CN106995419B - Fluorescent probe with aggregation-induced emission property and preparation method and application thereof - Google Patents
Fluorescent probe with aggregation-induced emission property and preparation method and application thereof Download PDFInfo
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- CN106995419B CN106995419B CN201610049647.2A CN201610049647A CN106995419B CN 106995419 B CN106995419 B CN 106995419B CN 201610049647 A CN201610049647 A CN 201610049647A CN 106995419 B CN106995419 B CN 106995419B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C—CHEMISTRY; METALLURGY
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
Abstract
The invention relates to a fluorescent probe with aggregation-induced emission (AIE) property and a general formula I and a preparation method thereof, wherein R is
1，R
2Independently is hydrogen, C
1‑18Alkyl, halogen, C
1‑18Alkyloxy, C
1‑18Alkylthio, aryl, heteroaryl; r
3，R
4，R
5Is C
1‑30An alkyl group; r
6Is a direct bond, C
1‑6An alkylene group; a. the
‑Is an anion. The fluorescent probe can realize the lightening detection of the anionic surfactant by forming the anion and cation aggregate in situ, and has high selectivity and high sensitivity to the anionic surfactant. In addition, the fluorescent probe can also be used for the fluorescent staining of bacteria, and has the advantages of low background noise, high staining efficiency, no need of extra washing and the like.
Description
Technical Field
The invention belongs to the field of surfactant detection, and particularly relates to a detection method of an anionic surfactant.
Background
Anionic surfactants, especially alkyl sulfonates, are the most important surfactants in commercial products and are produced in large quantities, such as: sodium Dodecylbenzenesulfonate (SDBS), Sodium Lauryl Sulfate (SLS), Sodium Dodecyl Sulfate (SDS), and the like. They are widely used in the cleaning, agricultural, cosmetic and pharmaceutical industries as detergents, emulsifiers, foaming agents and dispersants. Due to their wide range of applications, their residues in water bodies can pose serious hazards to the ecological environment, such as: inhibiting water bioactivity and accelerating the diffusion of oily pollutants. In addition, anionic surfactants may cause irritation and inflammation to the human eye and skin. In order to make the detection of anionic surfactants feasible even at home or in remote areas, it is highly desirable to develop an efficient detection method that is simple to operate and low in cost. Currently, in the literature, a large number of methods for detecting anionic surfactants have been reported, such as GC/LC-MS, colorimetry, potentiometry, immunoassay, capillary electrophoresis, and flow injection analysis. However, these methods also have the following disadvantages, such as: large fixed instruments are required, the operation is complicated, toxic chlorinated solvents are used, and in-situ analysis is difficult.
In recent years, fluorescence detection of anionic surfactants has shown significant advantages, such as: high sensitivity, simple operation and low cost. However, in the aggregation state, the fluorescent material with aggregation-induced fluorescence quenching (ACQ) property is easy to undergo self-quenching of fluorescence, and the high background fluorescence of the fluorescent material in the solution state can also obviously reduce the signal-to-noise ratio during the detection process.
In contrast to aggregation-induced fluorescence quenching (ACQ), fluorescent probes with aggregation-induced emission (AIE) properties emit very little light in dilute solutions, but very much light in the aggregate state. AIE probes have been widely used to detect metal ions, enzymes, DNA, etc. by forming luminescent aggregates. In addition, AIE molecules have been used to detect critical micelle concentrations, monitor micelle-encapsulated drug release, monitor micellar morphological transitions using fluorescence imaging, and the like.
The invention content is as follows:
the invention aims to provide a fluorescent probe with Aggregation Induced Emission (AIE) properties, which can be used for detecting anionic surfactants.
The invention also aims to provide a preparation method of the fluorescent probe and application thereof.
The purpose of the invention is realized based on the following technical scheme:
a compound of formula I:
wherein:
R
1，R
2independently is hydrogen, C
1-18Alkyl, halogen, C
1-18Alkyloxy, C
1-18Alkylthio, aryl, heteroaryl; r
3，R
4，R
5Independently is C
1-30An alkyl group; r
6Is a direct bond, C
1-6An alkylene group; a. the
-Is an anion.
The alkyl group can be a straight chain or branched chain alkyl group; for example, methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, and the like.
The aryl group means a monocyclic or polycyclic aromatic group having 6 to 20 carbon atoms, and representative aryl groups include: phenyl, naphthyl, anthracenyl, pyrenyl, and the like.
The heteroaryl group means a monocyclic or polycyclic heteroaromatic group having 1 to 20 carbon atoms and 1 to 4 heteroatoms selected from N, S, O, and representative heteroaryl groups include: pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, thiazolyl, indolyl, azanaphthyl, azaanthracenyl, azapyrenyl and the like.
In one embodiment, R
1，R
2Independently is hydrogen, C
1-6Alkyl, halogen, C
1-6Alkyloxy, C
1-6An alkylthio group.
In another embodiment, R
3，R
4，R
5At least one of them being C
8-
30Alkyl, more preferably C
10-
24Alkyl, preferably C
12-
22Alkyl, or C
14-
20An alkyl group.
In yet another embodiment, R
3，R
4，R
5One of the radicals being long-chain alkyl and the other two radicals being short-chain alkyl, e.g. R
3，R
4，R
5One group in (A) is C
8-
30Alkyl, more preferably C
10-
24Alkyl, preferably C
12-
22Alkyl, or C
14-
20An alkyl group; and the other two radicals are independently C
1-8Alkyl, more preferably C
1-6An alkyl group.
In yet another embodiment, R
6Is a direct bond, -CH
2-、-CH
2CH
2-、-CH
2CH
2CH
2-。
Preferably, the anion is a halide, perchlorate, sulfate, nitrate, hexafluorophosphate, or the like. The halide ion is preferably chloride.
Preferably, the formula I is the following formula (Ia):
wherein R is
1、R
2、R
3、R
4、R
5、A
-As defined above.
Preferably, in formula Ia, R
1Is hydrogen, R
2Is hydrogen, R
3，R
4Is methyl, R
5Is octadecyl, A
-Is an anion.
More preferably, the compound of formula I is selected from:
n- (3- (benzo [ d ] thiazol-2-yl) -4-hydroxybenzyl) -N, N-dimethyloctadecyl-1-ammonium chloride;
the invention also provides a preparation method of the compound shown in the formula I, which comprises the following steps of reacting a compound shown in the formula II with a compound shown in the formula III under the catalysis of protonic acid and the oxidation of an oxidizing agent to obtain the compound shown in the formula I:
wherein R is
1、R
2、R
3、R
4、R
5、R
6、A
-As defined above.
Preferably, the protonic acid is concentrated hydrochloric acid, and the oxidizing agent is hydrogen peroxide.
The compound of formula II can be obtained by a preparation method comprising: and (3) reacting the compound shown in the formula IV with the compound shown in the formula V to obtain the compound shown in the formula II.
Wherein R is
2、R
3、R
4、R
5、R
6、A
-As defined above.
The compounds of formula I according to the invention have aggregation-induced emission (AIE) properties. The structure comprises a 2- (2' -Hydroxyphenyl) Benzothiazole (HBT) molecular skeleton which is provided with an intramolecular six-membered ring hydrogen bond and a C-N single bond for connecting a benzothiazole unit and a substituted benzene unit. Due to the property of an excited proton intramolecular transfer process (ESIPT), the fluorescent probe has alcoholic emission and ketone emission, and has normal Stokes shift and large Stokes shift (more than 150nm) respectively. Since intramolecular hydrogen bonds are easily broken by polar solvents, the ratio of alcoholic to keto emissions is highly correlated with the polarity of the environment in which the molecule is located. In polar solvents, the compounds exhibit only alcoholic luminescence. And in the aggregation state, the molecular structures are closely arranged, the rotation of the C-N single bond in the molecule is inhibited, and the hydrogen bond in the molecule is protected from being interfered by the external environment. Since the fluorescence emission of the compounds is very sensitive to the polarity of the environment and the aggregation state of the molecules, they are particularly suitable as sensing materials for surfactants.
Compared to single cationic or anionic surfactant systems, mixed systems of surfactants with opposite charges tend to form anionic and cationic aggregates or micelles in solution even at concentrations well below the respective critical micelle concentration, due to the synergistic effect of electrostatic and hydrophobic properties that effectively reduces surface tension. Based on the characteristic that AIE dye emits light efficiently in an aggregation state, the invention utilizes positively charged amphiphilic probe molecules (compounds shown in formula I) with AIE property to detect anionic surfactant by forming anion and cation aggregates or micelles in situ. By the close intermolecular arrangement in the aggregate, intramolecular movement of the HBT backbone is restricted and intramolecular hydrogen bonds are protected from environmental interference (see fig. 1).
Furthermore, the invention also provides the application of the compound shown in the formula I in detecting anionic surfactants.
According to the invention, the anionic surfactants can be carboxylates, sulfates, sulfonates and phosphates. Preferably, the anionic surfactant is an anionic surfactant having a sulfonic acid group attached thereto, such as Sodium Dodecylbenzenesulfonate (SDBS), Sodium Dodecylsulfate (SDS), or the like.
The compounds of the present invention form anionic and cationic aggregates with anionic surfactants. In a polar solvent, when the concentration of the anionic/cationic micelle formed by the compound of the present invention and an anionic surfactant is higher than the critical micelle concentration, the compound of the present invention and the anionic surfactant form the anionic/cationic micelle. So that the polarity of the micro environment in which the compound of the invention is positioned is reduced. Thereby protecting hydrogen bonds within the molecule of the compound. The critical concentration of the anionic and cationic micelles in the mixed system is obviously lower than that of the anionic surfactant.
The compound provided by the invention can be used for monitoring the formation process of the anionic/cationic micelle, and has the advantages of high efficiency and sensitivity. The detection limit (3 delta/S) of the compound provided by the invention for the anionic surfactant is less than 0.08 mu M and is far lower than the national standard based on methylene blue spectrophotometry (the detection limit is about 0.14 mu M).
Further, based on the Excited State Intramolecular Proton Transfer (ESIPT) and intramolecular movement Restriction (RIM) mechanisms, the ketone/enol form of the compound of formula I emits light at a lower anionic surfactant concentration in a linear relationship with the anionic surfactant concentration, and thus, the concentration of the surfactant in water can be quantitatively determined from the ketone/enol form of the compound of formula I.
The invention also provides a method for detecting the anionic surfactant, which comprises the steps of adding the compound shown in the formula I into a solution of the anionic surfactant, and observing or detecting a fluorescence spectrum under fluorescence.
The compound has weak fluorescence emission in aqueous solution, but can emit strong fluorescence after being combined with an anionic surfactant to generate an aggregate, so that the compound can be used as an excellent lighting probe to realize the wash-free fluorescence imaging of bacteria surrounded by a negatively charged outer membrane.
Further, the invention provides the use of the compound of formula I in the washing-free fluorescent staining of bacteria.
According to the invention, the bacteria are bacteria having a negatively charged outer membrane envelope, such as E.coli, Bacillus subtilis, Staphylococcus aureus, etc.
The compounds of formula I according to the invention can be used to directly stain bacteria without further washing steps. And shows very low toxicity to bacteria, which is beneficial to the fluorescent staining experiment of the bacteria.
Because the outer membrane of the bacteria is composed of amphipathic molecules with negative charges, and the compound of the formula I is amphipathic molecules with positive charges, the compound can form strong affinity with the outer membrane of the bacteria through electrostatic and hydrophobic effects; in combination with the above, the intramolecular movement such as rotation of the compound is restricted, and the luminescence is significantly enhanced by the restriction of the intramolecular movement according to the mechanism of aggregation-induced luminescence. Whereas the compounds dissolved in the solution, which are not bound to the bacteria, cause weak luminescence due to intramolecular movement. It can be seen that the compound of the present invention emits strong fluorescence after binding to bacteria, but emits weak luminescence in solution, and only fluorescence signals on bacteria are observed at this high contrast. The traditional fluorescent material emits strong light in a dilute solution, so that the fluorescent material can be used for observing a fluorescent signal on bacteria only by centrifugally washing and removing fluorescent molecules in the dilute solution.
The invention also provides a method for washing-free fluorescent staining of bacteria, which comprises the step of co-culturing the compound shown in the formula I and the bacteria, and directly observing the fluorescence without separation, for example, observing by using a laser scanning confocal microscope.
The invention has at least the following beneficial effects:
1. the detection limit (3 delta/S) of the compound aiming at the anionic surfactant is less than 0.8 mu M, which is far lower than the national standard based on methylene blue spectrophotometry, and the compound can be directly detected in situ without extraction and concentration of toxic reagents such as chloroform and the like, so the operation is very convenient.
2. The compound can be used as an excellent lighting probe, and can be used for performing washing-free fluorescence imaging on bacteria without separation.
3. The compound of the invention is simple to prepare.
Drawings
Figure 1 shows HBT-C before and after SDBS addition
18The hypothetical stacking model of (1). Fluorescent probe HBT-C with AIE property
18Anionic surfactants were detected by micelle formation.
FIG. 2(A) shows HBT-C
18At H
2Normalized UV absorption spectra in a solution of O/DMSO (99:1, v/v); (B) is HBT-C
18At H
2Fluorescence emission spectra in solution and in solid state in O/DMSO (99:1, v/v). [ HBT-C
18]＝5μM；λ
ex＝334nm。
FIG. 3(A) shows HBT-C with increasing SDBS concentration
18(5. mu.M) in H
2Fluorescence emission profile in solution in O/DMSO (99:1, v/v); (B) HBT-C for increasing SDBS concentration
18A change in fluorescence emission intensity at 510nm and 450 nm; (C) HBT-C for increasing SDBS concentration
18Ratio of fluorescence emission intensities at 510nm and 450nm (I)
510/I
450) And (4) changing. Lambda [ alpha ]
ex＝334nm.
FIG. 4 shows HBT-C
18(5. mu.M) in H
2Fluorescence quantum yield in O/DMSO (99:1, v/v) as a function of SDBS concentration.
FIG. 5 shows HBT-C
18(A) normalized ultraviolet absorption and (B) fluorescence emission spectra at different SDBS concentrations (0,2.0, 8.0. mu.M). [ HBT-C
18]＝5μM；λ
ex＝334nm.
FIG. 6 shows HBT-C
18Ratio of fluorescence emission intensities at 510nm and 450nm (I)
510/I
450) As a function of SDBS concentration. Lambda [ alpha ]
exInside is a linear regression equation, 334nm.
FIG. 7(A) shows HBT-C
18At 510nm and 450nm (I)
510/I
450) The ratio of fluorescence emission intensity was plotted against the logarithm of the SDBS concentration; (B) PDI is 0.434 for micelle size as measured by dynamic light scattering; (C) and (D) is HBT-C
18(5. mu.M)/SDBS (8. mu.M) in H
2(C) SEM and (D) TEM images of micelles formed in O/DMSO (99:1, v/v) solution.
FIG. 8 shows HBT-C in the presence of different surfactants and ions
18Ratio of fluorescence emission intensities at 510nm and 450nm (I)
510/I
450) And (4) changing.
FIG. 9 is a photograph showing staining of E.coli in bright field and fluorescence. (HBT-C)
1810 μ M) scale bar 10 μ M.
Coli bacteria at different concentrations of HBT-C
18(0,10,20,40,60,80 and 100 μ M).
Detailed Description
The invention is further illustrated by the following specific examples and figures in the specification.
Example 1
The following compounds were specifically synthesized according to the following synthetic routes:
(1) synthesis of N- (3-formyl-4-hydroxybenzyl) -N, N-dimethyloctadecyl-1-ammonium chloride (Compound 3)
A mixture of 5- (chloromethyl) -2-hydroxy salicylaldehyde (340mg,2mmol) and N, N-dimethyloctadecan-1-amine (594mg,2mmol) was stirred under reflux for 5 hours. After completion of the reaction, the precipitate was filtered off and washed with ether (10 mL. times.2) and dried under vacuum to give the product compound 3 as a white solid in 90% yield (841mg).
1H NMR(CDCl
3,500MHz):δ11.26(br s,1H),8.16(d,J＝3.5Hz,1H),7.80(d,J＝8.5Hz,1H),7.09(d,J＝8.5Hz,1H),5.21(d,J＝8.5Hz,2H),3.47(t,J＝8.5Hz,2H),3.27(s,6H),1.26–1.25(m,32H),0.88(t,J＝7.0Hz,3H)；
13C NMR(d
6-DMSO,125MHz):190.0,162.3,140.1,132.9,122.5,118.8,118.0,65.3,63.1,48.7,31.3,29.0,29.0,28.9,28.76,28.66,28.5,25.8,22.1,21.7,13.9；HRMS(ESI):m/z[M-Cl]
+Calculated value is C
28H
50NO
2432.3836; the actual measurement is 432.3845.
(2) N- (3- (benzo [ d ])]Thiazol-2-yl) -4-hydroxybenzyl) -N, N-dimethyloctadecyl-1-ammonium chloride (HBT-C)
18) Synthesis of (2)
Concentrated HCl (37 wt.%, 100mg,1.0mmol) was added to a solution of N- (3-formyl-4-hydroxybenzyl) -N, N-dimethyloctadecyl-1-ammonium chloride (467mg,1.0mmol) and 2-aminobenzenethiol (150mg,1.2mmol) in methanol and the resulting mixture was stirred at room temperature for 10 min. Then adding H into the mixed solution
2O
2(30 wt.%, 113mg,1.0mmol) and further stirred at room temperature for 2 hours. After completion of the reaction, the solvent was evaporated, and the obtained residue was recrystallized from methylene chloride/N-hexane to obtain N- (3- (benzo [ d ]) as a white solid product]Thiazol-2-yl) -4-hydroxybenzyl) -N, N-dimethyloctadecyl-1-ammonium chloride (HBT-C)
18) (120mg, yield 21%)
1H NMR(CDCl
3,500MHz):δ12.87(br s,1H),8.28(s,1H),7.97(d,J＝8.0Hz,1H),7.85(d,J＝7.5Hz,1H),7.63(d,J＝8.0Hz,1H),7.49(t,J＝8.0Hz,1H),7.40(t,J＝8.0Hz,1H),7.15(d,J＝7.0Hz,1H),5.17(s,2H),3.47(t,J＝7.5Hz,2H),3.31(s,6H),1.26–1.23(m,32H),0.88(t,J＝7.0Hz,3H)；
13C NMR(CDCl
3,125MHz):168.2,159.7,136.8,133.7,132.7,126.9,126.0,122.1,121.8,118.8,118.2,117.3,67.0,63.8,49.6,31.9,29.69,29.65,29.6,29.4,29.4,29.3,26.4,22.9,22.7,14.1；HRMS(ESI):m/z[M-Cl]
+Calculated value is C
34H
53N
2537.3873 for OS; the actual measurement is 537.3880.
FIG. 2(A) shows HBT-C
18At H
2Normalized UV absorption spectra in a solution of O/DMSO (99:1, v/v); FIG. 2(B) shows HBT-C
18At H
2O/DFluorescence emission spectra in solution and in solid state of MSO (99:1, v/v). [ HBT-C
18]＝5μM；λ
exAs can be seen from the figure, in the solution state, only alcoholic luminescence at about 450nm is observed. This is due to environmental disturbances in aqueous solutions. In the case of a solid powder, strong luminescence with a large Stokes shift (176nm) is observed at 510nm, resulting from an Excited State Intramolecular Proton Transfer (ESIPT) process, and it can emit strong ketoluminescence because intramolecular hydrogen bonds are protected and its free motion is suppressed. The large Stokes shift (176nm) is a typical feature of the excited intramolecular proton transfer (ESIPT) process. HBT-C
18At H
2The quantum yields in O/DMSO (99:1 by volume) solution and in solid state were 0.2% and 50.5%, respectively, clearly confirming their AIE properties.
Example 2: detection of anionic surfactants
mu.L of HBT-C prepared in example 1 was added
18The DMSO (1.0mM) stock solution was added to 2.985mL of pure water containing varying levels of the anionic surfactant Sodium Dodecylbenzenesulfonate (SDBS), vortexed for 10 seconds, and fluorescence (excitation wavelength 334nm) was measured using a fluorescence spectrometer.
FIG. 3(A) shows HBT-C with increasing SDBS concentration
18(5. mu.M) in H
2Fluorescence emission profile in solution in O/DMSO (99:1, v/v); (B) HBT-C for increasing SDBS concentration
18A change in fluorescence emission intensity at 510nm and 450 nm; (C) HBT-C for increasing SDBS concentration
18Ratio of fluorescence emission intensities at 510nm and 450nm (I)
510/I
450) And (4) changing. Lambda [ alpha ]
ex＝334nm.
FIG. 4 shows HBT-C
18(5. mu.M) in H
2Fluorescence quantum yield in O/DMSO (99:1, v/v) as a function of SDBS concentration. As can be seen in the figure, HBT-C
18The fluorescence quantum yield of (a) increased from 0.2% to 8.5%. This is due to the formation of anionic and cationic aggregates and the protection of intramolecular hydrogen bonds.
FIG. 5 shows HBT-C
18(A) normalized ultraviolet absorption and (B) fluorescence emission spectra at different SDBS concentrations (0,2.0, 8.0. mu.M). [ HBT-C
18]＝5μM；λ
ex334nmIt can be seen that after SDBS is added, the absorption spectrum of the tailing peak at 370 nm-420 nm disappears, the hydrogen bonds in the molecule are protected, and strong ketone fluorescence is emitted.
FIG. 6 shows HBT-C
18Ratio of fluorescence emission intensities at 510nm and 450nm (I)
510/I
450) As a function of SDBS concentration. The luminescence ratio (I) detected at 450nm and 510nm can be seen from the graph
510/I
450) Gradually increasing compared to the amount of SDBS increase. Lambda [ alpha ]
exInside is a linear regression equation, 334nm. The signal-to-noise ratio based calculation method has a detection limit (3 delta/S) of 0.05 mu M for SDBS, which is much lower than the national standard based on methylene blue spectrophotometry (the detection limit is about 0.14 mu M).
FIG. 7(A) shows HBT-C
18At 510nm and 450nm (I)
510/I
450) The ratio of fluorescence emission intensity was plotted against the logarithm of the SDBS concentration; as shown, at HBT-C
18The critical concentration of the cationic micelle and the anionic micelle in the/SDBS mixed system is 2.39 mu M, which is obviously lower than that of the SDBS (1.2 mM).
Fig. 7(B) is the micelle particle size measured by Dynamic Light Scattering (DLS), PDI 0.434. As shown in the figure, the diameter of the micelle was 223.8nm on average.
FIGS. 7(C) and (D) are HBT-C
18(5. mu.M)/SDBS (8. mu.M) in H
2Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) images of micelles formed in O/DMSO (99:1, v/v) solution. The micelle is shown to be a spherical structure, the radius of the micelle is 90-200 nm, and the measured diameter of the micelle is slightly smaller compared with the DLS result, which is caused by the fact that the micelle is converted from a solution state to a dry state in the sample preparation process, and the solution is volatilized to cause the contraction of the micelle.
Example 3:
detection of different surfactants and ions
mu.L of HBT-C prepared in example 1 was added
182.985mL of a stock solution of DMSO (1.0mM) containing various surfactants and ions (sodium dodecylbenzene sulfonate (SDBS), Sodium Dodecyl Sulfate (SDS), Triton X-100, cetyltrimethylammonium bromide (CTAB) and simple ions (e.g., Na:. RTM.: Na)
+,K
+,Mg
2+,Ca
2+,Zn
2+NO
3 –,HCO
3 –,HCO
3 2–,PO
4 3–,SO
4 2–) ) was vortexed for 10 seconds and fluorescence (excitation wavelength 334nm) was measured with a fluorescence spectrometer. The results are shown in FIG. 8.
FIG. 8 shows HBT-C in the presence of different surfactants and ions
18Ratio of fluorescence emission intensities at 510nm and 450nm (I)
510/I
450) And (4) changing. The probe has high sensitivity to an anionic surfactant connected with a sulfonic group, and comprises the following components: sodium Dodecyl Benzene Sulfonate (SDBS), Sodium Dodecyl Sulfate (SDS), and Triton X-100, which is a neutral surfactant, and cetyltrimethylammonium bromide (CTAB), which is a cationic surfactant, and various simple cations (e.g., Na:. RTM.: Na:. RTM.)
+,K
+,Mg
2 +,Ca
2+,Zn
2+) Anions (e.g.: NO
3 –,HCO
3 –,HCO
3 2–,PO
4 3–,SO
4 2–) The response is weak.
Example 4: bacterial fluorescent staining
(1) Bacterial culture
Coli (e.coli, strain ATCC 15224) was purchased from VWR International, LLC. A single colony of the bacterium was inoculated in 5mL of LB medium and shaken at 37 deg.C (150 rpm). Thereafter, 1mL of the bacterial suspension was inoculated into 50mL of fresh medium and incubated at 37 ℃ with shaking for 5h to achieve mid-log growth.
(2) Fluorescent staining of coli
Coli was cultured at 37 ℃ for 12 hours, and the concentration of the bacteria was diluted to 10 by PBS
7CFU/mL, followed by HBT-C prepared with 10. mu.M of example 1
18The culture was carried out at 37 ℃ for 0.5 hour. Fluorescence imaging experiments (Leica TCS-SP8, Germany) were performed using a laser scanning confocal microscopy technique with excitation at 405nm and a filter at 508-558 nm. The results are shown in FIG. 9.
FIG. 9 is a photograph showing staining of E.coli in bright field and fluorescence. (HBT-C)
1810 μ M) scale bar 10 μ M, as shown in the figureIt can be seen that the fluorescence image of the escherichia coli is clearly visible under the laser scanning confocal microscope, and the influence of background noise is negligible. Thus HBT-C
18The probe can be used to directly stain bacteria without further isolation, washing steps.
(3) Antibacterial experiments
HBT-C prepared in example 1 was added at various concentrations
18Probe addition 1mL of bacteria concentration 10
7HBT-C was measured in LB medium (CFU/mL)
18The antibacterial activity of (1). The cells were incubated at 37 ℃ with shaking (150rpm) for 2h, and the resulting solution was diluted with 9mL of PBS. After rough mixing with a pipette, the solution was transferred to a 15mL tube and the bacteria were mixed for a further 3min with vortex shaking. Subsequently, the bacterial solution is diluted 10
0,10
1And 10
2After 10. mu.L of the diluted bacterial suspension was taken, the activity of the bacteria was evaluated by using an agar plate.
Coli bacteria at different concentrations of HBT-C
18(0,10,20,40,60,80 and 100 μ M). As can be seen from the figure, HBT-C
18The probe shows very low toxicity to bacteria, and is favorable for fluorescent staining experiments of the bacteria.
While the invention has been described in connection with preferred embodiments, the invention is not limited to the embodiments described above, and it should be understood that these embodiments are merely illustrative and not restrictive of the scope of the invention. Further, it will be appreciated that various changes or modifications of the invention may be made by those skilled in the art after reading the teachings herein without departing from the spirit of the invention, which equivalents fall within the scope of the invention as defined in the appended claims.
Claims (24)
1. A compound of formula Ia:
wherein:
R
1，R
2independently is hydrogen, C
1-6Alkyl, halogen, C
1-6Alkyloxy, C
1-6An alkylthio group; r
3，R
4，R
5Is C
1-30Alkyl, and R
3，R
4，R
5At least one of them being C
8-30An alkyl group; a. the
-Is an anion.
2. The compound of claim 1, wherein a
-Is halide ion, perchlorate ion, sulfate ion, nitrate ion, and hexafluorophosphate ion.
3. The compound of claim 1, wherein R
3，R
4，R
5At least one of them being C
10-24 alkyl groups.
4. The compound of claim 1, wherein R
3，R
4，R
5At least one of them being C
12-22An alkyl group.
5. The compound of claim 1, wherein R
3，R
4，R
5At least one of them being C
14-20An alkyl group.
6. The compound of claim 1, wherein R
3，R
4，R
5One group in (A) is C
8-30Alkyl radicals and the other two radicals being C
1-8An alkyl group.
7. The compound of claim 1, wherein R
3，R
4，R
5One of them is C
10-24Alkyl radicals and the other two radicals being C
1-8An alkyl group.
8. The compound of claim 1, wherein R
3，R
4，R
5One of them is C
12-22Alkyl radicals and the other two radicals being C
1-8An alkyl group.
9. The compound of claim 1, wherein R
3，R
4，R
5One of them is C
14-20Alkyl radicals and the other two radicals being C
1-8An alkyl group.
10. A compound according to any one of claims 6 to 9 wherein the other two groups are C
1-6An alkyl group.
11. A compound according to claim 1, wherein in formula Ia, R
1Is hydrogen, R
2Is hydrogen, R
3，R
4Is methyl, R
5Is octadecyl, A
-Is an anion.
12. The compound of claim 1, wherein the compound of formula Ia is selected from:
n- (3- (benzo [ d ] thiazol-2-yl) -4-hydroxybenzyl) -N, N-dimethyloctadecyl-1-ammonium chloride.
13. A process for the preparation of a compound according to any one of claims 1 to 12, comprising reacting a compound of formula IIa with a compound of formula III, under protonic acid catalysis and oxidation with an oxidizing reagent to give a compound of formula Ia:
wherein R is
1、R
2、R
3、R
4、R
5、A
-As defined in any one of claims 1 to 12.
14. The method according to claim 13, wherein the protonic acid is concentrated hydrochloric acid and the oxidizing agent is hydrogen peroxide.
15. Use of a compound according to any one of claims 1 to 12 for the detection of anionic surfactants.
16. The use according to claim 15, wherein the anionic surfactant is selected from the group consisting of carboxylates, sulfates, sulfonates, and phosphates.
17. Use according to claim 15, wherein the anionic surfactant is an anionic surfactant having a sulphonic acid group attached thereto.
18. The use according to claim 15, wherein the anionic surfactant is sodium dodecylbenzene sulfonate, sodium lauryl sulfate.
19. A method for detecting an anionic surfactant, comprising adding a compound according to any one of claims 1 to 12 to a solution of the anionic surfactant, and observing or detecting a fluorescence spectrum under fluorescence.
20. Use of a compound according to any one of claims 1 to 12 for leave-on fluorescent staining of bacteria.
21. The use of claim 20, wherein the bacterium is a bacterium having a negatively charged outer membrane envelope.
22. The use according to claim 20, wherein the bacteria are escherichia coli, bacillus subtilis, staphylococcus aureus.
23. A method for the leave-on fluorescent staining of bacteria comprising co-culturing a compound of any one of claims 1-12 with bacteria, whereby the fluorescent staining of the bacteria can be directly visualized without isolation.
24. The method of claim 23, wherein the observing is performed using a laser scanning confocal microscope.
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