapidly filtered using a Buchner funnel, the filter cake was rinsed with DCM, and the liquid was dried using a rotary evaporator. The crude product after spin drying was redissolved in DCM and extracted three times. The organic phase was dried over anhydrous Na2SO4. The organic phase was concentrated under reduced pressure and the product was purified by silica gel column chromatography with an eluent of PE:EA = 5:1 (v:v) to obtain an orange solid with a yield of 21%.

[0074] Compound B 1 H NMR spectrum Figure 1 As shown, 13 C NMR spectrum Figure 2 As shown in the high-resolution mass spectrum Figure 3The results show that 1 H NMR (400MHz, CDCl3) δ8.79(s,2H),7.34(s,2H),6.01(s,2H),2.56(s,6H),1.40(s,6H). 13 C NMR (101MHz, CDCl3) δ156.51,150.54,143.74,142.65,137.60,130.34,123.40,121.83,29.71,14.62. ESI-MS:calcd for 325.1562,found 326.1655[M] + H.

[0075] 2. Obtaining the fluorescent probe (using compound B obtained in step 1):

[0076] (1) B-8: In a 100 mL round-bottom flask, 97.5 mg of compound B (0.3 mmol) and 0.1 mL of bromooctane were dissolved in 20 mL of acetonitrile and refluxed for 24 h. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography using a 10:1 (v:v) ratio of DCM to MeOH as the eluent to obtain a dark red solid in a yield of 55%.

[0077] B-8 1 H NMR spectrum Figure 4 As shown, 13 C NMR spectrum Figure 5 As shown in the high-resolution mass spectrum Figure 6 The results show that 1 H NMR (400MHz, CDCl3) δ9.78 (d, J = 5.7Hz, 2H), 8.03 (d, J = 5.7Hz, 2H), 6.06 (s, 2H), 5.21 (t, J = 6.9Hz, 2H), 2.57 (s, 6H), 2.10 (s, 2H), 1.43 (s, 6H), 1.26 (dd, J = 16.5, 8.6Hz, 10H), 0.86 (t, J = 6.8Hz, 3H). 13 C NMR(151MHz,C DCl3)δ158.55,153.08,146.39,142.25,141.84,132.74,129.37,128.64,122.98,6 2.49,32.05,31.66,29.76,29.09,29.02,25.98,22.62,15.57,14.85,14.18,14.11. HRMS(E SI),calculated for(C 26 H 35BF2N3 + ):m / z[M] + :438.2887; found:m / z438.2900.

[0078] (2) B-14: In a 100-mL round-bottom flask, 97.5 mg of compound B and 0.1 mL of bromotetradecane were dissolved in 20 mL of acetonitrile and refluxed for 24 h. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography using a 10:1 (v:v) ratio of DCM to MeOH as the eluent to obtain a dark red solid in a 45% yield.

[0079] B-14 1 H NMR spectrum Figure 7 As shown, 13 C NMR spectrum Figure 8 As shown in the high-resolution mass spectrum Figure 9 The results show that 1 H NMR(400MHz, CDCl3)δ9.82(s,2H),8.04(s,2H),6.05(s,2H),5.19(s,2H),2 .56(s,6H),2.09(s,2H),1.42(s,6H),1.23(d,J=12.3Hz,20H),0.87(s,3H). 13 C NMR (151MHz, CDCl3) δ158.51,152.99,146.47,141.83,132.80,129.36,128.66,122.95,62.39,32 .07,31.98,29.74,29.71,29.66,29.54,29.48,29.41,29.10,25.99,22.74,15.53,14.83,14.18. HRMS(ESI),calculated for(C 32 H 47 BF2N3 + ):m / z[M] + :522.3826; found:m / z522.3830.

[0080] (3) B-18: In a 100-mL round-bottom flask, 97.5 mg of compound B and 0.1 mL of iodooctadecane were dissolved in 20 mL of acetonitrile and refluxed for 24 h. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography using a 10:1 (v:v) ratio of DCM to MeOH as the eluent to obtain a dark red solid in a 33% yield.

[0081] B-18 1 H NMR spectrum Figure 10 As shown, 13 C NMR spectrum Figure 11 As shown in the high-resolution mass spectrum Figure 12 The results show that 1 H NMR (400MHz, CDCl3) δ9.57(d,J=6.3Hz,2H),8.04(d,J=6.2Hz,2H),6.05(s,2H),5.11(t,J=7 .1Hz,2H),2.56(s,6H),2.09(s,2H),1.46(s,6H),1.29-1.25(m,30H),0.87(t,J=6.7Hz,3H). 13 C NMR (151MHz, CDCl3) δ158.65,153.31,145.93,141.90,132.49,129.34,128.60,123.01,77.31,77.10 ,76.89,62.95,32.00,31.74,29.92–29.61,29.61–29.30,29.06,25.99,22.76,16.02,14.87,14.19. ESI-MS:calcd for578.4452,found: 578.4462[M] + .

[0082] (4) B-22: In a 100 mL round-bottom flask, 97.5 mg of compound B and 0.1 mL of bromodocosane were dissolved in 20 mL of acetonitrile and refluxed for 24 h. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography using a 10:1 (v:v) ratio of DCM to MeOH as the eluent to obtain a dark red solid in a yield of 27%.

[0083] B-22 1 H NMR spectrum Figure 13 As shown, 13 C NMR spectrum Figure 14 As shown in the high-resolution mass spectrum Figure 15 The results show that 1 H NMR(400MHz, CDCl3) δ9.73(d,J=5.3Hz,2H),8.03(d,J=5.4Hz,2H),6.06(s,2H),5.21(t ,J=6.4Hz,2H),2.57(s,6H),1.43(s,6H),1.24(d,J=9.3Hz,38H),0.88(t,J=6.7Hz,3H). 13C NMR (151MHz, CDCl3) δ158.55,146.39,141.83,129.37,128.59,122.97,62.48,32.06,31.99, 29.80,29.79,29.73,29.69,29.56,29.49,29.43,29.11,26.01,22.76,15.56,14.84,14.19. HRMS(ESI),calculated for(C 40 H 63 BF2N3 + ):m / z[M] + :634.5078; found:m / z634.5085.

[0084] 3. Preparation of fluorescent probe mother solutions: Using ethanol as solvent, prepare 1 mM probe B-8 mother solution, probe B-14 mother solution, probe B-18 mother solution, and probe B-22 mother solution, respectively.

[0085] Example 2

[0086] The fluorescent probe stock solution prepared in Example 1 was used to fluorescently label bacteria:

[0087] 1. Bacterial species and culture:

[0088] (1) Salmonella enterica (ATCC 14028), Pseudomonas aeruginosa (BNCC 336458), Proteus vulgaris (BNCC 336633), Escherichia coli (ATCC 25922), Enterococcus faecalis (BNCC 102668), Listeria monocytogenes (BNCC 185986), Bacillus cereus (BNCC 1003930), and Staphylococcus aureus (ATCC 25923) were selected as representative bacteria for testing. The culture medium for Listeria monocytogenes (BNCC 185986) was brain heart infusion medium, while the other bacteria were cultured in LB broth, and the solid culture medium was NA nutrient agar.

[0089] (2) After the bacteria were cultured in liquid culture medium for 24 hours, the culture medium was centrifuged at 8000 rpm for 5 minutes, the supernatant was removed, and the culture medium was resuspended three times in PBS (10 mM, pH = 7.4). The absorbance of the bacteria at 600 nm was adjusted to 0.5 and the culture medium was set aside.

[0090] (3) Dead bacteria were obtained by killing live bacteria with 70% ethanol: After culturing the bacteria in liquid culture for 24 h, the culture was centrifuged at 8000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 70% ethanol. After incubation at 37°C and 120 rpm for 1 h, the supernatant was removed by centrifugation at 8000 rpm for 5 min, and the cells were resuspended three times in PBS (10 mM, pH = 7.4) to obtain a dead bacterial suspension.

[0091] 2. Fluorescent probe B-8 selectively responds to living and dead bacteria:

[0092] Experiments were conducted on Staphylococcus aureus (live bacteria), Staphylococcus aureus (dead bacteria), Escherichia coli (live bacteria), and Escherichia coli (dead bacteria).

[0093] Fluorescence intensity of fluorescent probe B-8 was used to detect bacteria: 30 μL of B-8 mother solution was added to 2970 μL of PBS as blank control group; 30 μL of B-8 mother solution was added to 2970 μL of PBS to resuspend the bacteria (bacterial concentration was about 1×10 8 The experimental group consisted of cells with 0.1% CFU / mL. The cells were incubated at room temperature for 20 min. The incubation solution was added to a cuvette, and the fluorescence signal intensity was measured using a fluorescence spectrophotometer with an excitation wavelength of 508 nm.

[0094] The response fluorescence spectrum is shown in Figure 2 Figure 16 As shown. Figure 16 It can be seen that the fluorescent signal of the blank control group of fluorescent probe B-8 is stronger, and the increase rate of fluorescence intensity in the experimental group is lower than that in the blank control group, indicating that the fluorescent probe B-8 has strong background interference and cannot achieve accurate detection under wash-free conditions.

[0095] 3. Fluorescent probe B-14 selectively responds to living and dead bacteria:

[0096] Experiments were conducted on Staphylococcus aureus (live bacteria), Staphylococcus aureus (dead bacteria), Escherichia coli (live bacteria), and Escherichia coli (dead bacteria).

[0097] Detection of bacteria using fluorescence intensity of fluorescent probe B-14: The method is the same as step 2.

[0098] Fluorescent probe B-14 laser confocal imaging of bacteria: The bacterial concentration was adjusted to 1×10 6CFU / mL, the bacterial suspension was then mixed with probe B-14 (final concentration: 10 μM) and incubated at room temperature for 20 min. Sytox deep red dye (purchased from Thermo Fisher Scientific, lot number S11381) was added to the bacterial suspension and incubated in the dark for 10 min. A 2 μL droplet of the combined solution was placed on a culture dish, covered with a thin layer of agarose gel, and observed using a laser confocal microscope. Probe B-14 excitation wavelength: 515 nm, emission wavelength: 530-600 nm; excitation wavelength: 635 nm, emission range: 650-750 nm.

[0099] The response fluorescence spectrum is shown in Figure 2 Figure 17 As shown. Figure 17 It can be seen that the fluorescence signal intensity of the fluorescent probe B-14 increased after incubation of Staphylococcus aureus (live bacteria), Staphylococcus aureus (dead bacteria), Escherichia coli (live bacteria), and Escherichia coli (dead bacteria) for 20 minutes. Figure 18 As shown. Figure 18 It can be seen that probe B-14 can effectively stain Staphylococcus aureus (live), Staphylococcus aureus (dead), Escherichia coli (live), and Escherichia coli (dead), and a bright green color can be seen under 515 nm excitation light.

[0100] 4. Fluorescent probe B-14 for no-wash fluorescence imaging of bacteria:

[0101] Select Salmonella enterica (G - ), Pseudomonas aeruginosa (G - ), Proteus vulgaris (G - ), Escherichia coli (G - ), Enterococcus faecalis (G + ), Listeria monocytogenes (G + ), Bacillus cereus (G + ) and Staphylococcus aureus (G + ) as representative.

[0102] Pipette 10 μL of B-14 mother solution into 990 μL of PBS to resuspend the bacteria (bacterial concentration 1×10 6 CFU / mL) and incubate at room temperature for 20 minutes. Washing off excess dye is not necessary. Slide preparation: Spot 20 μL of 0.3% agarose solution, add 10 μL of sample solution, and cover with a coverslip. Observe using a laser confocal microscope, using excitation at 515 nm and emission range from 550 to 650 nm.

[0103] Laser confocal imaging Figure 19 As shown. Figure 19The results show that the B-14 probe can image Gram-positive bacteria Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus, as well as Gram-negative bacteria Salmonella enterica, Pseudomonas aeruginosa, Proteus vulgaris, and Escherichia coli without washing. This indicates that the fluorescent B-14 probe can image a broad spectrum of bacteria without washing.

[0104] 5. Fluorescent probe B-18 selectively responds to live and dead bacteria:

[0105] Experiments were conducted on Staphylococcus aureus (live bacteria), Staphylococcus aureus (dead bacteria), Escherichia coli (live bacteria), and Escherichia coli (dead bacteria).

[0106] Detection of bacteria using fluorescence intensity of fluorescent probe B-18: The method is the same as step 2.

[0107] Fluorescent probe B-18 laser confocal imaging of bacteria: The method is the same as step 3.

[0108] The response fluorescence spectrum is shown in Figure 2 Figure 20 As shown. Figure 20 It can be seen that the fluorescent probe B-18 was incubated with live and dead Staphylococcus aureus and Escherichia coli for 20 minutes. Only the live Staphylococcus aureus increased in fluorescence signal intensity after incubation with B-18, and the fluorescence signal at 586nm increased by 53.2 times. Figure 21 As shown. Figure 21 It can be seen that B-18 has no specificity for live or dead Escherichia coli and dead Staphylococcus aureus, and only responds to live Staphylococcus aureus.

[0109] 6. Fluorescent probe B-18 for no-wash fluorescence imaging of bacteria:

[0110] Select Salmonella enterica (G - ), Pseudomonas aeruginosa (G - ), Proteus vulgaris (G - ), Escherichia coli (G - ), Enterococcus faecalis (G + ), Listeria monocytogenes (G + ), Bacillus cereus (G + ) and Staphylococcus aureus (G + ) as representative.

[0111] Pipette 10 μL of B-18 mother solution into 990 μL of PBS to resuspend the bacteria (bacterial concentration is about 1×10 6 CFU / mL) and incubate at room temperature for 20 minutes. Washing off excess dye is not necessary. Slide preparation: Spot 20 μL of 0.3% agarose solution, add 10 μL of sample solution, and cover with a coverslip. Observe using a laser confocal microscope, using excitation at 515 nm and emission range from 550 to 650 nm.

[0112] Laser confocal imaging Figure 22 As shown. Figure 22 As can be seen, bright fluorescence was observed after incubation of B-18 with four Gram-positive bacteria. However, fluorescence was barely visible when B-18 was incubated with Gram-negative bacteria. This indicates that the fluorescent probe B-18 can image Gram-positive bacteria without washing, but does not respond to Gram-negative bacteria.

[0113] 7. Fluorescent probe B-22 selectively responds to living and dead bacteria:

[0114] Experiments were conducted on Staphylococcus aureus (live bacteria), Staphylococcus aureus (dead bacteria), Escherichia coli (live bacteria), and Escherichia coli (dead bacteria).

[0115] Detection of bacteria using fluorescence intensity of fluorescent probe B-22: The method is the same as step 2.

[0116] The response fluorescence spectrum is shown in Figure 2 Figure 23 As shown. Figure 23 It can be seen that there is almost no fluorescent signal in the blank control group and experimental group of fluorescent probe B-22, indicating that due to its strong hydrophobicity, the fluorescent probe B-22 aggregates and causes fluorescence quenching, making it unable to bind to bacteria and unable to detect bacteria.

[0117] 8. Characterization of the no-wash properties of fluorescent probes B-14 and B-18:

[0118] 30 μL of each probe B-14 and probe B-18 stock solution was pipetted into 2970 μL of PBS, and the final concentration of the probe was 10 μM.

[0119] Dynamic light scattering particle size analyzer was used to detect the aggregation degree of fluorescent probes B-14 and B-18 in PBS. The results are as follows: Figure 24 shown. Figure 24 In the figure, (a) is the dynamic light scattering particle size distribution diagram of probe B-14 after aggregation in 1% (EtOH / PBS) system, and (b) is the dynamic light scattering particle size distribution diagram of probe B-18 after aggregation in 1% (EtOH / PBS) system.

[0120] The probes were fixed in an aqueous solution by freeze drying, and the aggregation morphology was observed by scanning electron microscopy. Figure 25 shown. Figure 25 In the figure, (a) is a scanning electron micrograph of probe B-14 after aggregation in a pure water system, and (b) is a scanning electron micrograph of probe B-18 after aggregation in a pure water system.

[0121] Figure 24 and Figure 25Results showed that fluorescent probes B-14 and B-18 exhibit low background fluorescence in aqueous solution, but their fluorescence signal recovers upon binding to their targets. This is because the probes aggregate into nanoparticles in aqueous solution, resulting in aggregation-induced fluorescence quenching. Binding of the probe-aggregated nanoparticles to the target disrupts the aggregation, restoring the fluorescence signal.

[0122] 9. Toxic effects of fluorescent probes B-14 and B-18 on bacterial growth under light-protected conditions:

[0123] Staphylococcus aureus was selected as a representative of Gram-positive bacteria, and Escherichia coli was selected as a representative of Gram-negative bacteria. Fluorescent probes B-14 and B-18 were taken as stock solutions, and the initial concentrations of the fluorescent probes B-14 and B-18 were adjusted to 80 μM. A volume of 20 μL was taken, and then an equal amount of ethanol solvent was added to a 96-well plate for dilution (probe concentrations were 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM, respectively). After the bacteria were cultured in LB broth for 24 hours, they were subcultured and diluted to a bacterial concentration of approximately 1×10 6 CFU / ml. Pipette 180 μL of the diluted bacterial solution into a 96-well plate and mix with the probe. Protect from light, incubate at 37°C, 120 rpm, and measure OD using a microplate reader after 24 hours. 600 .

[0124] See the results Figure 26 . Figure 26 In the figure, (a) shows the effect of probe B-14 on bacterial growth in the dark, and (b) shows the effect of probe B-18 on bacterial growth in the dark. The results show that fluorescent probes B-14 and B-18 have no toxic effect on bacteria in the dark.

[0125] Example 3

[0126] The fluorescent probe B-18 mother solution prepared in Example 1 was used to fluorescently label Staphylococcus aureus:

[0127] 1. Pick a single spot from the NA nutrient agar medium containing Staphylococcus aureus and transfer it to 10 mL LB medium. Incubate the culture in a shaker at 37°C for 16 hours. Transfer the bacteria to a sterile centrifuge tube and centrifuge at 8000 rpm for 5 minutes. Remove the supernatant. Resuspend the cells in PBS (10 mM, pH = 7.4) and calculate the OD 600 The value was adjusted to 0.1. Subsequently, the bacterial suspension was evenly mixed with the probe B-18 (final concentration was 10 μM), incubated at room temperature, and the fluorescence intensity at 578 nm after the probe bound to the bacteria was observed over time using a fluorescence spectrophotometer.

[0128] The fluorescence intensity of Staphylococcus aureus co-incubated with probe B-18 changes over time as shown in the figure below. Figure 27The results showed that the fluorescent probe B-18 could quickly respond to Staphylococcus aureus.

[0129] 2. Pick a single spot from the NA nutrient agar medium containing Staphylococcus aureus and transfer it to 10 mL of LB medium. Incubate the culture in a shaker at 37°C for 16 hours. Transfer the bacteria to a sterile centrifuge tube and centrifuge at 8000 rpm for 5 minutes. Remove the supernatant. Resuspend the bacteria in PBS (10 mM, pH = 7.4) and perform a gradient dilution of Staphylococcus aureus to 1 × 10 9 CFU / mL, 1×10 8 CFU / mL, 5×10 7 CFU / mL, 1×10 7 CFU / mL, 5×10 6 CFU / mL, 1×10 6 CFU / mL and 5×10 5 CFU / mL. No bacteria were added to the blank control group. The bacterial suspension was incubated with B-18 (10 μM) for 10 min and imaged using a fluorescence sensor.

[0130] After different concentrations of Staphylococcus aureus were incubated with probe B-18, the naked eye UV light was used as shown in the figure below. Figure 28 The results showed that after incubation with different concentrations of Staphylococcus aureus, B-18 emitted corresponding light under naked eye ultraviolet light. The fluorescent probe B-18 can realize a method for rapid and visual detection of Staphylococcus aureus concentration.

[0131] 3. Use mobile phone to measure the concentration of Staphylococcus aureus 5×10 5 ~1×10 9 The emission light of CFU / mL was captured. The solution color was extracted under UV irradiation (Photoshop, PS extracting brightness values). By collecting image brightness, a linear fit was performed between bacterial concentration and emission light brightness. Data analysis was used to construct a line between brightness and S. aureus concentration.

[0132] The relationship between brightness value and bacterial concentration is as follows Figure 29 The results show that the detection interval is 5×10 5 ~1×10 9 CFU / mL, R 2 =0.9902, showing a good linear relationship. The detection limit of fluorescent probe B-18 for Staphylococcus aureus was calculated to be 47 CFU / mL.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A wash-free fluorescent probe for specific identification of bacteria, characterized in that: The structural formula of the wash-free fluorescent probe is as follows: ， wherein n is 13 to 17, and X is selected from any one of I, Cl, and Br.

2. The wash-free fluorescent probe according to claim 1, characterized in that When n is 13, the bacteria are Gram-positive live bacteria, Gram-positive dead bacteria, Gram-negative live bacteria and Gram-negative dead bacteria.

3. The wash-free fluorescent probe according to claim 1, characterized in that When n is 17, the bacteria are viable Gram-positive bacteria.

4. The method for preparing a wash-free fluorescent probe according to any one of claims 1 to 3, wherein: The method comprises the following steps: dissolving compound B and alkyl halide in acetonitrile, refluxing, and purifying to obtain a wash-free fluorescent probe that specifically identifies bacteria; the structural formula of compound B is shown below: 。 5. The preparation method according to claim 4, characterized in that The alkyl halide is selected from any one of bromotetradecane, iodotetradecane, chlorotetradecane, bromopentadecane, iodopentadecane, chloropentadecane, bromohexadecane, iodohexadecane, chlorohexadecane, bromoheptadecane, iodoheptadecane, chloroheptadecane, bromooctadecane, iodooctadecane, and chlorooctadecane.

6. The preparation method according to claim 4, characterized in that The mass volume ratio of the compound B to the alkyl halide is 95-100 mg:0.1 mL.

7. The preparation method according to claim 4, characterized in that The reflux time is 20 to 26 hours; and the purification method is silica gel column chromatography purification.

8. The preparation method according to claim 4, characterized in that The synthesis steps of the compound B include: dissolving 4-pyridinecarboxaldehyde and 2,4-dimethylpyrrole in dichloromethane, adding trifluoroacetic acid to catalyze the reaction under nitrogen protection; adding DDQ after the reaction, stirring at room temperature; adding Et3N in an ice-water bath, stirring; adding BF3·Et2O dropwise in an ice-water bath, returning to room temperature after the addition, stirring; and filtering, spin drying, extracting, and purifying to obtain compound B.

9. A method for non-disease diagnosis or treatment by specifically detecting bacteria, characterized in that: The wash-free fluorescent probe according to any one of claims 1 to 3 is mixed with a sample solution, incubated in the dark, and then detected.

10. The method according to claim 9, characterized in that The detection method includes any one or more of fluorescence intensity detection, laser confocal imaging and ultraviolet light irradiation.

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