Triphenylamine amphiphilic fluorescent probe, preparation method and application thereof

Abstract

This invention relates to the field of fluorescent probe technology, specifically to a triphenylamine amphiphilic fluorescent probe, its preparation method, and its applications. The triphenylamine amphiphilic fluorescent probe is synthesized in three steps: bromination, coupling, and condensation to construct a D-π-A molecular structure, producing far-red light emission at 650-660 nm and exhibiting aggregation-induced emission (AIE) properties. The pyridine group endows it with hydrophilicity and cationic characteristics, while the triphenylamine group endows it with lipophilicity and AIE properties. It can specifically bind to bacterial surface anions, enabling wash-free, high-contrast real-time imaging, and can also disrupt bacterial biofilms to exert antibacterial effects. The probe has a MIC as low as 1.5625 μM against bacteria such as Staphylococcus aureus and Escherichia coli, can be co-stained with acridine orange for verification, is compatible with conventional detection equipment, and achieves integrated bacterial diagnosis and treatment, solving the problems of fluorescence quenching and low emission peaks in traditional probes.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, specifically to a triphenylamine amphiphilic fluorescent probe, its preparation method, and its application. Background Technology

[0002] Bacterial infections are a major challenge in the medical field today. Rapid and accurate diagnosis and treatment of bacterial infections are crucial for preventing disease transmission and improving treatment outcomes. Therefore, developing novel fluorescent probes that integrate bacterial diagnosis and treatment has significant clinical implications. Cationic fluorescent probes with aggregation-induced emission (AIE) properties offer unique advantages and promising applications as an emerging fluorescence imaging tool. They exhibit high selectivity and sensitivity, interacting with anionic components on bacterial surfaces to achieve accurate imaging and diagnosis. Furthermore, due to their cationic nature, cationic fluorescent probes also hold the potential for therapeutic applications in bacterial infections.

[0003] Compared to cationic fluorescent probes with AIE properties, organic fluorescent probes currently widely used for fluorescent staining of bacteria and bacterial biofilms, such as acridine orange, suffer from fluorescence quenching and low emission peaks, limiting their application range. Furthermore, organic fluorescent probes used for fluorescent staining of bacteria and bacterial biofilms are generally ineffective in antibacterial therapy, making it difficult to achieve the dual functions of efficient imaging and bacterial biofilm disruption. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a triphenylamine amphiphilic fluorescent probe, its preparation method, and its applications. In this probe, triphenylamine acts as an electron donor, a cationic indole group as an electron acceptor, and a carbon-carbon double bond as a π-bridge, forming a D-π-A type molecular structure. This extends the molecular conjugation length, producing far-red light emission at 650nm~660nm, significantly distinct from the biological autofluorescence background, enabling high-contrast, wash-free real-time imaging. By protonating the pyridine group to form a stable cation, the probe specifically targets the anionic components on the bacterial surface, achieving rapid and selective localization in complex biological environments. This avoids the drawbacks of traditional probes, such as the need for light-protected operation and susceptibility to environmental interference. Thus, it solves the problems of fluorescence quenching, low emission peaks, difficulty in imaging, and damage to bacterial biofilms associated with traditional fluorescent probes.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a triphenylamine amphiphilic fluorescent probe, the structure of which is shown below: ; Wherein, R1 is one of the following structures: ; R2 can be one of the following structures: .

[0006] In a preferred embodiment of the present invention, the triphenylamine amphiphilic fluorescent probe has one of the following structures: , , .

[0007] A second objective of this invention is to provide a method for preparing the above-mentioned triphenylamine amphiphilic fluorescent probe, comprising the following steps: S1. Using 4-(diphenylamino)benzene-1-carboxaldehyde and N-bromosuccinimide as starting materials, a bromination reaction is carried out in a reaction system with the first solvent to obtain the first intermediate. The structure of the first intermediate is shown below:

[0008] .

[0009] S2. Under a protective gas atmosphere, a coupling reaction is carried out using the first intermediate and 4-pyridineboronic acid as raw materials in a reaction system of base, second solvent, and palladium-based catalyst to obtain the second intermediate; the structure of the second intermediate is shown below: .

[0010] S3. Using the second intermediate and indole iodide as raw materials, a condensation reaction is carried out in a reaction system with a Lewis base catalyst and a third solvent; or after the condensation reaction, an iodoalkane is added to undergo an alkylation reaction to obtain a triphenylamine amphiphilic fluorescent probe.

[0011] In a preferred embodiment of the present invention, when a condensation reaction is carried out using a second intermediate and indole iodide as raw materials to obtain a triphenylamine amphiphilic fluorescent probe, the indole iodide is 1,2,3,3-tetramethyl-3H-indole iodide, the molar ratio of the second intermediate to 1,2,3,3-tetramethyl-3H-indole iodide is 1:1.1, the molar ratio of the second intermediate to 1,2,3,3-tetramethyl-3H-indole iodide is 1:1.1, the molar ratio of the second intermediate to a Lewis base catalyst is 1:1.1, and the Lewis base catalyst is sodium acetate.

[0012] In a preferred embodiment of the present invention, when a triphenylamine amphiphilic fluorescent probe precursor is obtained after a condensation reaction, an iodoalkane is added to the precursor to undergo an alkylation reaction to obtain a triphenylamine amphiphilic fluorescent probe, the iodoalkane is iodomethane, and the molar ratio of the triphenylamine amphiphilic fluorescent probe precursor to iodomethane is 0.25:1.5.

[0013] In a preferred embodiment of the present invention, a condensation reaction is carried out using a second intermediate and indole iodide as raw materials to obtain a triphenylamine amphiphilic fluorescent probe precursor. When an iodoalkane is added to the precursor to undergo an alkylation reaction to obtain the triphenylamine amphiphilic fluorescent probe, the indole iodide is 1,1,2,3-tetramethyl-1H-benzoindole iodide, and the iodoalkane is iodomethane. The molar ratio of the second intermediate to 1,1,2,3-tetramethyl-1H-benzoindole iodide is 1:1.1, the molar ratio of the second intermediate to the Lewis base catalyst is 1:1.1, and the Lewis base catalyst is sodium acetate. The mass-to-volume ratio of the triphenylamine amphiphilic fluorescent probe precursor to iodomethane is 0.5 g:1 mL.

[0014] In a preferred embodiment of the present invention, the molar ratio of 4-(diphenylamino)benzyl-1-carboxaldehyde and N-bromosuccinimide is 8:7.5, and the first solvent is tetrahydrofuran. In a preferred embodiment of the present invention, the molar ratio of the first intermediate to 4-pyridineboronic acid is 5:7.5; the molar ratio of the first intermediate to the base is 1:6, and the base is potassium carbonate; the molar ratio of the first intermediate to the palladium-based catalyst is 10:1, and the palladium-based catalyst is tetrakis(triphenylphosphine)palladium; the second solvent is an aqueous solution of 1,4-dioxane, and the protective gas is nitrogen.

[0015] A third objective of this invention is to provide an application of the above-mentioned triphenylamine amphiphilic fluorescent probe in bacterial diagnostic and therapeutic materials.

[0016] In a preferred embodiment of the present invention, the concentration of the triphenylamine amphiphilic fluorescent probe solution is 0.1 μM to 100 μM, and the triphenylamine amphiphilic fluorescent probe solution is a mixture of triphenylamine amphiphilic fluorescent probe and dimethyl sulfoxide.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides a triphenylamine amphiphilic fluorescent probe. In this probe, triphenylamine acts as an electron donor, a cationic indole group acts as an electron acceptor, and a carbon-carbon double bond forms a π-bridge, creating a D-π-A molecular structure. Triphenylamine provides the core fluorescent framework, and the pyridine group is protonated to form a stable cation, endowing the probe with amphiphilic characteristics. This allows it to specifically recognize anionic components on the bacterial surface, thereby enhancing its binding ability to bacteria. The aldehyde group condenses with indole iodide to form a cyanine dye conjugated system, extending the molecular conjugation length and generating far-red light emission at 650nm~660nm, effectively avoiding interference from biological autofluorescence. The above probe specifically recognizes and binds to anionic components on the bacterial surface. Based on its amphiphilic design, it ensures rapid and selective localization in complex biological environments, while avoiding the shortcomings of traditional probes that require light-protected operation and are susceptible to environmental interference. Furthermore, by emitting near-infrared fluorescence of 650nm~660nm when the probe accumulates on the surface of bacteria with high water content, it is significantly different from the biological autofluorescence background, realizing high-contrast, wash-free real-time imaging, thereby solving the problems of fluorescence quenching and low emission peak of traditional fluorescent probes, as well as difficulty in imaging and destruction of bacterial biofilms.

[0018] 2. The triphenylamine amphiphilic fluorescent probes provided by this invention prepare compounds TIPC1~TIPC3 through a specific three-step synthetic pathway. By utilizing the structural characteristics of the triphenylamine core and the precise introduction of functional groups, long-wavelength emission, aggregation-induced emission, and amphiphilic design are achieved. This overcomes the problems of traditional fluorescent probes in bacterial diagnosis, such as low emission peaks, susceptibility to fluorescence quenching requiring light-protected operation, and the inability to simultaneously achieve efficient imaging and antibacterial therapy. Therefore, it provides a new technical solution for wash-free imaging and simultaneous treatment of bacteria.

[0019] 3. This invention provides the application of a triphenylamine amphiphilic fluorescent probe as a bacterial diagnostic and therapeutic material. After binding to bacteria, the probe can disrupt the bacterial biofilm structure, exerting a direct antibacterial effect, thereby simultaneously inhibiting bacterial growth during the diagnostic process, achieving the goal of integrated diagnosis and treatment. The probe is used in solution form with a concentration that can be flexibly adjusted from 0.1 μM to 100 μM, and is compatible with conventional equipment such as laser confocal microscopes and fluorescence spectrophotometers. In biofilm detection, imaging can be achieved after only 30 minutes of co-incubation with bacteria, and it can be verified by co-staining with acridine orange (AO). The washing procedure is simplified (only a light wash with PBS buffer is required). Attached Figure Description

[0020] Figure 1 This is the proton NMR spectrum of TIPC1 of the present invention.

[0021] Figure 2 This is the carbon NMR spectrum of TIPC1 of the present invention.

[0022] Figure 3This is a diagram showing the inhibitory effect of TIPC1 of the present invention on the film-forming bacteria of Staphylococcus aureus and Escherichia coli. Figure 3 Figure (a) shows the inhibitory effect of TIPC1 on the biofilm growth of Staphylococcus aureus, and Figure (b) shows the inhibitory effect of TIPC1 on the biofilm growth of Escherichia coli.

[0023] Figure 4 This invention presents a graph showing the inhibition of Staphylococcus aureus biofilm growth by different concentrations of TIPC1 observed using CLSM. Figure 4 Figure a shows the negative control; Figure b shows the inhibition of Staphylococcus aureus biofilm growth by TIPC1 at a concentration of 1 / 2 MIC; Figure c shows the inhibition of Staphylococcus aureus biofilm growth by TIPC1 at a concentration of MIC; and Figure d shows the inhibition of Staphylococcus aureus biofilm growth by TIPC1 at a concentration of 2 MIC.

[0024] Figure 5 This is a graph showing the inhibition of biofilm growth of Escherichia coli by different concentrations of TIPC1 observed by CLSM in this invention. Figure 5 Figure a shows the negative control; Figure b shows the inhibition of biofilm growth of Escherichia coli by TIPC1 at a concentration of 1 / 2 MIC; Figure c shows the inhibition of biofilm growth of Escherichia coli by TIPC1 at a concentration of MIC; and Figure d shows the inhibition of biofilm growth of Escherichia coli by TIPC1 at a concentration of 2 MIC.

[0025] Figure 6 This is an image showing the imaging effect of TIPC1 and AO co-staining on biofilms of Gram-positive bacteria according to the present invention. Figure 6 Image a1 shows the imaging effect of AO on the Staphylococcus aureus membrane, image a2 shows the imaging effect of TIPC1 on the Staphylococcus aureus membrane, image a3 is a combined image of images a1 and a2, image b1 shows the imaging effect of AO on the Listeria monocytogenes membrane, image b2 shows the imaging effect of TIPC1 on the Listeria monocytogenes membrane, and image b3 is a combined image of images b1 and b2.

[0026] Figure 7 This is an image showing the effect of TIPC1 and AO co-staining on the biofilm of Gram-negative bacteria according to the present invention. Figure 7 Image a1 shows the imaging effect of AO on the Escherichia coli membrane, image a2 shows the imaging effect of TIPC1 on the Escherichia coli membrane, image a3 is a combined image of images a1 and a2, image b1 shows the imaging effect of AO on the Salmonella enteritidis membrane, image b2 shows the imaging effect of TIPC1 on the Salmonella enteritidis membrane, and image b3 is a combined image of images b1 and b2.

[0027] Figure 8This is an image showing the imaging effect of TIPC1 and AO co-staining on the biofilm of fungi according to the present invention. Figure 8 Image a1 shows the imaging effect of AO on the Candida albicans capsule, image a2 shows the imaging effect of TIPC1 on the Candida albicans capsule, and image a3 is a combined image of images a1 and a2.

[0028] Figure 9 The images show the fluorescence spectra of the triphenylamine amphiphilic fluorescent probes of Examples 1 to 3 of this invention in different solvents. Figure 9 Figure (a) shows the fluorescence spectrum of Example 1, Figure (b) shows the fluorescence spectrum of Example 1, and Figure (c) shows the fluorescence spectrum of Example 1. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] It should be noted that the technical terms used in this invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0031] Traditional organic fluorescent probes, such as acridine orange, suffer from fluorescence quenching and low emission peaks when used for fluorescent staining of bacteria and bacterial biofilms, limiting their application range. Furthermore, existing fluorescent probes do not effectively combine bacterial imaging and antimicrobial therapy, failing to achieve the dual functions of efficient wash-free imaging and bacterial biofilm disruption. Therefore, there is an urgent need to develop a novel fluorescent probe that can simultaneously meet the requirements of efficient bacterial imaging and antimicrobial therapy, while possessing good luminescent properties and stability.

[0032] Based on this, firstly, the present invention provides a triphenylamine amphiphilic fluorescent probe, the structure of which is shown below: ; Wherein, R1 is one of the following structures: ; R2 can be one of the following structures: .

[0033] The triphenylamine amphiphilic fluorescent probe is compound TIPC1, compound TIPC2, or compound TIPC3. Its structure is shown below:

[0034] TIPC1, TIPC2, TIPC3.

[0035] It should be noted that in this structure, triphenylamine is the electron donor (D), the cationic indole group is the electron acceptor (A), and the carbon-carbon double bond is the π-bridge, forming a D-π-A type molecular structure. This causes a red shift in the absorption and emission wavelengths, which can effectively distinguish it from biological background fluorescence and improve imaging results.

[0036] Secondly, this invention provides a method for preparing the above-mentioned triphenylamine amphiphilic fluorescent probe, comprising the following steps: S1. Using 4-(diphenylamino)benzene-1-carboxaldehyde and N-bromosuccinimide as raw materials, a first reaction is carried out in a reaction system with a first solvent to obtain a first intermediate; the first intermediate is TPA-Br-CHO, and the first reaction is a bromination reaction.

[0037] The specific synthesis process of TPA-Br-CHO is as follows: 4-(diphenylamino)benzene-1-carboxaldehyde is dissolved in anhydrous tetrahydrofuran, N-bromosuccinimide (NBS) is added, and the reaction is carried out at 0°C for 12 hours. After removing the tetrahydrofuran by vacuum evaporation, the mixture is separated by silica gel column chromatography using a petroleum ether / dichloromethane volume ratio of 5:1 to obtain TPA-Br-CHO. The synthetic route is shown below:

[0038] .

[0039] The reaction mechanism of the first reaction is as follows: 4-(diphenylamino)benzene-1-carboxaldehyde, as the core fluorescent backbone of the triphenylamine derivative, reacts with N-bromosuccinimide in the first solvent to generate the first intermediate. In this process, N-bromosuccinimide selectively introduces a bromine atom at the ortho position of the aldehyde group to form a bromoaldehyde intermediate, providing site specificity for subsequent coupling reactions, while ensuring that the structural basis of the cationic group is established.

[0040] S2. Under a protective gas atmosphere, using the first intermediate and 4-pyridineboronic acid as raw materials, a second reaction is carried out in a reaction system of base, second solvent, and palladium-based catalyst to obtain a second intermediate, which is TPA-PY-CHO; the second reaction is a coupling reaction. The reaction conditions for the second reaction include: carrying out under a protective gas atmosphere, a pH value of 10-11 (alkaline), a temperature of 115°C, and monitoring the reaction progress by thin-layer chromatography. The base includes potassium carbonate.

[0041] The specific synthesis process of TPA-PY-CHO is as follows: TPA-Br-CHO, 4-pyridineboronic acid, potassium carbonate, and tetrakis(triphenylphosphine)palladium are added to a mixed solvent of water and 1,4-dioxane, wherein the volume ratio of water to 1,4-dioxane is 3:4. The reaction is carried out at 115℃ under nitrogen protection, and the reaction progress is monitored by thin-layer chromatography. After the reaction is complete, the mixture is cooled to room temperature, and the organic phase is removed by vacuum evaporation. After extraction with dichloromethane, the organic layer is evaporated under vacuum to obtain the crude product TPA-PY-CHO. The synthetic route is shown below:

[0042] .

[0043] In the second reaction, the first intermediate TPA-Br-CHO reacts with 4-pyridineboronic acid, a base, a second solvent, and a palladium-based catalyst to generate the second intermediate. Specifically, the brominated derivative of TPA-Br-CHO undergoes efficient coupling with 4-pyridineboronic acid under the action of a base and a palladium-based catalyst, introducing a pyridine group. This group can be protonated in a physiological environment to form a stable cation, endowing the probe with amphiphilic characteristics, enabling it to specifically recognize anionic components on the bacterial surface, thereby enhancing its binding ability to bacteria.

[0044] S3. Using the second intermediate and indole iodide as raw materials, a condensation reaction is carried out in a reaction system with a Lewis base catalyst and a third solvent; or after the condensation reaction, an iodoalkane is added to undergo an alkylation reaction to obtain a triphenylamine amphiphilic fluorescent probe.

[0045] The indole iodide is 1,2,3,3-tetramethyl-3H-indole iodide or 1,1,2,3-tetramethyl-1H-benzoindole iodide.

[0046] The specific synthetic process for TIPC1 is as follows: 1,2,3,3-tetramethyl-3H-indole iodide, TPA-PY-CHO, and sodium acetate are dissolved in acetic acid. The mixture is stirred at 70°C for 12-24 hours. The solvent is removed under reduced pressure to obtain the crude product. The crude product is then purified by silica gel chromatography using a dichloromethane and methanol mixture with a volume ratio of 15:1 to obtain the black solid TIPC1. The synthetic route is shown below:

[0047] .

[0048] During the synthesis of TIPC1, the molar ratio of the second intermediate TPA-PY-CHO to 1,2,3,3-tetramethyl-3H-indole iodide was 1:1.1, the molar ratio of TPA-PY-CHO to the Lewis base catalyst, sodium acetate, was 1:1.1, and the mass-to-volume ratio of TPA-PY-CHO to the third solvent, acetic acid, was 350 mg:5 mL.

[0049] The specific synthetic process for TIPC2 is as follows: Iodomethane is added to the triphenylamine amphiphilic fluorescent probe precursor obtained after the condensation reaction, resulting in an alkylation reaction to yield the triphenylamine amphiphilic fluorescent probe. The triphenylamine amphiphilic fluorescent probe precursor is TIPC1, the molar ratio of the triphenylamine amphiphilic fluorescent probe precursor to iodomethane is 0.25:1.5, the alkylation reaction temperature is 70℃, and the reaction time is 12 h. The synthetic route is shown below:

[0050] .

[0051] The specific synthetic process for TIPC3 is as follows: 1,1,2,3-tetramethyl-1H-benzoindole iodide, TPA-PY-CHO, and sodium acetate were dissolved in acetic acid and stirred at 70°C for 12-24 hours. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography using a dichloromethane and methanol mixture with a volume ratio of 15:1 to obtain the black solid RETIPC3. Subsequently, RETIPC3 was dissolved in acetonitrile, injected into CH3I, and stirred at 70°C for 12 hours to obtain the triphenylamine amphiphilic fluorescent probe TIPC3. The synthetic route is shown below:

[0052] , .

[0053] In the third reaction, the second intermediate undergoes a condensation reaction with 1,2,3,3-tetramethyl-3H-indole iodide under acid catalysis to generate the target compound TIPC1. Compound TIPC2 is obtained by methylation of TIPC1 and exhibits better water solubility than TIPC1. Compound TIPC3 is obtained by condensation of the second intermediate with 1,1,2,3-tetramethyl-1H-benzoindole iodide under acid catalysis, followed by methylation, and also exhibits better water solubility than TIPC1. During this process, the aldehyde group of the second intermediate condenses with the indole iodide to form a cyanine dye conjugated system, extending the molecular conjugation length and generating far-red light emission at 650nm~660nm, effectively avoiding interference from biological autofluorescence. Simultaneously, based on the aggregation-induced emission mechanism, this compound significantly enhances the fluorescence signal when aggregated in a high-water environment on the bacterial surface, achieving wash-free imaging, and exerts antibacterial therapeutic functions by disrupting the bacterial biofilm structure through cation exchange.

[0054] It should be noted that this invention constructs compounds TIPC1~TIPC3 through a three-step chemical reaction to realize their role as materials for bacterial diagnosis and treatment. Through the synergistic effect of the above three-step reaction, compounds TIPC1~TIPC3 achieve long-wavelength emission, aggregation-induced emission, and amphiphilic design, solving the problems of traditional fluorescent probes such as low emission peaks, easy fluorescence quenching requiring light-avoidance operation, and inability to simultaneously achieve efficient imaging and antibacterial therapy.

[0055] Finally, this invention provides the application of the above-mentioned triphenylamine amphiphilic fluorescent probe in bacterial diagnostic and therapeutic materials.

[0056] The application process is as follows: the triphenylamine amphiphilic fluorescent probe is brought into contact with bacteria, and bacterial diagnosis is performed by detecting the fluorescent signal generated by the fluorescent probe. Bacterial treatment is achieved through the antibacterial properties of the fluorescent probe.

[0057] This invention combines the unique cationic properties of a triphenylamine amphiphilic fluorescent probe with an aggregation-induced emission mechanism, thereby achieving simultaneous detection and intervention of bacterial infections. Specifically, the probe can specifically recognize and bind to anionic components on the bacterial surface. Its amphiphilic design ensures rapid and selective localization in complex biological environments, while avoiding the drawbacks of traditional probes, such as the need for light-protected operation and susceptibility to environmental interference. Furthermore, by emitting near-infrared fluorescence at 650nm–660nm when the probe aggregates on the surface of bacteria with high water content, significantly distinguishing it from the biological autofluorescence background, it achieves high-contrast, wash-free real-time imaging, overcoming the application limitations caused by the low emission peak of traditional fluorescent probes. More importantly, after binding to bacteria, the probe can disrupt the bacterial biofilm structure, exerting a direct antibacterial effect, thereby simultaneously inhibiting bacterial growth during the diagnostic process and achieving the goal of integrated diagnosis and treatment.

[0058] The bacteria include Gram-positive or Gram-negative bacteria. Gram-positive bacteria include Staphylococcus aureus and Listeria monocytogenes, while Gram-negative bacteria include Escherichia coli and Salmonella enteritidis.

[0059] The triphenylamine amphiphilic fluorescent probe is used in solution form at a concentration of 0.1 μM to 100 μM.

[0060] This invention, through the aforementioned technical solution, effectively addresses the problems of insufficient diagnostic accuracy, unstable treatment effects, and limited applicability by limiting the range of bacterial types and probe concentrations. First, the clear classification of Gram-positive and Gram-negative bacteria allows the probe to adapt to a wider range of bacterial detection needs, enhancing the method's universality. Second, by strictly controlling the probe concentration, not only is the sensitivity of the fluorescence signal improved, but the safety of the probe in antibacterial therapy is also ensured. Furthermore, combining the above technical solution with the aforementioned method for preparing triphenylamine amphiphilic fluorescent probes further enhances the probe's comprehensive performance in bacterial imaging and anti-infective therapy. For example, in practical applications, the probe can rapidly achieve efficient, wash-free imaging of bacteria by interacting with anionic components on the bacterial surface, while simultaneously disrupting bacterial biofilms, thus achieving an integrated diagnostic and therapeutic effect.

[0061] The following specific examples will provide further explanation.

[0062] In this invention, N-bromosuccinimide is abbreviated as NBS, and dimethyl sulfoxide is abbreviated as DMSO.

[0063] Example 1 A triphenylamine amphiphilic fluorescent probe, TIPC1, has the following structural formula: .

[0064] The preparation method of the above-mentioned triphenylamine amphiphilic fluorescent probe TIPC1 includes the following steps: S1. Dissolve 2.19 g (equivalent to 8.0 mmol) of 4-(diphenylamino)benzene-1-carboxaldehyde in 60 mL of anhydrous tetrahydrofuran, and add 1.33 g (equivalent to 7.5 mmol) of NBS. React at 0 °C for 12 h. After the reaction is complete, remove the tetrahydrofuran by evaporation under reduced pressure, and separate by silica gel column chromatography using a petroleum ether / dichloromethane system with a volume ratio of 5:1 to obtain TPA-Br-CHO.

[0065] S2. 1.76 g (equivalent to 5.0 mmol) of TPA-Br-CHO, 0.92 g (equivalent to 7.5 mmol) of 4-pyridineboronic acid, 4.15 g (equivalent to 30.0 mmol) of potassium carbonate, and 0.58 g (equivalent to 0.5 mmol) of Pd(PPh3)4 were added to 100 mL of a mixed solvent consisting of water and 1,4-dioxane in a volume ratio of 3:4. The reaction was then carried out at 115 °C under nitrogen protection, and the reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the mixture was cooled to room temperature, and the organic phase was removed by evaporation under reduced pressure. After extraction with dichloromethane, the organic layer was evaporated under reduced pressure to obtain TPA-PY-CHO.

[0066] S3. Dissolve 331 mg (equivalent to 1.1 mmol) of 1,2,3,3-tetramethyl-3H-indole iodide, 350 mg (equivalent to 1 mmol) of TPA-PY-CHO, and 187 mg (equivalent to 1.1 mmol) of sodium acetate in 5 mL of acetic acid to obtain a mixed solution. Stir at 70 °C for 12 h, and remove the solvent under reduced pressure to obtain the crude product. Finally, purify the crude product by silica gel chromatography (dichloromethane:methanol = 15:1) to obtain the triphenylamine amphiphilic fluorescent probe, i.e., the black solid TIPC1.

[0067] The yield of TIPC1 was 337 mg, representing a yield of 53.2%. TIPC1 was characterized using 1H and 1C NMR spectra, as shown below. Figure 1 and Figure 2 As shown, the results are as follows: 1 H NMR (400 MHz, DMSO) δ 8.65 (d, J = 6.1 Hz, 2H), 8.36 (d, J = 16.1 Hz, 1H), 8.11 (d, J = 9.0 Hz, 2H), 7.92 – 7.87 (m, 2H), 7.84(ddd, J = 7.3, 3.8, 2.4 Hz, 2H), 7.77 (dd, J = 4.7, 1.6 Hz, 2H), 7.64 – 7.54(m, 2H), 7.52 – 7.41 (m, 3H), 7.34 – 7.23 (m, 5H), 7.03 (d, J = 8.9 Hz, 2H),4.07 (s, 3H), 1.77 (s, 6H). 13 C NMR (101 MHz, DMSO) δ 181.44, 172.52, 153.26,152.23, 150.76, 149.58, 146.88, 146.49, 145.60, 143.68, 142.39, 133.85,133.23, 130.73, 129.36, 128.84, 127.85, 127.15, 126.61, 126.33, 123.27,121.33, 120.29, 115.09, 110.08, 52.15, 34.43, 26.14, 21.55. TIPC1 was characterized by mass spectrometry, and the results are as follows: ESI HRMS (m / z): calcd for C36H32N3+ M+ ​​506.2591 found 506.2592.

[0068] Example 2 The structure of a triphenylamine amphiphilic fluorescent probe, TIPC2, is shown below: .

[0069] The preparation method of the above-mentioned triphenylamine amphiphilic fluorescent probe TIPC2 includes the following steps: 158 mg (equivalent to 0.25 mmol) of TIPC1 prepared in Example 1 and 213 mg (equivalent to 1.5 mmol) of iodomethane were dissolved in 10 mL of acetonitrile. The mixture was stirred at 70 °C for 12 h. After the reaction was completed and cooled to room temperature, ethyl acetate was added, and a black solid precipitated out. The precipitate was then filtered and repeatedly washed with ethyl acetate to obtain a black solid TIPC2 with a yield of 112 mg, representing a yield of 57.8%.

[0070] Example 3 The structure of TIPC3, a triphenylamine amphiphilic fluorescent probe, is shown below: .

[0071] The preparation method of the above-mentioned triphenylamine amphiphilic fluorescent probe TIPC3 includes the following steps: S1. Dissolve 2.19 g (equivalent to 8.0 mmol) of 4-(diphenylamino)benzene-1-carboxaldehyde in 60 mL of anhydrous tetrahydrofuran, and add 1.33 g (equivalent to 7.5 mmol) of NBS. React at 0 °C for 12 h. After the reaction is complete, remove the tetrahydrofuran by evaporation under reduced pressure, and separate by silica gel column chromatography using a petroleum ether / dichloromethane system with a volume ratio of 5:1 to obtain TPA-Br-CHO.

[0072] S2. 1.76 g (equivalent to 5.0 mmol) of TPA-Br-CHO, 0.92 g (equivalent to 7.5 mmol) of 4-pyridineboronic acid, 4.15 g (equivalent to 30.0 mmol) of potassium carbonate, and 0.58 g (equivalent to 0.5 mmol) of Pd(PPh3)4 were added to 100 mL of a mixed solvent consisting of water and 1,4-dioxane in a volume ratio of 3:4. The reaction was then carried out at 115 °C under nitrogen protection, and the reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the mixture was cooled to room temperature, and the organic phase was removed by evaporation under reduced pressure. After extraction with dichloromethane, the organic layer was evaporated under reduced pressure to obtain TPA-PY-CHO.

[0073] S3. 1.16 g (equivalent to 3.3 mmol) of 1,1,2,3-tetramethyl-1H-benzoindole iodide, 1.05 g (equivalent to 3 mmol) of TPA-PY-CHO, and 0.27 g (equivalent to 3.3 mmol) of sodium acetate were dissolved in 10 mL of acetic acid to obtain a mixed solution. The solution was then stirred at 70 °C for 12 h, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (dichloromethane:methanol = 15:1) to give 0.85 g of black solid RETIPC3, with a yield of 41.5%.

[0074] S4. Dissolve 0.50 g (equivalent to 0.73 mmol) of RETIPC3 in 2 mL of acetonitrile, and add 1 mL (equivalent to 16 mmol) of CH3I. Stir at 70 °C for 12 h. After the reaction is complete and cooled to room temperature, add ethyl acetate, and a black solid precipitates. Filter the precipitate and wash repeatedly with ethyl acetate to obtain the triphenylamine amphiphilic fluorescent probe TIPC3. TIPC3 is a black solid with a yield of 303 mg, representing a yield of 50.3%.

[0075] The structure and performance of the triphenylamine amphiphilic fluorescent probes of Examples 1 to 3 were tested.

[0076] The fluorescence emission spectroscopy test procedure was as follows: 6 mg of the triphenylamine amphiphilic fluorescent probe from Example 1, 7 mg of the triphenylamine amphiphilic fluorescent probe from Example 2, and 8 mg of the triphenylamine amphiphilic fluorescent probe from Example 3 were dissolved in 10 mL of DMSO to prepare a 1 mM stock solution. 10 μL of the stock solution was added to 1 mL of solvent. The solvent could be methanol, ethanol, dimethyl sulfoxide, dimethylformamide, acetonitrile, tetrahydrofuran, dioxane, or pure water. Subsequently, a fluorescence spectrophotometer was used to measure the fluorescence intensity of different triphenylamine amphiphilic fluorescent probes in each solvent.

[0077] Figure 9 The images show the fluorescence spectra of the triphenylamine amphiphilic fluorescent probes of Examples 1 to 3 of this invention in different solvents. Figure 9 Figure (a) shows the fluorescence spectrum of Example 1, Figure (b) shows the fluorescence spectrum of Example 1, and Figure (c) shows the fluorescence spectrum of Example 1. Figure 9As shown, TIPC1 exhibits the highest fluorescence intensity in ethanol. The emission wavelengths in ethanol are all between 650 nm and 660 nm. TIPC1 shows a significant redshift in methanol and ethanol. Compared to other solvents, TIPC1 is redshifted by 10 nm. These results suggest that ethanol is the preferred solvent for TIPC1, enabling it to exhibit the strongest fluorescence intensity and optimal emission wavelength. TIPC2 and TIPC3 also show the highest fluorescence intensity in ethanol. The emission wavelengths in ethanol are all between 650 nm and 660 nm. TIPC3 exhibits relatively strong fluorescence intensity in ethanol. TIPC2 and TIPC3 show significant redshifts in methanol and ethanol. Compared to other solvents, TIPC1 is redshifted by 10 nm, while TIPC2 and TIPC3 are redshifted by 50 nm. TIPC3 shows a redshift of approximately 20 nm in DMSO. These results suggest that ethanol is the preferred solvent for TIPC2 and TIPC3, enabling them to exhibit the strongest fluorescence intensity and optimal emission wavelength. Furthermore, different solvents had varying degrees of influence on the fluorescence properties of the compounds, indicating that the solvent environment may affect the molecular structure or conformation of the compounds, thereby affecting their fluorescence characteristics.

[0078] Next, in vitro antibacterial tests were conducted. The minimum inhibitory concentration (MIC) of the compound was determined using the broth dilution method and the plate coating method. Furthermore, the effects of the compound on biofilm formation and disruption were evaluated using biofilm inhibition and disruption assays in 96-well polystyrene culture plates. The specific procedures are as follows:

[0079] (1) Bacterial culture and preparation: The bacteria used were Staphylococcus aureus ATCC25923, Escherichia coli O157:H7, Listeria monocytogenes X99, Candida albicans ATCC10231, Pseudomonas aeruginosa PAO1, and Salmonella enteritidis CMCC50746.

[0080] Under aseptic conditions, pick a single colony from the inoculated plate and inoculate it into 5 mL of liquid culture medium. Incubate in a shaker at 37°C and 180 rpm until the logarithmic phase is reached, then harvest the bacterial stock solution.

[0081] (2) Determination of minimum inhibitory concentration (MIC): The minimum inhibitory concentration (MIC) of the compound was determined using the broth dilution method in 96-well polystyrene culture plates. First, the bacterial stock solution was diluted to 1 × 10⁻⁶. 5 CFU / mL ~5×10 5 Add CFU / mL to a compound solution that has been serially diluted twofold (using DMSO as the solvent). Incubate in a constant-temperature shaker for at least 24 hours, and visually determine the minimum inhibitory concentration (MIC) (the concentration at which bacteria no longer grow).

[0082] (3) Biofilm formation inhibition test: Dilute the bacteria to 1×10⁻⁶ 5 CFU / mL ~5×10 5 After adding CFU / mL, DMSO solutions of different concentrations of the compound were added, and the mixtures were incubated in 96-well polystyrene plates at 37°C for at least 36 hours. Planktonic bacteria were washed with phosphate-buffered saline (PBS) buffer, dried at 60°C, fixed with methanol for 15 minutes, and then dried again. 200 μL of 0.05% crystal violet staining solution was added for 15 minutes. After removing the crystal violet, excess dye was washed away with PBS buffer, and 200 μL of 95% (v / v) ethanol was added for destaining in a shaker for 15 minutes. The OD570 was measured using a microplate reader to assess the inhibitory effect of the compound on biofilm formation.

[0083] (4) Observation of biofilm damage using laser confocal microscopy (CLSM): Staphylococcus aureus and Escherichia coli were diluted to 1×10⁻⁶ using liquid culture medium. 5 CFU / mL ~5×10 5 After adding CFU / mL, DMSO solutions of different probe concentrations were added to achieve probe concentrations of 0, 0.5 MIC, MIC, and 2 MIC in the bacterial growth environment. After thorough mixing, the glass-bottomed culture dishes were placed in a constant temperature incubator at 37°C and incubated for at least 36 hours. After incubation, the planktonic bacteria and fluorescent probe were gently washed with phosphate-buffered saline (PBS) solution, retaining the bacterial biofilm attached to the glass bottom. The biofilm was then stained with acridine orange (AO) for 5 minutes under dark conditions, followed by washing away excess dye with PBS. After drying in an oven, the biofilm was directly imaged using a laser confocal microscope to assess the effect of the compound on biofilm disruption.

[0084] (5) Co-staining with bacterial biofilm fluorescent staining agent AO: Using glass-bottomed culture dishes, after adding liquid culture medium, Staphylococcus aureus ATCC25923, Escherichia coli O157:H7, Listeria monocytogenes X99, Candida albicans ATCC10231, Pseudomonas aeruginosa PAO1, and Salmonella enteritidis CMCC50746 were inoculated separately. Suitable incubation temperatures and environments were maintained for at least 36 hours. The dishes were then slowly rinsed with PBS to remove airborne bacteria and residual drugs. 0.1 mg / mL of AO and 0.1 mg / mL of the compound were added to PBS solutions, respectively. After incubation in the dark for 30 minutes, the bottom of the culture dishes was rinsed with PBS to remove excess AO and the compound. Finally, images were taken using a laser confocal microscope (CLSM).

[0085] Table 1 shows the MICs of TIPC1-TIPC3 against various bacteria. As can be seen from Table 1, TIPC1-TIPC3 exhibit strong bactericidal activity, with MIC values ​​against Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes ranging from 1.5625 μM to 6.25 μM. However, their activity against Pseudomonas aeruginosa and Salmonella enteritidis is weaker, with MIC values ​​greater than 100 μM. Notably, TIPC3 shows good bactericidal activity against Salmonella enteritidis, with a MIC value of 25 μM. In conclusion, TIPC1-TIPC3 demonstrate strong bactericidal activity against various bacteria, especially Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes.

[0086] Table 1. MIC (μM) of TIPC1~TIPC3 against various bacteria. Figure 3 This is a diagram showing the inhibitory effect of TIPC1 of the present invention on the film-forming bacteria of Staphylococcus aureus and Escherichia coli. Figure 3 Figure (a) shows the inhibitory effect of TIPC1 on the biofilm growth of Staphylococcus aureus, and Figure (b) shows the inhibitory effect of TIPC1 on the biofilm growth of Escherichia coli. Figure 3 It can be seen that the inhibitory effect on biofilm growth is related to the drug concentration. At the minimum inhibitory concentration, TIPC1 inhibited the biofilm growth of Staphylococcus aureus and Escherichia coli by 21.32% and 27.97%, respectively.

[0087] Figure 4 This invention presents a graph showing the inhibition of Staphylococcus aureus biofilm growth by different concentrations of TIPC1 observed using CLSM. Figure 4 Figure a shows the negative control; figure b shows the inhibition of Staphylococcus aureus biofilm growth by TIPC1 at a concentration of 1 / 2 MIC; figure c shows the inhibition of Staphylococcus aureus biofilm growth by TIPC1 at a concentration of MIC; and figure d shows the inhibition of Staphylococcus aureus biofilm growth by TIPC1 at a concentration of 2 MIC. Figure 4 It was observed that, for Staphylococcus aureus, the negative control group showed dense biofilm growth and extensive coverage. However, with increasing TIPC1 concentration, the biofilm density gradually decreased. At 1 / 2 MIC concentration, the biofilm coverage decreased. After reaching the MIC concentration, the biofilm coverage decreased significantly. Furthermore, the bacterial count also decreased accordingly, resulting in a sparse biofilm. Ultimately, at 2 MIC concentration, there was no significant bacterial aggregation, and only single bacterial colonies were observed.

[0088] Figure 5This is a graph showing the inhibition of biofilm growth of Escherichia coli by different concentrations of TIPC1 observed by CLSM in this invention. Figure 5 Figure a shows the negative control; figure b shows the inhibition of TIPC1 biofilm growth in *E. coli* at a concentration of 1 / 2 MIC; figure c shows the inhibition of TIPC1 biofilm growth in *E. coli* at a concentration of MIC; and figure d shows the inhibition of TIPC1 biofilm growth in *E. coli* at a concentration of 2 MIC. Figure 5 It was found that TIPC1 also exhibits a dose-dependent effect on the biofilm growth of *E. coli*. After reaching the MIC concentration, the *E. coli* biofilm became sparse, and an increase in individual bacteria was observed. Furthermore, at a concentration of 2 MIC, the number of *E. coli* bacteria was generally lower than that of *Staphylococcus aureus*.

[0089] Figure 6 This image shows the imaging effect of TIPC1 and AO co-staining on biofilms of Gram-positive bacteria according to the present invention. The Gram-positive bacteria include Staphylococcus aureus and Listeria monocytogenes. Figure 6 Image a1 shows the imaging effect of AO on the Staphylococcus aureus membrane; image a2 shows the imaging effect of TIPC1 on the Staphylococcus aureus membrane; image a3 is a combined image of images a1 and a2; image b1 shows the imaging effect of AO on the Listeria monocytogenes membrane; image b2 shows the imaging effect of TIPC1 on the Listeria monocytogenes membrane; image b3 is a combined image of images b1 and b2. Figure 6 It can be seen that both TIPC1 and AO successfully stained the membranes of Staphylococcus aureus and Listeria monocytogenes, and the results showed a high degree of overlap, proving that TIPC1 can be applied to imaging the membranes of Staphylococcus aureus and Listeria monocytogenes.

[0090] Figure 7 This image shows the imaging effect of TIPC1 and AO co-staining on biofilms of Gram-negative bacteria according to the present invention. The Gram-negative bacteria include *Escherichia coli* and *Salmonella enteritidis*. Figure 7 Image a1 shows the imaging effect of AO on the E. coli biofilm; image a2 shows the imaging effect of TIPC1 on the E. coli biofilm; image a3 is a combined image of images a1 and a2; image b1 shows the imaging effect of AO on the Salmonella enteritidis biofilm; image b2 shows the imaging effect of TIPC1 on the Salmonella enteritidis biofilm; image b3 is a combined image of images b1 and b2. Figure 7 It can be seen that both TIPC1 and AO successfully stained the membranes of Escherichia coli and Salmonella enteritidis, and the results showed a high degree of overlap, proving that TIPC1 can be applied to the imaging of the membranes of Escherichia coli and Salmonella enteritidis.

[0091] Figure 8This is an image showing the biofilm imaging effect of TIPC1 and AO co-staining of fungi according to the present invention. The fungus is Candida albicans. Figure 8 Image a1 shows the imaging effect of AO on the Candida albicans capsule, image a2 shows the imaging effect of TIPC1 on the Candida albicans capsule, and image a3 is a combined image of images a1 and a2. Figure 8 It can be seen that both TIPC1 and AO successfully stained the Candida albicans capsule and were highly overlapping, proving that TIPC1 can be applied to Candida albicans capsule imaging.

[0092] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range, as well as any value between the two endpoints, can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended scope of protection is intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of this invention.

[0093] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of protection of this invention and its equivalents, this invention also intends to include these modifications and variations.

Claims

1. A triphenylamine amphiphilic fluorescent probe, characterized in that, The structure of the triphenylamine amphiphilic fluorescent probe is shown below: ； Wherein, R1 is one of the following structures: ； R2 can be one of the following structures: 。 2. The triphenylamine amphiphilic fluorescent probe according to claim 1, characterized in that, The triphenylamine amphiphilic fluorescent probe has one of the following structures: 、 、 。 3. A method for preparing the triphenylamine amphiphilic fluorescent probe according to claim 1 or claim 2, characterized in that, Includes the following steps: Using 4-(diphenylamino)benzene-1-carboxaldehyde and N-bromosuccinimide as raw materials, a bromination reaction was carried out in a reaction system with a first solvent to obtain a first intermediate; the structure of the first intermediate is shown below: ； Under a protective gas atmosphere, a coupling reaction was carried out using a first intermediate and 4-pyridineboronic acid as raw materials in a reaction system of base, second solvent, and palladium-based catalyst to obtain a second intermediate; the structure of the second intermediate is shown below: ； Using a second intermediate and indole iodide as raw materials, a condensation reaction is carried out in a reaction system with a Lewis base catalyst and a third solvent; or after the condensation reaction, an iodoalkane is added to undergo an alkylation reaction to obtain a triphenylamine amphiphilic fluorescent probe.

4. The method for preparing the triphenylamine amphiphilic fluorescent probe according to claim 3, characterized in that, When a condensation reaction is carried out using a second intermediate and indole iodide as raw materials to obtain a triphenylamine amphiphilic fluorescent probe, the indole iodide is 1,2,3,3-tetramethyl-3H-indole iodide, the molar ratio of the second intermediate to 1,2,3,3-tetramethyl-3H-indole iodide is 1:1.1, the molar ratio of the second intermediate to the Lewis base catalyst is 1:1.1, and the Lewis base catalyst is sodium acetate.

5. The method for preparing the triphenylamine amphiphilic fluorescent probe according to claim 3, characterized in that, When a triphenylamine amphiphilic fluorescent probe precursor is obtained after the condensation reaction, an iodoalkane is added to the precursor to undergo an alkylation reaction to obtain the triphenylamine amphiphilic fluorescent probe. In this case, the iodoalkane is iodomethane, and the molar ratio of the triphenylamine amphiphilic fluorescent probe precursor to iodomethane is 0.25:1.

5.

6. The method for preparing the triphenylamine amphiphilic fluorescent probe according to claim 3, characterized in that, A condensation reaction was carried out using a second intermediate and indole iodide as raw materials to obtain a triphenylamine amphiphilic fluorescent probe precursor. When an iodoalkane was added to the precursor and an alkylation reaction was performed to obtain the triphenylamine amphiphilic fluorescent probe, the indole iodide was 1,1,2,3-tetramethyl-1H-benzoindole iodide, and the iodoalkane was iodomethane. The molar ratio of the second intermediate to 1,1,2,3-tetramethyl-1H-benzoindole iodide was 1:1.1, the molar ratio of the second intermediate to the Lewis base catalyst was 1:1.1, and the Lewis base catalyst was sodium acetate. The mass-to-volume ratio of the triphenylamine amphiphilic fluorescent probe precursor to iodomethane was 0.5 g:1 mL.

7. The method for preparing the triphenylamine amphiphilic fluorescent probe according to claim 3, characterized in that, The molar ratio of 4-(diphenylamino)benzene-1-carboxaldehyde to N-bromosuccinimide is 8:7.5, and the first solvent is tetrahydrofuran.

8. The method for preparing the triphenylamine amphiphilic fluorescent probe according to claim 3, characterized in that, The molar ratio of the first intermediate to 4-pyridineboronic acid is 5:7.5; the molar ratio of the first intermediate to the base is 1:6, and the base is potassium carbonate; the molar ratio of the first intermediate to the palladium-based catalyst is 10:1, and the palladium-based catalyst is tetrakis(triphenylphosphine)palladium; the second solvent is an aqueous solution of 1,4-dioxane, and the protective gas is nitrogen.

9. The application of the triphenylamine amphiphilic fluorescent probe according to claim 1 or claim 2 in bacterial diagnostic and therapeutic materials.

10. The application of the triphenylamine amphiphilic fluorescent probe according to claim 9 in bacterial diagnostic and therapeutic materials, characterized in that, The concentration of the triphenylamine amphiphilic fluorescent probe solution is 0.1 μM to 100 μM. The triphenylamine amphiphilic fluorescent probe solution is a mixture of triphenylamine amphiphilic fluorescent probe and dimethyl sulfoxide.

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