CN115466210A - Fluorescent probe for detecting outer membrane vesicles and application thereof - Google Patents

Fluorescent probe for detecting outer membrane vesicles and application thereof Download PDF

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CN115466210A
CN115466210A CN202211154664.4A CN202211154664A CN115466210A CN 115466210 A CN115466210 A CN 115466210A CN 202211154664 A CN202211154664 A CN 202211154664A CN 115466210 A CN115466210 A CN 115466210A
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fluorescence
outer membrane
fluorescent probe
membrane vesicles
gram
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赵恩贵
陈斯杰
辜美佳
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Shenzhen Graduate School Harbin Institute of Technology
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    • C—CHEMISTRY; METALLURGY
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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Abstract

The invention belongs to the technical field of detection, and aims to develop a fluorescent probe capable of being used for OMV research. The material emits no fluorescence or weak fluorescence in buffer solution or MOPS culture solution. In the presence of gram-negative bacteria, they emit little or no fluorescence. When it binds to OMVs, fluorescence increases. Facilitates rapid identification of OMV production and determination of optimal conditions for inducing OMV production. Has important significance for researching OMV.

Description

Fluorescent probe for detecting outer membrane vesicles and application thereof
Technical Field
The invention belongs to the technical field of detection, and aims to develop a fluorescent probe capable of being used for OMV research.
Background
Outer Membrane Vesicles (OMVs) are mostly spherical protrusions produced by gram-negative bacteria. The OMV is loaded with biomolecules including lipopolysaccharide, flagellin, peptidoglycan, enzyme, DNA, RNA, etc. These biomolecules enable OMVs to participate in many important biological processes, including bacterial pathogenicity, message transmission, quorum sensing, horizontal gene transmission, and response to stressful environments. In addition, OMV plays an important role in regulating and controlling host immunity, so that the OMV can be regulated to induce immunity, and is further applied to vaccine adjuvants. OMVs are also of great interest in the development of cancer vaccines and immunotherapy of cancer.
The size of OMV is 20-250nm. Such small dimensions make it very difficult to observe OMVs in experiments.
Currently, proteomics (1) and particle tracking technology (2) are commonly used in OMV research. However, both techniques require lengthy differential centrifugation or ultrafiltration, making the study of OMVs very difficult (3-5).
Fluorescence techniques have good sensitivity, but most of the fluorescence currently has strong fluorescence in solution, thus creating a strong background during the study of OMVs. This strong background makes quantitative analysis of OMVs very difficult and also reduces the sensitivity of the analysis.
Reference documents:
1.W.Elhenawy，M.O.Debelyy，M.F.Feldman，mBio 2014，5，e00909-00914.
2.M.J.H.Gerritzen，D.E.Martens，R.H.Wijffels，M.Stork，J.Extracell.Vesicles 2017，6，1333883.
3.R.Dehinwal，D.Cooley，A.V.Rakov，A.S.Alμgupalli，J.Harmon，O.Cunrath，P.Vallabhajosyula，D.Bμmann，D.M.Schifferli，mBio 2021，12，e0086921；
4.A.J.Manning，M.J.Kuehn，BMC Microbiol.2011，11，258；
5.A.Bauwens，L.Kunsmann，H.Karch，A.Mellmann，M.Bielaszewska，Antimcrob.Agents Chemother.2017，61，e00937-00917.
disclosure of Invention
In view of the problems of the prior art, it is an object of the present invention to develop a fluorescent probe that can be used for OMV studies. The fluorescent probe has low fluorescent background, the fluorescence of the fluorescent probe is enhanced after the fluorescent probe is combined with the OMV, the generation of the OMV can be sensitively detected, and factors generated by the OMV can be induced by high-flux screening.
A fluorescent probe for detecting outer membrane vesicles adopts a compound with a chromophore of the following formula, and is applied to the detection of Outer Membrane Vesicles (OMVs),
Figure BDA0003856787640000021
wherein A is 1 ，A 2 ，A 3 ，A 4 Is independently selected from
Figure BDA0003856787640000022
And A is 1 ，A 2 ，A 3 ，A 4 At least one of which is
Figure BDA0003856787640000023
At least one is
Figure BDA0003856787640000024
Wherein A is 1 ，A 2 ，A 3 ，A 4 May be the same or different;
wherein n is from 0 to 100, preferably from 1 to 50, more preferably from 1 to 10;
R 1 ，R 2 which may be the same or different, may be any anion, including fluoride, chloride, bromide, iodide, hexafluorophosphate particles, acetate, and the like. The change of the anion does not affect the protection content of the present invention.
As a preferred embodiment of the present invention, the compound is selected from
Figure BDA0003856787640000025
Compared with other compounds capable of serving as fluorescent probes, the compound has low fluorescence background, the fluorescence of the compound is enhanced after the compound is combined with OMV, and the generation of the OMV can be sensitively detected.
In a preferred embodiment of the present invention, the compounds are in solution with little or no fluorescence.
In a preferred embodiment of the present invention, the compounds emit no light or weak light in the buffer or MOPS medium.
As a preferred embodiment of the present invention, when gram-negative bacteria are stained with these compounds in a buffer or MOPS medium, the gram-negative bacteria do not emit light or emit very weak light.
As a preferred embodiment of the present invention, incubation with these compounds results in an increase in the fluorescence of the solution when the gram negative bacteria produce OMVs under stimulatory conditions, including chemical stimuli, temperature stimuli, physical stimuli, and the like.
As a preferred embodiment of the present invention, the chemical stimulus comprises an antibiotic stimulus, including but not limited to ampicillin, kanamycin, aztreonam, chloramphenicol.
As a preferred embodiment of the invention, the compounds exhibit enhanced fluorescence when bound to OMVs.
As a preferred embodiment of the present invention, OMV-secreting gram-negative bacteria are also bound by these compounds and fluorescence enhanced.
As a preferred embodiment of the present invention, these compounds are used in concentrations ranging from 0.01 micromolar to 10 millimolar. Preferably from 0.1 micromoles to 1 millimole. More preferably from 1 micromole to 500 micromoles.
As a preferred embodiment of the present invention, there is no limitation on the kind of OMV-producing gram-negative bacteria.
In a preferred embodiment of the present invention, the gram-negative bacteria are selected from EcN (e.coli Nissle 1917).
In a preferred embodiment of the present invention, the OMVs are produced by bacteria, which may be gram-positive or gram-negative bacteria.
The beneficial effects of the invention compared with the prior art comprise:
the invention uses a new class of fluorescent materials to detect the presence of OMVs. The material emits no fluorescence or weak fluorescence in buffer solution or MOPS culture solution. In the presence of gram-negative bacteria, it emits little or no fluorescence. When it binds to OMVs, fluorescence increases. Thus, OMV production can be detected based on changes in its fluorescence intensity, and various factors capable of inducing OMV production, including antibiotic treatment of various species/concentrations, etc., can be screened at high throughput. Facilitates rapid identification of OMV production and determination of optimal conditions for inducing OMV production. Has important significance for researching OMV.
Drawings
FIG. 1 shows the change in fluorescence intensity of OTM under different conditions, OD 600 Concentration representative of EcN (e.coli Nissle 1917), wherein:
fluorescence photograph of 5 hours after EcN culture in the presence of 10. Mu. Mol/L OTM;
fluorescence photographs of EcN in the presence of 10. Mu. Mol/L OTM after 5 hours of culture under 1. Mu.g/mL ampicillin;
fluorescence photograph of EcN in the presence of 10. Mu. Mol/L of the compound after culturing for 5 hours under 1. Mu.g/mL ampicillin, removing the supernatant and redispersing the cell mass;
fluorescence photograph of supernatant in the presence of 10. Mu. Mol/L of compound after 5 hours of EcN incubation under 1. Mu.g/mL ampicillin;
5. fluorescence in the presence of 10. Mu. Mol/L of compound with only ampicillin. Experiments showed that ampicillin induced OMV production, and that OMVs in the supernatant emitted fluorescence.
FIG. 2 relative fluorescence intensity of EcN in the presence of 10. Mu. Mol/L of OTM after 5 hours of incubation at different concentrations of antibiotic. I.C. A 0 If no antibiotics exist, after EcN is cultured for five hours, the fluorescence intensity of OTM of 10 mu mol/L is added; i is the fluorescence intensity I of 10. Mu. Mol/L OTM added after five hours of EcN incubation in the presence of different concentrations of antibiotics d Fluorescence intensity of OTM of 10. Mu. Mol/L in MOPS-only medium.
Detailed Description
The invention will be further illustrated, but is not limited, by the following examples and the accompanying drawings.
Example 1
Figure BDA0003856787640000041
The molecular structure of the compound employed.
OTM characterization data
1 H NMR(CD 3 OD，400MHz)：δ(TMS，ppm)8.85(d，1H，J＝6Hz，Ar)，8.24(d，1H，J＝6Hz，Ar)，8.03-7.99(m，1H，CH＝C)，7.82-7.79(m，2H，Ar)，7.82-7.68(m，2H，Ar)，7.50-7.43(m，3H，Ar)，7.13-6.89(m，14H，Ar)，6.72-6.66(m，2H，Ar)，4.63(t，2H，J＝8Hz，alkyl)，4.05-4.03(m，2H，alkyl)，3.80-3.77(m，2H，alkyl)，3.68-3.49(m，11H，alkyl)，3.20(s，9H，alkyl)，2.60-2.52(m，2H，alkyl)，1.89ppm(s，6H，alkyl)； 13 C NMR(125MHz，CD 3 OD)：δ(TMS，ppm)180.05(RCOO)，159.18，159.08，156.23，145.57，145.44，145.36，145.30，145.24，144.40，143.34，142.65，141.05，138.91，137.58，135.37，133.69，133.65，133.20，132.51，132.46，130.18，128.95，128.90，128.85，128.76，128.45，127.75，127.59，127.27，127.19，125.49，123.59，115.03，114.88(Ar)，72.99，72.97，71.76，71.59，71.42，71.40，70.85，68.53，63.91，59.13，58.14，53.94，26.00，24.24(alkyl).HRMS(ESI-Q-TOF，m/z)calcd for(C 52 H 58 N 2 O 4 2+ )[(M-2(CH 3 COO)) 2+ ]387.2193，found 387.2167.
Example 2
Fluorescence when compound OTM of example 1 was added to different cultures containing EcN (e.coli Nissle 1917):
specific results refer to FIG. 1. Variation of fluorescence intensity of OTM under different conditions, OD in the graph 600 Represents the concentration of EcN (e.coli Nissle 1917).
Fluorescence photograph in the presence of 10. Mu. Mol/L of OTM after 5 hours of EcN (37 ℃ MOPS medium);
fluorescence photographs of EcN in the presence of 10. Mu. Mol/L OTM after 5 hours of culture under 1. Mu.g/mL ampicillin;
fluorescence photograph of EcN in the presence of 10. Mu. Mol/L of the compound after culturing for 5 hours under 1. Mu.g/mL ampicillin, removing the supernatant and redispersing the cell mass;
fluorescence photograph of supernatant in the presence of 10. Mu. Mol/L of compound after 5 hours of EcN incubation under 1. Mu.g/mL ampicillin;
5. fluorescence in the presence of 10. Mu. Mol/L of compound with only ampicillin.
Experiments showed that ampicillin induced OMVs production, and that in the supernatant OMVs emitted fluorescence.
In FIG. 1, 2 contains both the supernatant and the cells, so the fluorescence is strongest, and 3 contains cells, the cell structure is changed after OMV is generated, and the fluorescence is also enhanced; 4. binding of OMVs to the probe was present in the supernatant, resulting in a significant increase in fluorescence over 5.
Example 2
Fluorescence when compound OTM of example 1 was added to antibiotic conditions containing EcN (e.coli Nissle 1917) at different concentrations:
specific results refer to FIG. 2. The relative fluorescence intensity of EcN (37 ℃ MOPS medium) in the presence of 10. Mu. Mol/L OTM after 5 hours of incubation at different concentrations of antibiotic.
Wherein, I 0 If no antibiotics exist, after the EcN is cultured for five hours, the fluorescence intensity of OTM of 10 mu mol/L is added; i is the fluorescence intensity of OTM added with 10 mu mol/L after EcN is cultured for five hours in the presence of antibiotics with different concentrations; I.C. A d The fluorescence intensity of OTM was 10. Mu. Mol/L in the case of MOPS-only medium.
As shown in FIG. 2, the concentration range of ampicillin is 0.25-5. Mu.g/mL, which can induce OMV more efficiently than kanamycin, thus demonstrating that the fluorescent probe of the present invention can rapidly identify the antibiotic-induced condition.
The foregoing is a further detailed description of the invention in connection with specific preferred embodiments and it is not intended to limit the invention to the specific embodiments described. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (10)

1. A fluorescent probe for detecting outer membrane vesicles, which is characterized in that a compound with a chromophore of the following formula is adopted and applied to Outer Membrane Vesicles (OMVs) detection,
Figure FDA0003856787630000011
wherein A is 1 ，A 2 ，A 3 ，A 4 Is independently selected from
Figure FDA0003856787630000012
And A is 1 ，A 2 ，A 3 ，A 4 At least one of which is
Figure FDA0003856787630000013
At least one is
Figure FDA0003856787630000014
Wherein A is 1 ，A 2 ，A 3 ，A 4 May be the same or different;
wherein n is from 0 to 100, preferably from 1 to 50, more preferably from 1 to 10;
R 1 ，R 2 which may be the same or different, may be any anion, including fluoride, chloride, bromide, iodide, hexafluorophosphate particles, acetate, and the like.
2. The outer membrane vesicle fluorescence probe of claim 1, wherein the compounds exhibit low or no fluorescence in solution.
3. The fluorescent probe for detecting outer membrane vesicles as claimed in claim 1, wherein the compounds do not emit light or emit weak light in buffer or MOPS (3- (N-morpholino) propanesulfonic acid) culture solution.
4. The fluorescent probe for detecting outer membrane vesicles as claimed in claim 1, wherein when gram-negative bacteria are stained with the compounds in buffer or MOPS medium, the gram-negative bacteria do not emit light or emit very weak light.
5. The fluorescent probe for detecting outer membrane vesicles as claimed in claim 1, wherein the solution fluorescence is enhanced by incubation with gram-negative bacteria under stimulating conditions, including chemical stimulation, temperature stimulation, physical stimulation, etc., to produce OMVs.
6. The fluorescent probe for detecting outer membrane vesicles of claim 1, wherein the compounds have increased fluorescence when bound to OMVs.
7. The fluorescent probe for detecting outer membrane vesicles as claimed in claim 1, wherein OMV-secreting gram-negative bacteria are also bound by the compounds and fluorescence is enhanced.
8. A fluorescent probe for the detection of outer membrane vesicles according to claim 1, wherein the compounds are used at a concentration in the range of 0.01 micromolar to 10 millimolar; preferably 0.1 micromoles to 1 millimole; more preferably from 1 micromole to 500 micromoles.
9. The fluorescent probe for detecting outer membrane vesicles according to claim 5 or 7, wherein the species of OMV-producing gram-negative bacteria is not limited.
10. The outer membrane vesicle fluorescence probe of claim 1, wherein the OMVs are produced by bacteria, and the bacteria are gram-positive bacteria or gram-negative bacteria.
CN202211154664.4A 2022-09-21 2022-09-21 Fluorescent probe for detecting outer membrane vesicles and application thereof Pending CN115466210A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104974745A (en) * 2014-04-07 2015-10-14 香港科技大学深圳研究院 Amphiphilic illuminant with aggregation induced emission characteristics and applications thereof
CN107001927A (en) * 2014-11-21 2017-08-01 香港科技大学 AIE illuminophores and its production method for bacterium imaging, killing, photodynamic therapy and antibiotic-screening

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104974745A (en) * 2014-04-07 2015-10-14 香港科技大学深圳研究院 Amphiphilic illuminant with aggregation induced emission characteristics and applications thereof
CN107001927A (en) * 2014-11-21 2017-08-01 香港科技大学 AIE illuminophores and its production method for bacterium imaging, killing, photodynamic therapy and antibiotic-screening

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WEI XIONG，等: "Pyridinium-substituted tetraphenylethylene salt-based photosensitizers by varying counter anions: a highly efficient photodynamic therapy for cancer cell ablation and bacterial inactivation", J. MATER. CHEM. B, vol. 8, no. 24, pages 5234 - 5244 *

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Inventor after: Zhao Engui

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Inventor before: Gu Meijia