CN111595829B - Method for selectively imaging capsular bacteria by using tetrastyrene derivatives - Google Patents
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Abstract
The invention relates to a method for selectively imaging capsular bacteria by using a tetrastyrene derivative. The method comprises the design and synthesis of a compound which takes tetraphenyl vinyl as a framework and has aggregation-induced emission properties and positive charges, wherein the probe with two positive charges has better membrane penetration capacity and can effectively stain bacteria with thick capsules, and the single-charge probe can only stain common bacteria. Meanwhile, the regulation of the emission spectrum of the probe molecule is realized by connecting different electron donating and accepting groups, the method selectively images the capsular bacteria by utilizing the combination of single and double charges, which is different from the charge number, the difference of fluorescence signals and the membrane penetrating capability of the compound, and the problem that the capsular bacteria is difficult to stain is solved. The compound involved in the method has good light stability and fluorescence imaging capability; can be used for fluorescence imaging and dynamic visual tracking of bacteria; the capsular bacteria can be selectively identified, and the method has potential application value in realizing rapid imaging diagnosis of pathogens.
Description
Technical Field
The invention relates to a method for selectively imaging capsular bacteria through tetraphenyl ethylene derivatives, which comprises a series of tetrastyrene compounds with aggregation-induced emission properties and capable of staining capsular bacteria and a method for selectively imaging capsular bacteria through different combinations of charge numbers, fluorescence signal differences and membrane penetrating capacity of the compounds.
Background
In recent years, fluorescence technology has received much attention due to its high sensitivity and simplicity. However, most conventional fluorescent probes have an aggregation-induced quenching (ACQ) effect. Furthermore, the photobleaching of probes during dynamic visualization and long-term monitoring greatly limits their application in microbial detection. The main aggregation-induced emission material at present uses tetraphenyl vinyl as a main structure, and an electron-withdrawing group is introduced to form an electron donor-acceptor structure to adjust the fluorescence property of the material, so that the material has excellent light stability and large stokes shift (Advanced Biosystems 2018,1800074, AdvMater2017,29(28), Small 2017,13 (41)), and is widely applied to biological detection and imaging.
Microorganisms such as bacteria and fungi are ubiquitous and are closely related to human life and health. Many diseases are also caused by microorganisms, and therefore detection of microorganisms is also becoming increasingly important. Traditional microbial detection methods include smear and bacterial culture methods, Polymerase Chain Reaction (PCR) methods, enzyme-linked immunosorbent assay (ELISA) methods and the like, and the traditional methods generally consume long time and require professional operation, thus preventing the methods from being widely popularized; recently, new methods such as molecular biology, chemical sensors, mass spectrometry, etc. have been developed, and these methods have a short time, but the detection apparatus is expensive and complicated to operate, and require a complicated preliminary pretreatment process. In recent years, fluorescence imaging technology has attracted more and more attention due to its high sensitivity, easy operation and real-time monitoring, and has been widely used in the fields of microbial detection, detection of proteins and metal ions, detection and diagnosis of tumor diseases, and the like (ACS appl. Mater. interfaces 2018,10(25), 21619-.
Capsular polysaccharide is a special structure on the surface of some bacteria, is a layer of colloidal substance on the surface of the bacterial cell wall, and is also an important virulence factor of the bacteria. Due to the existence of capsular polysaccharide, the bacteria can resist the severe environment, protect the bacteria from phagocytosis of macrophages, and maintain the survival ability of the bacteria in cells. Also, the presence of capsular polysaccharides makes it difficult for the bacteria with pods to be stained by conventional methods and dyes (molecular medical Microbiology 2015,33-53, j. curr. protoc. microbiol.2009, Appendix 3I.). There is therefore a need for a simple and convenient method to identify bacteria with thick capsules.
Disclosure of Invention
The invention aims to provide a series of tetra-styrene compounds with aggregation-induced emission properties and capable of staining capsular bacteria and a method for realizing multi-probe multichannel selective imaging of the capsular bacteria through different combinations of charge numbers, fluorescence signal differences and membrane penetration capacity of the compounds. The problem that the fluorescent dye is difficult to stain the bacteria with thick capsules and the problem of fluorescence imaging in the prior art are solved, and a novel method for detecting the capsular bacteria is provided.
The technical method for solving the technical problem comprises the following steps: the aggregation-induced emission material with different fluorescence signals and different positive charge recognition groups is designed by using tetraphenyl ethylene as a framework and regulating the fluorescence spectrum property of the material, so that cell membranes with negative charges on the surface of bacteria are induced to generate different electrostatic interactions, and probes with more positive charges have stronger membrane penetrating capacity and effectively stain capsular bacteria; meanwhile, the double-charge fluorescent probe has stronger membrane penetrating capability and can dye bacteria with a membrane, the single-charge fluorescent probe can only dye common bacteria, through the characteristic, the fluorescence spectrum property of the material is designed and adjusted, the probes with larger emission wavelength difference and different membrane penetrating capability are used, and the multi-probe multi-channel selective recognition of capsular bacteria and fluorescence imaging are realized through the fluorescence intensity analysis in different channels and the single-double charge combination.
The structure of a series of tetraphenyl vinyl compounds with aggregation-induced emission properties, which can stain capsular bacteria, is contained in a group selected from any structural formula:
wherein R is1、R2Are each selected from the following structures:
wherein R ═ ClO3 -，Cl-，I-Or Br-These resemble negatively charged groups.
In the single charge aggregation-induced emission molecular structure, the single charge aggregation-induced emission molecular structure comprises a tetraphenylethylene main structure and an R1Structure R2is-H.
In the double charge aggregation-induced emission molecular structure, a main structure of tetraphenylethylene is contained, and R is contained1And R2Structure R1And R2Similarly, molecules with two positive charges on one side are exemplified below:
the invention relates to a method for realizing multi-probe multi-channel selective imaging of capsular bacteria through different combinations of charge number, fluorescence signal difference and membrane penetrating capability of a compound, which mainly comprises the steps of designing and adjusting fluorescence spectrum properties of materials, using probes with larger emission wavelength difference and different membrane penetrating capability, and realizing multi-probe multi-channel selective identification of capsular bacteria and fluorescence imaging through fluorescence intensity analysis in different channels and single-double charge combination. The fluorescent molecular structure involved in the method is not limited to the single-double charge aggregation-induced emission molecule designed above. The kit comprises a series of tetrastyrene derivatives with aggregation-induced emission properties of non-staining capsular bacteria with one positive charge and a series of tetrastyrene derivatives with aggregation-induced emission properties of staining capsular bacteria with two positive charges, and the selected two fluorescent molecules have obvious difference in emission wavelength and can be used for multichannel imaging identification of capsular bacteria.
The capsular bacteria involved in the invention comprise the following: any bacteria having a capsular structure, such as Streptococcus pneumoniae, Klebsiella pneumoniae, Neisseria meningitidis, Bacillus anthracis, Cryptococcus, and the like.
The invention also provides a preparation method of the tetraphenyl vinyl compound with aggregation-induced emission properties.
The invention also provides application of the tetraphenyl vinyl compound with aggregation-induced emission properties in bacterial fluorescence imaging.
The invention also provides application of the single-double single-charge probe combination in selective recognition of capsular bacteria.
The invention also provides application of the double-charge probe in visually tracking macrophage phagocytosis capsular bacteria.
The implementation of a series of tetraphenyl vinyl compounds with aggregation-induced emission properties capable of staining capsular bacteria and the method for selectively identifying capsular bacteria through different combinations of optical properties and membrane penetration capacity of the compounds of the invention has the following beneficial effects: the tetraphenyl vinyl compound capable of staining the capsular bacteria has strong membrane penetrating capacity, excellent water solubility and good light stability, and can effectively stain the capsular bacteria and perform fluorescence imaging and dynamically visualize the process of phagocytosis of the capsular bacteria by macrophages; the capsular bacteria can be identified by single and double charge combinations through the difference of the charge number, optical property and membrane penetrating capability of the compound, so that the problem that the capsular bacteria are difficult to stain by common dyes is solved, and a novel method for identifying the capsular bacteria is provided.
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FIG. 1 is a scheme for synthesizing a tetraphenylvinyl compound having aggregation-induced emission properties in an example of the present invention.
FIG. 2 is a graph showing the ultraviolet absorption intensity curve and the fluorescence intensity curve of the compound in the example of the present invention.
FIG. 3 is a graph showing fluorescence intensity before and after single-charge probes stained Klebsiella pneumoniae, respectively, in an example of the present invention.
FIG. 4 is a graph showing the comparison of fluorescence intensity before and after the single-charge probe stains Klebsiella pneumoniae, respectively, in the example of the present invention.
FIG. 5 is a fluorescence imaging photograph of separately staining capsular bacteria, which represents Klebsiella pneumoniae and common bacteria, with a single double-charge probe in the example of the present invention.
FIG. 6 shows a commercial probe Syto according to an embodiment of the present inventionTMAnd 9, FITC and Phrodo Red stain capsular bacteria respectively to represent fluorescence imaging photographs of Klebsiella pneumoniae and common general bacteria.
FIG. 7 is a fluorescent image photograph of selectively recognizing capsular bacteria and Klebsiella pneumoniae by single-charge probe combination in the example of the invention.
FIG. 8 is a fluorescent image photograph of macrophage phagocytic bacteria visual tracking using dual-charge probe labeled Klebsiella pneumoniae in the present example.
Detailed Description
The series of tetraphenyl vinyl compounds with aggregation-induced emission properties that can stain capsular bacteria and the method for specifically identifying capsular bacteria by different combinations of charge number, difference in fluorescence signal and membrane penetration ability of the compounds of the invention are further illustrated below with reference to the accompanying drawings and examples:
the novel series of tetraphenyl vinyl compounds with aggregation-induced emission properties capable of staining capsular bacteria of the present invention and methods for multi-probe multichannel selective imaging of capsular bacteria by different combinations of charge number, fluorescence signal differences, and membrane penetration ability of the compounds are illustrated by the following examples. It should be noted that the tetraphenyl vinyl compound prepared below is only one or more of those represented by each structure in the structural formula protected in the claims, but the aggregation-induced emission of the tetraphenyl vinyl compound protected by the present invention is not limited thereto; the single and double charge probes selected for selective recognition of capsular bacteria by different combinations of optical properties and membrane penetrating ability of the compounds used below are only one or several of the combination options claimed in the claims, but the combination of single and double charges claimed in the present invention is not limited thereto; the capsular bacteria selected below represent Klebsiella pneumoniae only one of the claimed capsular bacteria, but the capsular bacteria to which the invention relates are not limited thereto.
Example (b): synthetic tetraphenyl vinyl compound with aggregation-induced emission property
Structural formula (xvi):
the synthetic process is shown in the synthetic route shown in figure 1.
(1) Synthesis of TPE-Py+: the reactants 1, 2-lutidine-1-iodide (55.95mg, 0.24mmol) and TPE-CHO (50mg, 0.12mmol) were first dissolved in anhydrous ethanol (30mL) and a few drops of triethylamine were added. The reaction mixture is then reacted with N2Reflux under protection for 48 hours. After the reaction mixture was cooled to room temperature, the mixture was washed with water and CH2Cl2Extraction was carried out three times. Then Na is added2SO4Drying. The crude product was purified by silica gel column using CH2Cl2The MeOH/MeOH mixture (10: 1v/v) served as eluent to give a yellow powder (23.50mg, yield: 31%).1H NMR(500MHz,Chloroform-d)δ9.32(d,J＝6.2Hz,1H),8.34(t,J＝8.0Hz,1H),8.25(d,J＝8.4Hz,1H),7.80(t,J＝6.9Hz,1H),7.59(d,J＝15.7Hz,1H),7.49(d,J＝7.9Hz,2H),7.34(s,1H),7.14(q,J＝7.6Hz,5H),7.08–6.91(m,6H),6.67(dd,J＝12.5,8.3Hz,4H),4.58(s,3H),3.77(s,6H).13C NMR(126MHz,Chloroform-d)δ158.50,158.38,153.29,148.49,146.47,144.66,144.42,143.63,141.99,138.10,135.86,135.76,132.70,132.61,132.30,131.94,131.57,131.38,131.24,128.37,127.96,126.50,125.43,125.25,115.42,113.31,113.06,55.21,55.12,47.50.HR-MS(ESI，positive)found C36H32NO2(M-I)510.24，calcd for 510.2429。
(2) Synthesis of TPE-N+: the reactant TPE-CO3Br (100mg, 0.19mmol) and Me3N (33.55mg, 0.57mmol) was dissolved in anhydrous THF (30 mL). Then the mixture is added to N2Stir at room temperature for 2 days with protection. After completion of the reaction, the mixture was filtered and washed with excess THF solution to give a white solid (79.88mg, yield 83%).1H NMR(400MHz,Methanol-d4)δ7.12–7.06(m,3H),7.02–6.97(m,2H),6.95–6.88(m,6H),6.73–6.62(m,6H),4.06(t,J＝5.7Hz,2H),3.73(d,J＝7.8Hz,6H),3.63–3.56(m,2H),3.20(s,9H),2.28(dq,J＝11.5,5.6Hz,2H).13C NMR(101MHz,Methanol-d4)δ158.23,136.47,132.26,132.19,131.01,127.30,125.78,113.37,112.70,112.64,64.11,54.18,54.16,52.31,52.27,52.23,48.24,48.03,47.82,47.61,47.39,47.18,46.97,22.94.HR-MS(ESI,positive)found C34H38NO3(M–Br)508.2849.calcd for 508.2852.
(3) Synthesis of TPE-Pn++: the reaction of 4-methyl-1- (3- (trimethylamino) propyl) pyridine-1-bromide (41.89mg, 0.12mmol) and TPE-CHO (50mg, 0.12mmol) was dissolved in anhydrous ethanol (30mL) with piperidine as the catalyst. Then the mixture is added to N2Reflux overnight with protection. After the mixture was cooled to room temperature, the solvent was evaporated under reduced pressure. MeOH was used as a washThe crude product was purified by column on silica gel to obtain red powder (46.67mg, yield: 52%).1HNMR(400MHz，CH3OH)δ8.97(d，J)＝6.4Hz，2H)，8.25(d，J＝6.4Hz，2H)，7.96(d，J＝16.2Hz，1H)，7.58(d，J＝8.0Hz，2H)，7.43(d，J＝16.2)Hz，1H)，7.26-6.90(m，11H)，6.86-6.62(m，4H)，4.74(t，J＝7.7Hz，2H)，3.79(s，6H)，3.73-3.62(m，2H)，3.30(s，9H)，2.79-2.60(m，2H)。13C NMR(101MHz，CH3OH)δ143.93,141.91,132.35,132.27,131.86,131.09,127.58,123.93,121.93,112.89,112.71,62.44,56.56,54.21,52.63,48.25,48.03,47.82,47.61,47.39，47.18,46.97,24.74.HR-MS(ESI，positive)found C47H44N2O2(M-2Br)/2298.17，calcd for 289.1697。
(4) Synthesis of TPE-Py++: 1, 4-Dimethylpyridinium iodide (206.8mg, 0.88mmol) and THE-2CHO (100.00mg, 0.24mmol) were dissolved in anhydrous ethanol (50mL) and a few drops of triethylamine were added to THE mixture. Mixing the mixture in N2Reflux under protection for 48 hours. After the reaction mixture was cooled to room temperature, the resulting mixture was washed with water and CH2Cl2Extraction was carried out three times. Then Na is added2SO4The mixture is dried. The crude product was purified by silica gel column using CH2Cl2MeOH/MeOH mixture (15: 1v/v) as eluent gave a yellow powder (40.76mg, yield: 21%).1H NMR(400MHz,DMSO-d6)δ8.86(d,J＝6.3Hz,4H),8.21(d,J＝6.7Hz,2H),8.02–7.29(m,8H),7.10–6.79(m,10H),6.74(d,J＝8.8Hz,4H),4.29(d,J＝19.1Hz,6H),3.75–3.67(m,6H).13C NMR(101MHz,DMSO-d6)δ158.14,152.40,145.80,145.02,144.44,140.11,135.21,133.20,132.18,132.08,131.59,127.90,127.84,127.78,123.39,113.36,55.19,55.02,55.01,47.27,46.93.HR-MS(ESI,positive)found C44H44N2O2(M-2I)/2314.1540,calcd for 314.1539.
Example (b): cultivation of bacteria
First, a single colony of Klebsiella pneumoniae (K.pneumoconiae) on a solid nutrient broth medium was transferred to 10mL of a liquid LB medium, andthe culture was carried out at 37 ℃ for 12 hours. Measuring Optical Density (OD) at 600nm by Using an ultraviolet Spectrophotometer600) To determine the bacterial concentration. Then, the bacteria were diluted to OD600The bacteria were transferred to a 1.5mL EP tube as 0.5. The bacteria were centrifuged at 10,000rpm for 5 minutes. After centrifugation, the supernatant was removed and washed 3 times with Phosphate Buffered Saline (PBS). The precipitated klebsiella pneumoniae was resuspended in PBS for later use. The conditions and procedures for culture of staphylococcus aureus (s. aureus), escherichia coli (e. coli) and candida albicans (c. albicans) were the same as for klebsiella pneumoniae.
Example (b): fluorescence imaging of bacteria
For fluorescence imaging of bacteria, an initial concentration of 10 was first prepared using dimethylsulfoxide-2M probe solution, then using phosphate buffer solution to the initial probe solution diluted to a concentration of 20M ready for use. Then, 200 μ L of the treated bacterial solution and an equal amount of probe solution were added to the sterilized EP tube and incubated at room temperature for 30 minutes. Thereafter, 10. mu.L of the stained bacteria were transferred to a clean glass slide and fixed by covering with a cover slip. Images were collected under a Confocal Laser Scanning Microscope (CLSM) using a 405nm laser and a 100 fold mirror.
Example (b): hybrid bacterial imaging
Equal amounts of E.coli and Klebsiella pneumoniae were mixed in PBS. Then, 200. mu.L of the bacterial mixture and an equal amount of TPE-N were added+The probe solutions were added together to sterilized EP tubes and incubated at room temperature for 1 hour. Then, 200uL of the TPE-Py probe was added++The solution was incubated at room temperature for 1 hour. The bacteria were harvested by centrifugation (10000rpm, 3 min) and resuspended in PBS. 10 μ L of the staining bacteria solution was transferred to a clean glass slide and fixed by covering with a cover slip. Images were collected by confocal laser scanning microscopy and excited using a 405nm laser. The procedure for imaging the mixed bacteria staphylococcus aureus and klebsiella pneumoniae was the same as for escherichia coli and klebsiella pneumoniae.
Example (b): macrophage phagocytosis of klebsiella pneumoniae
First, 200. mu.L of the suspension was centrifugedAnd adding the same amount of TPE-Py probe into the Klebsiella pneumoniae++Solution (20. mu. mol). Incubate at room temperature for 30 minutes. The bacteria were harvested by centrifugation (1000rpm, 3 minutes). The supernatant was removed, resuspended in 100. mu.L of PBS, and then mixed by adding 900. mu.L of DMEM. Finally, 1mL of the prepared bacterial solution was added to Raw 264.7 cells and incubated at 37 ℃ for 30 minutes. Images of bacterial infection were collected at different times by confocal microscopy using a 405nm laser and a 40 fold mirror. TPE-Pn++Probe-labeled Klebsiella pneumoniae with TPE-Py++The same conditions as the probes infected Raw 264.7 cells.
Example (b): the ultraviolet spectral property and the fluorescence spectral property of the compound are characterized
The spectral properties of the synthesized probes were investigated. As shown in FIG. 2a, the single charge probe TPE-N is in glycerol solution+(20. mu.M) and TPE-Py+The maximum absorption peaks (20. mu.M) were 320nm and 416nm, respectively. Double-charge probes (TPE-Py) in contrast to Single-charge probes++And TPE-Pn++) The absorption of the compound has obvious red shift, and the maximum absorption peaks are respectively 425nm and 440 nm. The absorption peaks change mainly because the D- π -A structure successfully shifts the absorption of the doubly charged probe to more red wavelengths, due to the difference in its donor-acceptor structure. All four probes had aggregation-induced emission activity and showed significantly enhanced fluorescence emission. FIG. 2b, TPE-Py++，TPE-Pn++And TPE-Py+Mainly emitting at 550nm to 700nm, and TPE-N+Emission of 430nm to 570 nm. TPE-N in comparison with other similar compounds+The emission range of (a) is short and the emission wavelengths are also significantly different.
Example (b): spectral property characterization of single-double charge staining capsular bacteria representing Klebsiella pneumoniae
Mixing the washed and centrifuged Klebsiella pneumoniae with a probe TPE-Py++，TPE-Pn++，TPE-Py+,TPE-N+After incubation for half an hour, the fluorescence intensity of the incubated solution was measured with a UV spectrophotometer and compared to the fluorescence intensity of the probe alone in PBS. Co-incubated bis with Klebsiella pneumoniae, as shown in FIGS. 3 and 4The fluorescence of the charged probe increases significantly, while the fluorescence of the singly charged probe solution does not change significantly. These experiments clearly show that the double charge probe (TPE-Py)++And TPE-Pn++) It penetrates the capsule more easily and can stain klebsiella pneumoniae effectively.
Example (b): the bacterial imaging ability of the aggregation-induced emission property of the tetraphenyl vinyl compound is characterized.
The designed tetraphenyl vinyl compounds with aggregation-induced emission properties have excellent water solubility and low background signal in aqueous solution, so that the compounds are effective candidates for bacterial leave-on imaging. The probes were incubated with the bacteria for 30 minutes and images of the bacteria were viewed by CLSM. As shown in fig. 5, the dual charged probe can efficiently interact with klebsiella pneumoniae and produce red fluorescence in bacteria. By way of comparison, we co-cultured the probes with other microorganisms, including E.coli as a representative of gram-negative bacteria, S.aureus as a representative of gram-positive bacteria, and Candida albicans as a representative of fungi. The results indicate that the singly charged probe (TPE-Py)+And TPE-N+) Can effectively stain non-capsulated microorganisms such as Escherichia coli, Staphylococcus aureus and Candida albicans. But klebsiella pneumoniae is difficult to stain. In contrast, the double charge probe (TPE-Py)++And TPE-Pn++) All microorganisms including klebsiella pneumoniae were successfully stained. Images show that klebsiella pneumoniae has a unique capsular structure, making it difficult to penetrate with a singly charged probe. Also, to demonstrate that the designed probe has excellent membrane penetration ability, it was compared with the commercial probe FITC, Syto TM9 and Phrodo red. As shown in fig. 6, all three commercial probes were effective in staining non-capsulated microorganisms, but FITC and Phrodo red were almost unable to stain klebsiella pneumoniae. Only with Syto TM9 the incubated klebsiella pneumoniae showed fluorescence. The results show that the Klebsiella pneumoniae has a more complex and thicker capsular structure than other microorganisms, and can effectively resist foreign substances, so that the Klebsiella pneumoniae is difficult to interact with fluorescent dyes. The designed double-charge probe has stronger membrane penetrating capability and effectively dyes bacteria with capsules and performs fluorescence imaging.
Example (b): fluorescence imaging characterization of single and double charge combinations for selective recognition of capsular bacteria
Double charge probe (TPE-Py)++And TPE-Pn++) It stains Klebsiella pneumoniae and exhibits strong fluorescence, whereas a singly charged probe can only interact with bacteria without a capsule. The single and double charge probe combination was used to identify klebsiella pneumoniae based on the differences in the interaction between the single and double charge probes and klebsiella pneumoniae. TPE-N+Emission is different from that of double-charge TPE-Py++Fluorescent emission of (2). TPE-N+Blue fluorescence at about 475nm under excitation at 405nm, and doubly charged TPE-Py++It has red fluorescence near 650nm under 405nm excitation. The two probes were added to a mixed bacteria (staphylococcus aureus + klebsiella pneumoniae, escherichia coli + klebsiella pneumoniae) in sequence and incubated together, then recorded with CLSM. As shown in FIG. 7, only Staphylococcus aureus and Escherichia coli stained in TPE-N were observed+The emission channel (Ex 405nm, Em 420-470nm) has blue fluorescence. Klebsiella pneumoniae did not show any fluorescence on this channel. For TPE-Py++The emission channel (Ex 405nm, Em 650 + 700nm), Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae all showed significant fluorescence. When images on different emission channels were merged, Klebsiella pneumoniae only appeared TPE-Py++While the other microorganisms (staphylococcus aureus and escherichia coli) showed combined colors (blue and red). Klebsiella pneumoniae fluoresces only in the emission range of the dual charge probe, and escherichia coli and staphylococcus aureus can show fluorescence in both channels. And we can clearly see the difference in fluorescence color and intensity from the merged image, plots d and h are plotted along the intensity distribution of the white and green squares in the plot. Klebsiella pneumoniae in TPE-N+Fluorescence under the channel is very weak, while in TPE-Py++The fluorescence on the channel is strong. However, Escherichia coli and Staphylococcus aureus in TPE-N+And TPE-Py++The channel has strong fluorescence. Thus, capsular bacteria can be stained by assaying combinations of single and double chargesMethods of fluorescence data identify capsular bacteria.
Example (b): the doubly charged probe marks bacteria for carrying out fluorescence imaging capability characterization of macrophage phagocytosis bacteria visual tracking.
Using TPE-Py++And TPE-Pn++The fluorescent probe is used for marking Klebsiella pneumoniae, and a mouse cell line Raw 246.7 is selected as a model of macrophage. After labelling klebsiella pneumoniae with a dual charge probe, klebsiella pneumoniae was co-incubated with Raw 246.7 cells. The CLSM was used to observe the overall process of phagocytosis of klebsiella pneumoniae by macrophage Raw 246.7. As shown in FIG. 8, after 30 minutes of co-incubation, Klebsiella pneumoniae was free in solution. Within 1 hour, the Klebsiella pneumoniae gradually approached the Raw 246.7 cells. Within 2 hours, Raw 246.7 cells began to phagocytose klebsiella pneumoniae. After 4 hours, a large number of klebsiella pneumoniae were phagocytosed by the Raw 246.7 cells and were mainly present in the cytoplasm of the Raw 246.7 cells. After 6 hours of culture, the cytoplasm of Raw 246.7 cells was filled with klebsiella pneumoniae, while the whole cells were slightly swollen and showed strong fluorescence. After six hours of laser irradiation, the marked klebsiella pneumoniae can still be observed under bright fluorescence. This indicates TPE-Pn++Has excellent light stability and is suitable for biological imaging application. These results indicate that the double charge probe (TPE-Py)++And TPE-Pn++) Can be used as a fluorescent probe to mark the capsular bacteria and directly observe the position and the shape of the capsular bacteria in the process of macrophage phagocytosis.
It should be understood to be apparent to one of ordinary skill in the art. Modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention, and all such modifications and variations are intended to be included within the scope of the following claims.
Claims (3)
1. A method for selectively imaging a capsular bacterium by using a tetrastyrene derivative comprises a series of tetrastyrene positively charged compounds which can stain the capsular bacterium and have aggregation-induced emission properties; methods of selectively imaging capsular bacteria by different combinations of charge number, fluorescence signal difference and transmembrane capacity of the compounds;
the compound takes tetraphenylethylene as a skeleton to design aggregation-induced emission molecules with recognition groups with different positive charges, and the molecular structure of the compound contains a group selected from any one of the following structural formulas:
wherein R is1、R2Are each selected from the following structures:
wherein R ═ ClO3 -,Cl-，I-Or Br-These are similarly negatively charged groups;
the single charge aggregation-induced emission molecular structure comprises a tetraphenylethylene main structure and an R1Structure R2is-H;
the double charge aggregation-induced emission molecular structure comprises a tetraphenylethylene main structure and R1And R2Structure R1And R2Similarly, molecules with two positive charges on one side are exemplified below:
the probe has aggregation-induced emission property, and the double-charge probe has stronger membrane penetrating capability and can stain capsular bacteria; the capsular bacteria is selectively identified and fluorescence imaging is carried out through designing and adjusting the differential single-double charge combination of the fluorescence property and the membrane penetrating capability of the material.
2. The method for selectively recognizing capsular bacteria through a tetrastyrene derivative according to claim 1, wherein: the capsular bacteria comprise the following: streptococcus pneumoniae, Klebsiella, Neisseria meningitidis, Bacillus anthracis, Cryptococcus having a capsular structure.
3. The method for selectively recognizing capsular bacteria through a tetrastyrene derivative according to claim 1, wherein: the compounds with positive charges of the tetrastyrene with the aggregation-induced emission property can be applied to the preparation of fluorescent materials, the fluorescence imaging of bacteria and the visualization tracking imaging of bacteria invading cells.
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