WO2016078603A1

WO2016078603A1 - Aie luminogens for bacteria imaging, killing, photodynamic therapy and antibiotics screening, and their methods of manufacturing

Info

Publication number: WO2016078603A1
Application number: PCT/CN2015/095002
Authority: WO
Inventors: Benzhong Tang; Engui Zhao
Original assignee: The Hong Kong University Of Science And Technology
Priority date: 2014-11-21
Filing date: 2015-11-19
Publication date: 2016-05-26
Also published as: CN107001927B; CN107001927A

Abstract

The synthesis of fluorescent molecules with aggregation-induced emission (AIE) characteristics and application as fluorescent probes for cellular imaging, killing, and antibiotic screening, wherein the cells are bacteria or mammalian cells. The fluorescent molecules generate reactive oxygen species (ROS) upon light irradiation. A probe comprising fluorescent molecules exhibiting AIE phenomenon, wherein the probe is used for bacterial and biological study. A method of imaging and quantifying bacterial. A method of killing cells. A method of high throughput antibiotics screening and determining bacteria resistance. A method of photodynamic therapy. A method of determining critical micelle concentration.

Description

AIE LUMINOGENS FOR BACTERIA IMAGING, KILLING, PHOTODYNAMIC THERAPY AND ANTIBIOTICS SCREENING, AND THEIR METHODS OF MANUFACTURING

RELATED APPLICATIONS

The present patent application claims priority to provisional U.S. Patent Application No.62/123,596 filed November 21, 2014, which was filed by the inventors hereof and is incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present subject matter relates to the development of luminogens with aggregation induced emission (AIE) characteristics and use of these materials in bacteria, mammalian cell, and related biological study. In particular, the present subject matter is associated with preparation of AIE luminogens and magnetite for use as fluorescent probes for bacteria quantification, imaging, killing, antibiotics screening, and photodynamic therapy (PDT) .

BACKGROUND

With the advancement of human living standards, hygiene and food safety issues have gained increasing attention from both research scientists and the general public, owing to the close relation to human health. Antibiotics are the most widely used materials for pathogen treatment, due to high effectiveness in inhibiting bacteria growth and low interference to mammalian cells. The rapid emergence of antibiotic resistance, however, has urged scientists to develop new antibiotics, which is generally a long-term and costly process. Investigating both new bacteria killing methods, where the bacteria have difficulty developing resistance to the methods, and new methods for screening antibiotics have arisen as alternative pathways to solve the antibiotics resistance issue.

PDT, which utilizes photosensitizers to produce toxic reactive oxygen species (ROS) for tumor elimination for localized pathogen elimination has been thus proposed and applied. Currently, the most-widely used materials for PDT are porphyrin and phenothiazium. Conjugated polymers are gaining attention as new photosensitizers. However, because most of the materials are coplanar or extremely hydrophobic in nature, strong π-π or hydrophobic interactions may take place, which lead to chromophore aggregation, decreased bacterial killing efficiency, and a fluorescence quenching effect.

SUMMARY

The present subject matter is directed to a species of propeller-shaped molecules which demonstrate increased emission upon aggregation, and the phenomenon is termed as AIE. Systematic studies have shown restriction of intramolecular motion is the main cause for the AIE effect. The AIE phenomenon is of both scientific value and practical application. Owing to biocompatibility, photostability, and selectivity, the AIE materials have been applied to cell and bacteria imaging, cell apoptosis detection, chemotherapy, and drug delivery. Some AIE chromophores are capable of light-induced ROS generation, and may be applied to mammalian cell and bacteria imaging and killing studies, as well as high throughput antibiotics screening.

In the present subject matter, fluorescent molecules with AIE characteristics are designed and synthesized. Tetraphenylethene (TPE) and silole are functionalized with trimethylamine and triethylamine through different linkages, such as an ether, ester, alkyl chain, amide, or any combination thereof, to yield fluorescent molecules with AIE properties. The AIE active molecules are then applied to mammalian cell and bacteria imaging, PDT, and high throughput screening.

Due to the water solubility of TPE-Bac and the typical AIE characteristics of TPE-Bac, the imaging process of bacteria may be simplified. For instance, the washing process may be eliminated, which enables TPE-Bac to be used in antibiotics screening studies. In addition to developing new methods for bacterial killing, AIE materials may be applied to high throughput antibiotics screening. Taking advantage of low background emission and high emission efficiency when bounded to the targets, screening of antibiotics could take place in a simple, fast fashion.

In an embodiment, the present subject matter relates to an AIE luminogen comprising fluorescent molecules comprising a backbone structure of:

wherein

at least one of R, R′, R″, R″′, R″″, and R″″′ is independently selected from the group consisting of

R₁, R₂, and R₃are independently selected from the group consisting of H, C_nH_2n+1, OC_nH_2n+1, and salts thereof；

R, R′, R″, R″′, R″″, and R″″′ are independently selected from the group consisting of H, C_nH_2n+1, OC_nH_2n+1, (OC₂H₄) _n, C₆H₅, C₁₀H₇, C₁₂H₉, OC₆H₅, OC₁₀H₇, and OC₁₂H₉； and n ＝ 0 to 20.

Another embodiment of the present subject matter relates to a probe comprising fluorescent molecules exhibiting AIE phenomenon, wherein the fluorescent molecules comprise a backbone structure of:

wherein

R₁, R₂, and R₃ are independently selected from the group consisting of H, C_nH_2n+1, OC_nH_2n+1, and salts thereof；

Another embodiment of the present subject matter relates to a method of imaging and quantifying bacteria comprising introducing the AIE luminogen to a sample； and detecting bacteria by observing production of fluorescence from aggregation； wherein the bacteria is quantified by observing emission intensity.

Another embodiment of the present subject matter relates to a method of killing cells, comprising introducing the AIE luminogen to a sample containing cells in the presence of normal light； wherein the AIE luminogen generates ROS upon light irradiation； wherein exposure to light kills the cells； and wherein the cells are bacteria or mammalian cells.

Another embodiment of the present subject matter relates to a method of high throughput antibiotic screening and determining bacteria resistance, comprising introducing the AIE luminogen to a sample containing an antibiotic； evaluating the antibiotic based on an emission intensity of the AIE luminogen； wherein bacteria in the sample turns on emission of the AIE luminogen； wherein rapid bacteria growth indicates ineffective antibiotics and inhibited bacteria growth indicates effective antibiotics.

Another embodiment of the present subject matter relates to a method of photodynamic therapy, comprising introducing the AIE luminogen to a sample containing a tumor, wherein the AIE luminogen is a photosensitizer； and eliminating the tumor by PDT, wherein the photosensitizer produces light-induced toxic reactive species.

Another embodiment of the present subject matter relates to a method of determining critical micelle concentration (CMC) , comprising introducing the AIE luminogen of claim 1 to a solvent； and determining the CMC by evaluating fluorescence changes, wherein no emission indicates concentration below CMC and emission is turned on when concentration approaches CMC.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows UV spectra of TPE-Bac in DMSO.

FIG. 2A shows PL spectra of TPE-Bacin THF and THF/DMSO mixtures with different THF fractions (f_THF) . Concentration: 50 μM； excitation wavelength: 405 nm.

FIG. 2B shows a plot of relative PL intensity (I/I₀) versus the composition of the THF/DMSO mixture of TPE-Bac. Inset image: photographs of THF/DMSO mixtures of TPE-Bac with f_THF of 0％and 100％taken under 365 nm UV irradiation.

FIG. 3A shows PL spectra of aqueous solutions of TPE-Bacwith different concentrations. Excitation wavelength: 405 nm.

FIG. 3B shows a plot of PL intensity versus the solution concentration.

FIG. 4shows particle size of aggregates of TPE-Bac formed in aqueous solution. Concentration: 0.4 mM. Inset SEM image of the particles.

FIG. 5A shows PL spectra of mixtures of SOSG (5 μM) and TPE-Bac (5 μM) after normal light irradiation for different times. Excitation wavelength: 505 nm.

FIG. 5B shows a plot of relative PL intensity (I/I₀) at 530 nm versus the irradiation time. [SOSG] ＝ 5 μM and [TPE-Bac] ＝ 5 μM. Excitation wavelength: 505 nm.

FIG. 6A shows a bright field of S. epidermidis incubated with 10 μM of TPE-Bac for 10 min. Excitation wavelength: 460-490 nm.

FIG. 6B shows fluorescence images of S. epidermidis incubated with 10 μM of TPE-Bac for 10 min. Excitation wavelength: 460-490 nm.

FIG. 6C shows bright field images of E. coli incubated with 10 μM of TPE-Bac for 10 min. Excitation wavelength: 460-490 nm.

FIG. 6D shows fluorescence images of E. coli incubated with 10 μM of TPE-Bac for 10 min. Excitation wavelength: 460-490 nm.

FIG. 7A shows bright-field images of S. epidermidis with 10 μM TPE-Bac for 10 min followed by normal light exposurefor 10 min, and staining with 1.5 μM PI for 10 min. Excitation wavelength: 510-550 nm.

FIG. 7B shows fluorescence images of S. epidermidis with 10 μM TPE-Bac for 10 min followed by normal light exposurefor 10 min, and staining with 1.5 μM PI for 10 min. Excitation wavelength: 510-550 nm.

FIG. 7C shows bright-field images of S. epidermidis incubated in the dark for 10 min followed by normal light exposurefor 10 min, and staining with 1.5 μM PI for 10 min. Excitation wavelength: 510-550 nm.

FIG. 7D shows fluorescence images of S. epidermidis incubated in the dark for 10 min followed by normal light exposurefor 10 min, and staining with 1.5 μM PI for 10 min. Excitation wavelength: 510-550 nm.

FIG. 7E shows bright-field images of E. coli incubated with 10 μM TPE-Bac for 10 min followed by normal light exposurefor 10 min, and staining with 1.5 μM PI for 10 min. Excitation wavelength: 510-550 nm.

FIG. 7F shows fluorescence images of E. coli incubated with 10 μM TPE-Bac for 10 min followed by normal light exposurefor 10 min, and staining with 1.5 μM PI for 10 min. Excitation wavelength: 510-550 nm.

FIG. 7G shows bright-field images of E. coli incubated in the dark for 10 min followed by normal light exposurefor 10 min, and staining with 1.5 μM PI for 10 min. Excitation wavelength: 510-550 nm.

FIG. 7H shows fluorescence images of E. coli incubated in the dark for 10 min followed by normal light exposurefor 10 min, and staining with 1.5 μM PI for 10 min. Excitation wavelength: 510-550 nm.

FIG. 8 shows bacteria viability evaluated with plate count method. The bacteria were irradiated with normal light for 1 h prior to quantification.

FIG. 9 shows killing efficiency of TPE-Bac on E. coli and S. epiderimidis in the absence and presence of normal light irradiation for different times. The bacteria were incubated with 10 μM of TPE-Bac for 10 min prior to light irradiation.

FIG. 10A shows plates of E. coli incubated in the dark for 1 h in the absence of TPE-Bac, followed by further incubation for 24 h.

FIG. 10B shows plates of E. coli with light irradiation for 1 h in the absence of TPE-Bac, followed by further incubation for 24 h.

FIG. 10C shows plates of E. coli treated with 10 μM of TPE-Bac for 10 min, followed by storage in dark for 1 h and then further incubated for 24 h.

FIG. 10D shows plates E. coli treated with 10 μM of TPE-Bac for 10 min, followed by irradiation with normal light for 1 h then further incubated for 24 h.

]FIG. 11A shows plates of S. epidermidis incubated in the dark for 1 h in the absence of TPE-Bac, followed by further incubation for 24 h.

FIG. 11B shows plates of S. epidermidis with light irradiation for 1 h in the absence of TPE-Bac, followed by further incubation for 24 h.

FIG. 11C shows plates of S. epidermidis treated with 10 μM of TPE-Bac for 10 min, followed by storage in dark for 1 h and then further incubated for 24 h.

FIG. 11D shows plates of S. epidermidis treated with 10 μM of TPE-Bac for 10 min, followed by irradiation with normal light for 1 h then further incubated for 24 h.

FIG. 12A shows SEM images of S. epidermidis incubated in the dark for 10 min. The bacteria were then stored in dark for another 1 h.

FIG. 12B shows SEM images of S. epidermidis with 10 μM of TPE-Bac for 10 min. The bacteria were then exposed to normal light for 1 h.

FIG. 12C shows SEM images of E. coli incubated in the dark for 10 min. The bacteria were then stored in dark for another 1 h.

FIG. 12D shows SEM images of E. coli incubated with 10 μM of TPE-Bac for 10 min. The bacteria were then exposed to normal light for 1 h.

FIG. 13A shows agar plates sprayed with S. epidermidis followed by storage in dark for 1 h and then incubation for 24 h for successive 6 times. First time.

FIG. 13B shows agar plates sprayed with S. epidermidis followed by storage in dark for 1 h and then incubation for 24 h for successive 6 times. Second time.

FIG. 13C shows agar plates sprayed with S. epidermidis followed by storage in dark for 1 h and then incubation for 24 h for successive 6 times. Third time.

FIG. 13D shows agar plates sprayed with S. epidermidis followed by storage in dark for 1 h and then incubation for 24 h for successive 6 times. Fourth time.

FIG. 13E shows agar plates sprayed with S. epidermidis followed by storage in dark for 1 h and then incubation for 24 h for successive 6 times. Fifth time.

FIG. 13F shows agar plates sprayed with S. epidermidis followed by storage in dark for 1 h and then incubation for 24 h for successive 6 times. Control group.

FIG. 14A shows agar plates containing 10 μM of TPE-Bac sprayed with bacteria. The plates were first sprayed with S. epidermidis irradiated with normal light for 1 h, and then incubated for 24 h. First cycle.

FIG. 14B shows agar plates containing 10 μM of TPE-Bac sprayed with bacteria. The plates were first sprayed with S. epidermidis irradiated with normal light for 1 h, and then incubated for 24 h. Second cycle.

FIG. 14C shows agar plates containing 10 μM of TPE-Bac sprayed with bacteria. The plates were first sprayed with S. epidermidis irradiated with normal light for 1 h, and then incubated for 24 h. Third cycle.

FIG. 14D shows agar plates containing 10 μM of TPE-Bac sprayed with bacteria. The plates were first sprayed with S. epidermidis irradiated with normal light for 1 h, and then incubated for 24 h. Fourth cycle.

FIG. 14E shows agar plates containing 10 μM of TPE-Bac sprayed with bacteria. The plates were first sprayed with S. epidermidis irradiated with normal light for 1 h, and then incubated for 24 h. Fifth cycle.

FIG. 14F shows agar plates containing 10 μM of TPE-Bac sprayed with bacteria. The plates were first sprayed with S. epidermidis irradiated followed by storage in the dark for 1 h, and then incubated for 24 h.

FIG. 15A shows agar plates containing 10 μM of TPE-Bac sprayed with S. epidermidis. The plates were first irradiated with normal light for 1 h, sprayed with bacteria and then incubated for 24 h.

FIG. 15B shows agar plates containing 10 μM of TPE-Bac sprayed with S. epidermidis. The plates were first stored in the dark for 1 h, sprayed with bacteria and then incubated for 24 h.

FIG. 16 shows the strategy for antibiotic screening.

FIG. 17 shows UV spectra of water solution of TPE-Bac1 4×10^-5 M.

FIG. 18A shows PL spectra of TPE-Bac1in H₂O/DMSO mixtures with different water fractions (f_w) . Concentration: 50 μM； excitation wavelength: 405 nm

FIG. 18B shows a plot of PL intensity versus the composition of the H₂O/DMSO mixtures of TPE-Bac1 (50 μM) . Inset: Photographs of H₂O/DMSO mixtures of TPE-Bac1with different water fractions taken under 365 nm UV irradiation at concentrations of 50 μM.

FIG. 19A shows PL spectra of TPE-Bac1in water with different concentrations. Excitation wavelength: 405 nm.

FIG. 19B shows a plot of PL intensity versus the concentrations of TPE-Bac1； inset: photographs of TPE-Bac1 with different concentrations taken under handheld UV irradiation

FIG. 20 shows particle size measured by Zeta potential particle size analyzer of 0.4 mM TPE-Bac1 in aqueous solution. Inset: TEM image of the particles formed.

FIG. 21A shows a bright field image of S. epidermidis incubated with 10 μM of TPE-Bac1 for 10 min. Excitation wavelength: 330-385 nm.

FIG. 21B shows fluorescence images of S. epidermidis incubated with 10 μM of TPE-Bac1 for 10 min. Excitation wavelength: 330-385 nm.

FIG. 21C shows bright field images of E. coli incubated with 10 μM of TPE-Bac1 for 10 min. Excitation wavelength: 330-385 nm.

FIG. 21D shows fluorescence images of E. coli incubated with 10 μM of TPE-Bac1 for 10 min. Excitation wavelength: 330-385 nm.

FIG. 22 shows a change in PL intensity of TPE-Bac1with EtOH fraction in the presence/absence of 10⁸ CFU/mL S. epidermidis. Excitation wavelength: 430 nm

FIG. 23 shows PL spectra of MOPS/EtOH (v/v, 8/2) mixture of TPE-Bac1 with/without 10⁸ CFU/mL S. epidermidis. Excitation wavelength: 430 nm.

FIG. 24 shows a change in PL intensity of TPE-Bac1 with the concentration of S. epidermidis in MOPS/EtOH (v/v, 8/2) mixture. Excitation wavelength: 430 nm.

FIG. 25 shows evaluation of ampicillin effectiveness on S. epidermidis. S. epidermidis was firstly incubated with different concentrations of ampicillin, followed by quantification with TPE-Bac1 in MOPS/EtOH (v/v, 8/2) mixture. Excitation wavelength: 430 nm.

FIG. 26 shows evaluation of the effectiveness of different antibiotics on S. epidermidis. S. epidermidis was firstly incubated with different concentrations of different antibiotics, followed by quantification with TPE-Bac1 in MOPS/EtOH (v/v, 8/2) mixture. Excitation wavelength: 430 nm.

FIG. 27 shows the UV spectrum of TPE-Bac2 in ethanol. Concentration: 10 μM.

FIG. 28Ashows PL spectra of TPE-Bac2 in ethanol/hexane mixtures with different hexane fractions (f_h) . Concentration: 10 μM； excitation wavelength: 355 nm.

FIG. 28B showsaplot of relative PL intensity (I/I₀) of TPE-Bac2 at 502 nm versus the composition of the ethanol/hexane mixtures of TPE-Bac2. I₀ ＝ PL intensity in pure hexane solution (f_h ＝ 0) . Inset: fluorescent photo of ethanol/hexane mixtures of TPE-Bac2 at f_h ＝ 0 and 99 vol％taken under 365 nm UV illumination from a hand-held UV lamp.

FIG. 29 showsfluorescent photos of ethanol/hexane mixtures of TPE-Bac2 with different hexane fractions (f_h) taken under 365 nm UV illumination from a hand-held UV lamp.

FIG. 30 shows the size distributions of nanoaggregates of TPE-Bac2 and in ethanol/hexane mixtures with 90％hexane fraction. Concentration: 10 μM.

FIG. 31A shows the bright-field image of E. coli.

FIG. 31B shows the fluorescence image of E. coli incubated with 10 μMTPE-Bac2 for 2 h.

DETAILED DESCRIPTION

Definitions

The following definitions are provided for the purpose of understanding the present subject matter and for constructing the appended patent claims.

It is noted that, as used in this specification and the appended claims, the singular forms “a, ” “an” and “the” include plural references unless the context clearly dictates otherwise.

“Aggregation-induced emission” means the fluorescence/phosphorescence is turned on upon aggregation formation or in the solid state. When molecularly dissolved, the material is nonemissive. However, the emission is turned on when the intramolecular rotation is restricted.

“Emission intensity” means the magnitude of fluorescence/phosphorescence normally obtained from a fluorescence spectrometer or fluorescence microscopy measurement.

“Luminogen” means a molecule which exhibits luminescence.

“Chromophore” means the part of a molecule responsible for its color.

“Fluorophore” means a molecule which exhibits fluorescence.

Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges, percentage ranges, or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the described subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use “comprising” language； however, it will be understood by one of skill in the art, that in some specific instances, an embodiment can alternatively be described using the language “consisting essentially of” or “consisting of. ”

For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about. ” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Abbreviations

AIE: aggregation-induced emission

CFU: colony forming unit

CMC: critical micelle concentration

DCM: dichloromethane

DMF: dimethylformamide

DMSO: dimethylsulfoxide

DNA: deoxyribonucleic acid

EtOH: ethyl alcohol

FL: fluorescence

HRMS: high-resolution mass spectroscopy

LB: luria broth

MALDI-TOF: matrix assisted laser desorption ionization time-of-flight

MOPS: 3- (N-morpholino) propanesulfonic acid

NMR: nuclear magnetic resonance

PDI: polydispersity index

PDT: photodynamic therapy

PI: propidium iodide

PL: photoluminescence

PBS: phosphate-buffered saline

ROS: reactive oxygen species

SEM: scanning electron microscopy

SOSG: singlet oxygen sensor green

TEM: transmission electron microscopy

THF: tetrahydrofuran

TPE: tetraphenylethene

TPE-Bac: 4- (2- (4'- (1-phenyl-2, 2-bis (4- (undecyloxy) phenyl) vinyl) - [1, 1'-biphenyl] -4-

yl) vinyl) -1- (3- (trimethylammonio) propyl) pyridin-1-ium bromide

TPE-Bac1: 4- ( (1E) -2- (4'- (1, 2-diphenyl-2- (4- (undecyloxy) phenyl) vinyl) - [1, 1'-

biphenyl] -4-yl) vinyl) -1- (3- (trimethylammonio) propyl) pyridin-1-ium

bromide

TPE-Bac2: 1, 2-bis {9, 9-bis [6- (N, N, N-trimethylammonium) hexyl] -2-fluorenyl} -1, 2-

diphenylethene tetrabromide

UV: ultraviolet

wherein

wherein

In an embodiment, the fluorescent molecules are applied to bacteria imaging. The emission of fluorescent molecules can be turned on both the Gram positive and negative bacteria. Due to the AIE characteristics and water solubility of these molecules, no washing procedure is needed due to the weak background emission. The chromophores generate ROS upon light irradiation, enabling them to be used for both mammalian cell and bacteria therapy. In the presence of normal light, the bacteria and mammalian cell are killed efficiently. The agar plates containing the AIE materials may be reused several times for bacteria killing.

In an embodiment, the AIE materials are applied to high throughput antibiotic screening studies. In the presence of effective antibiotics, bacteria growth will be inhibited. In the presence of ineffective antibiotics, however, the bacteria will grow rapidly. The bacteria “turns on” the emission of AIE materials, and based on the emission intensity, the antibiotic effect may be evaluated.

One exemplary fluorescent molecule according to the present subject matter is TPE-Bac. In an embodiment, one synthetic route to prepare TPE-Bac is shown below:

Through the cross McMurry coupling reaction between 4, 4’ -dihydroxybenzophone (1) and 4-bromobenzophenone (2) , the TPE core (3) was built. Following Williamson ether synthesis, TPE with two undecane groups (4) was obtained. Suzuki coupling reaction with 4-bromobenzaldehyde (5) readily gives 6 in high yield. TPE-Bac was generated by the aldol condensation between a double-charged pyridinium salt (7) and TPE with aldehyde functionality (6). All the intermediates were obtained in medium to high yield. ¹NMR, ¹³NMR and high resolution mass (HRMS) were utilized to characterize the intermediates and TPE-Bac, and satisfactory results corresponding to their structures were obtained.

In an embodiment, TPE, which is constituted by aromatic rings, is hydrophobic in nature. The double charged pyridinium salt group renders TPE with good hydrophilicity, making TPE-Bac readily soluble in polar solvents such as DMSO, methanol, and ethanol. In non-polar solvent such as THF, hexane, and chloroform, TPE-Bac is insoluble. TPE-Bac may dissolve in water at low concentrations, but forms micelles at higher concentrations.

In an embodiment, when dissolved in DMSO, TPE-Bac shows an absorption maximum at 428 nm, (FIG. 1) around 100 nm red-shifted compared with TPE alone. The pyridinium salt group is a strong electron-withdrawing group, while the ether groups are medium electron-donating groups. The D-Ainteraction across the TPE core may facilitate the electron movement and lower the energy gap, leading to the red-shifted absorption maximum. The tail of the absorption peak extends to around 550 nm, which almost covers the full UV, blue, and green light regions, making TPE-Bac a good candidate for photodynamic therapy.

In an embodiment, as shown in FIG. 2, the PL spectrum of TPE-Bac in DMSO shows a small emission peak centered around 520 nm. The PL spectrum remained unchanged with the gradual increase in THF fraction. The THF solution of TPE-Bac, however, shows a distinctively different emission profile: a strong emission peak at 641 nm is easily identified from the PL spectrum. The emission intensity of TPE-Bac in THF solution was more than 53 times higher than its DMSO solution, and the difference is easily distinguished by naked eyes when irradiated with a hand-held UV lamp (as shown in the inset picture of FIG. 2B) . The results clearly demonstrate TPE-Bac is AIE-active, and decorating TPE with the water-soluble functional group does not change the AIE characteristics of TPE-Bac.

In an embodiment, the long alkyl chains and the double charged pyridinium salt add to the hydrophobicity and hydrophilicity of TPE, respectively, making TPE-Bac an amphiphilic molecule. TPE-Bac may be dissolved in water at low concentrations. At high concentrations, however, micelle-formation takes place. Taking advantage of the enhanced emission of TPE-Bac upon aggregation, the critical micelle concentration (CMC) is easily determined by following the fluorescence changes. When the concentration is below CMC, TPE-Bac will molecularly present in solvent and no emission could be observed. When the concentration is approaching CMC, the intermolecular interaction will take place to restrict intramolecular motion, thus turning on the emission of TPE-Bac. As shown in FIG. 3, when the concentration of TPE-Bac is below 0.001 mM, increasing concentration did not influence the PL intensity of TPE-Bac much. However, the intensity increased dramatically when the concentration of TPE-Bac reached 0.001 mM. Plotting the PL intensity versus the dye concentration generates two lines, the intersection of which determines the CMC to be 0.01 mM.

In an embodiment, the formation of nanoaggregates at a concentration higher than CMC was verified by the Zeta potential particle size analyzer (FIG. 4) . Particles with effective diameters of 219 nm may be detected with PDI of 0.278. The presence of nanoaggregates is further proved with the TEM measurement. As shown in the inset image of FIG. 4, black spots with the size of 100-200 nm are clearly observed in the TEM image.

In an embodiment, TPE-Bac is different in that it has two longer alkyl chains, but the chromophore is the same. There should not be too much difference in the ROS generation. To verify this hypothesis, singlet oxygen sensor green (SOSG) was utilized for detection of the singlet oxygen generation, which is among the ROS generated. SOSG is non-emissive, but oxidation by the singlet oxygen turns on its emission at around 530 nm. As show in FIG. 5, irradiating SOSG alone or TPE-Bac alone with normal light did not change the fluorescence intensity very much. However, in the presence of both SOSG and TPE-Bac, with the increase in irradiation time, the intensity at 530 nm increased accordingly with the irradiation time, indicating the generation of singlet oxygen.

In an embodiment, TPE-Bac is applied to bacteria imaging. As shown in FIG. 6, after 10 min incubation with 10 μM of TPE-Bac, the bacteria is clearly imaged by TPE-Bac. Thanks to the AIE characteristics and water solubility of TPE-Bac, without a washing procedure, the background emission from TPE-Bac is still very low. This may simplify the imaging process and decrease the loss of bacteria during the washing process.

In an embodiment, the killing effect of TPE-Bac on both Gram positive and negative bacteria was tested with the aid of propidium iodide (PI) . PI is a cell membrane impermeable fluorescent bioprobe. For living bacteria, the cell membrane is intact and PI could not enter the bacteria, thus the living bacteria are unstained. For dead bacteria, however, the damaged cell membrane will open the access for PI to approach its DNA, thus selectively lighting up the dead bacteria. The bacteria were first incubated with TPE-Bac for 10 min. To exclude the interference of TPE-Bac in the solution, the bacteria were washed by centrifuging and removal of the supernatant.

Afterwards, the bacteria were dispersed again in PBS solution, followed by normal light irradiation for 10 min and staining with PI. For comparison, a control group was also included, in which the bacteria were only exposed to normal light, but without incubation with TPE-Bac. As shown in FIG. 7 the Gram Positive bacteria S. epidermidis agglomerated together after treatment with both TPE-Bac and the light. This may be because these treatments may destroy the integrity of the bacteria cell membrane and expose the hydrophobic part of the membrane, which may lead to the aggregation of the bacteria.

After PI staining, red emission is clearly observed from the bacteria, indicating the bacteria membrane is destroyed. In the control group, however, no agglomeration of PI emission is observed. The killing effect on Gram negative bacteria is not as extensive when compared with the killing effect on Gram positive bacteria. As shown in FIG. 7, the change in bacteria morphology is also observed, but relatively low PI emission from the bacteria was observed because Gram negative bacteria has another outer membrane, which serves as an additional protecting layer to the bacteria.

In an embodiment, the killing efficiency of TPE-Bac with the plate-count methods was evaluated. Before studying the effect of TPE-Bac, the effect of light alone was investigated. As shown in FIG. 8, after 1 h irradiation, both E. coli and S. epidermidis remained healthy and no obvious decrease in bacteria viability was observed. Then, the effect of TPE-Bac was incorporated.

In anembodiment, as shown in FIG. 9, when E. coli were treated with TPE-Bac and then stored in the dark for 10 min, the bacteria viability decreased to around 70％, demonstrating the dark toxicity of TPE-Bac towards E. coli. Further increasing the storage time in the dark did not exert further killing effect on the bacteria. For S. epidermidis, without light irradiation, the bacteria viability was around 50％. Light irradiation may significantly increase the killing efficiency on both Gram positive and negative bacteria. The viability of TPE-Bac treated Gram positive and negative bacteria were 40％and 45％, respectively, after 10 min of normal light irradiation, both of which dropped below 10％after 30 min of light irradiation and less than 1％after 1 h of normal light irradiation.

In an embodiment, to gain a direct impression of the killing effect, the images of plates for the quantification of the killing effect on E. coliare shown in FIG. 10. Without treatment, the bacteria grew healthily on the plate. Light irradiation alone does not have an obvious effect on bacteria viability (FIG. 10B) , and both the size and number of coloniesare similar to that in FIG. 10A. Treatment with TPE-Bac alone decreased E. coli to some extent, but still there are many colonies on the plate (FIG. 10C) , suggesting the inefficient killing of TPE-Bac on E. coli in the dark. In the presence of both TPE-Bac and light irradiation, E. coli was killed effectively and almost no colonies existed on the plate, a good sign that almost all the bacteria was killed. Similar effects could be observed on the S. epidermidis (FIG. 11) . These results strongly proved that together with light irradiation, TPE-Bac may kill both Gram positive and Gram negative bacteria effectively.

In an embodiment, bacteria viability changes may also followed by tracking morphological changes. Both E. coli and S. epidermidis were treated with TPE-Bac and irradiated with normal light for 1 h, followed by drying, and imaging under SEM. As shown in FIG. 12, without treatment (FIG. 12A and 12C) , the morphology of both Gram positive and negative bacteria are quite regular. Even when the bacteria overlap, a clear border is resolved, clearly indicating the healthy state of the bacteria. After treatment (FIG. 12B and 12D) , however, the bacteria shrunk and fusion took place, making the morphology completely different from FIG. 12A and 12C. The SEM results clearly demonstrated the presence of TPE-Bac and light irradiation together may lead to the morphology change and lead to bacteria death.

In an embodiment, the performance of TPE-Bac for bacteria killing encouraged further investigation of the reusability in bacterial killing. To simply the experiment process, S.epidermidis was utilized for the demonstration. TPE-Bac was added into an agar plate, S. epidermidis was then sprayed onto the agar plate, followed by normal light irradiation for 1 h and further culture in the 37℃ incubator for 24 h to allow the bacteria to grow. In the control group (FIG. 13A) , after 24 h of incubation, bacteria grew into small colonies. In the experimental group (FIG. 14A) , however, no colonies were observed. The experiment plate was then sprayed with new bacteria to repeat the process. After four times of sprayingthe bacteria, still no colony formation was observed. In the fifth time, the amount of S. epidermidis was doubled and still no bacteria grew on the plate. Note that five times is not the limit of inhibiting bacteria growth. For the agar plate with only TPE-Bac, S. epidermidis grew to form colonies (FIG. 14G) , even though the amount was less than the one without any treatment. Interestingly, if the plate containing TPE-Bac was first irradiated and then bacteria were sprayed on the plate, there was still no colony formation on the plate after 24 h of incubation (FIG. 15) . As such, some toxics to the bacteria are generated by the normal light-induced ROS generation, which may still kill the bacteria.

In an embodiment, materials with AIE characteristics are applied to high throughput antibiotics screening. The working mechanism of the detection is shown in FIG. 16. In the presence of ineffective antibiotics, bacteria growth will not be inhibited, and the concentration of bacteria will be very high. In the presence of effective antibiotics, however, the bacteria growth will be inhibited and the concentration of bacteria will be very low. After incubation with AIE materials, the concentration of bacteria will be in a linear relationship with the fluorescence intensity. Based on the fluorescence intensity, the effectiveness of antibiotics could be determined.

In an embodiment, TPE-Bac1 was selected as the material for antibiotic screening. The structure of TPE-Bac1 is shown below:

]In an embodiment, as shown in FIG. 17, when dissolved in water, TPE-Bac1 shows an absorption maximum at 400 nm, which is around 100 nm red-shifted compared with TPE alone, similar to TPE-Bac.

In an embodiment, as shown in FIG. 18, the PL spectrum of TPE-Bac1 in DMSO is basically non-emissive. The PL spectrum remained unchanged with the gradual increase in water fraction. When the water fraction increased to 40％, the PL intensity increased accordingly with the water fraction. A strong emission peak at 577 nm is easily identified from the PL spectrum. The difference in fluorescence intensity of the water solution and DMSO solution is easily distinguished by naked eyes when irradiated with a hand-held UV lamp (as shown in the inset picture of FIG. 18B) . The results clearly demonstrate TPE-Bac1 is AIE-active. Furthermore, decorating TPE with the water-soluble functional group does not change the AIE characteristics of TPE-Bac1.

In an embodiment, TPE-Bac1 is dissolved in water at low concentrations. However, at high concentrations, micelle-formation takes place. Taking advantage of the enhanced emission of TPE-Bac1 upon aggregation, the critical micelle concentration (CMC) is easily determined by following the fluorescence changes. When the concentration is below CMC, TPE-Bac1 will molecularly present in solvent and no emission is observed. When the concentration is approaching CMC, the intermolecular interaction will take place to restrict intramolecular motion, thus turning on the emission of TPE-Bac1. As shown in FIG. 19, when the concentration of TPE-Bac1 is below 0.001 mM, increasing concentration does not influence the PL intensity of TPE-Bac1 very much. The intensity increased dramatically when the concentration of TPE-Bac1 reached 0.001 mM. Plotting the PL intensity versus the dye concentration generates two lines, the intersection of which determines the CMC to be 0.02 mM.

In an embodiment, the formation of nanoaggregates at a concentration higher than the CMC was verified by the Zeta potential particle size analyzer (FIG. 20) . Particles with effective diameters of 240 nm are detected with PDI of 0.202. The presence of nanoaggregates was further proved with the TEM measurement. As shown in the inset image of FIG. 20, black spots with the size of around 100 nm are clearly observed in the SEM image.

In an embodiment, TPE-Bac1 isapplied to bacteria imaging. As shown in FIG. 21, after 10 min incubation with 10 μM of TPE-Bac1, the bacteria is clearly imaged by TPE-Bac1. Thanks to the AIE characteristics and water solubility of TPE-Bac, and without a washing procedure, the background emission from TPE-Bac is still very low. This may simplify the imaging process and decrease the loss of bacteria during the washing process.

In an embodiment, ethanol was added to increase the solubility of TPE-Bac1 and reduce the background emission in the mixture (FIG. 22) . Different fractions of ethanol were tested and 20％was determined to be the optimal fraction with a high signal-to-noise ratio and stable signal.

In an embodiment, the PL spectra of MOPS/ethanol mixtures (v/v, 8/2) without and with 10⁸ CFU/mL S. epidermidis was evaluated (FIG. 23) . In the presence of bacteria, the emission is around 14 times higher than that without the bacteria. The difference is easily identified by naked eyes, as shown in the inset image of FIG. 23.

In an embodiment, the standard curve of PL intensity versus S. epidermidis concentration was collected. As shown in FIG. 24, with the increase in S. epidermidis concentration, the fluorescence intensity increased linearly, indicative of the workability of using TPE-Bac1 for antibiotics screening.

In an embodiment, the effect of ampicillin on S. epidermidis was evaluated utilizing this method. As shown in FIG. 25, without ampicillin, the fluorescence intensity of TPE-Bac1 was very high, due to the high concentration of bacteria. With the increasing concentration of ampicillin, the fluorescence intensity dropped dramatically, indicating effective antibiotics inhibit S. epidermidis, thus decreasing fluorescence intensity. From this curve, the IC₅₀is determined to be less than 1μg/mL, indicating ampicillin works effectivelyto inhibit S.epidermidis growth.

In an embodiment, more antibiotics were tested with TPE-Bac1. As shown in FIG. 26, these antibiotics inhibit the growth of S. epidermidis to different extents. From the curve, ampicillin, kanamycin, colistin, streptomycin are very effective antibiotics with IC₅₀ lower than 1μg/mL, while stectinomycin is less effective, and gramicidin is ineffective.

In an embodiment, TPE-Bac2 was used for bacteria imaging. The structure of TPE-Bac2 is shown below:

In an embodiment, compound 9 was synthesized by Friedel–Crafts acylation of fluorene (8) and benzoyl chloride in the presence of AlCl₃ as catalyst. It was then coupled with 1, 6-dibromoheaxane in basic solution to afford 10. Compound 11 was subsequently synthesized by McMurry coupling of 8 catalyzed by TiCl₄ and Zn. Treatment of 11 with trimethylamine finally furnished the desirable product TPE-Bac2 in 83％yield. In an embodiment, one synthetic route to prepare TPE-Bac2 is shown below:

In an embodiment, TPE-Bac2 absorbs at 280 and 350 nm in the UV spectra (FIG. 27) .

In an embodiment, TPE-Bac2 is AIE active: its ethanol solution gives almost no light upon photoexcitation, but its nano-aggregates in the ethanol–hexane mixture with 80％hexane content are strong green emitters (FIG. 28A-B) .

In an embodiment, the different in fluorescence intensity in ethanol/hexane with different hexane fractions can be easily differentiated by naked eyes under UV lamp irradiation (FIG. 29) .

In an embodiment, DLS analysis reveals TPE-Bac2 forms nanoparticlesin ethanol/hexane mixtures with 90％hexane fraction (FIG. 30) .

In an embodiment, TPE-Bac2 may stain bacteria after incubation (FIG. 31A-B) .

EXAMPLES

The following examples are illustrative of the presently described subject matter and are not intended to be limitations thereon.

Materials:

LB agar, LB broth, potassium phosphate dibasic anhydrous, and sodium phosphate were purchased from USB Co., while Singlet Oxygen Senor Green (SOSG) was purchased from Invitrogen. Zinc dust, titanium tetrachloride, 4-hydroxy benzophenone, 4-bromobenzophenone, 1-bromoundecane, piperidine, propidium iodide, potassium carbonate, 4-formylphenylboronic acid, and tetrakis (triphenylphosphine) palladiumwerepurchased from Sigma-Aldrich and used as received. THF was purified by distillation from sodium benzophenone ketyl immediately prior to use. 1- (3-Trimethylammoniopropyl) -4-methylpyridinium dibromide was synthesized according to the Yan, et al. (P. Yan, A. Xie, M. Wei, L. M. Loew, J. Org. Chem. 2008, 73, 6587) method. Other reagents used, such as dimethyl sulfoxide, potassium chloride, and sodium chloride were purchased from Sigma-Aldrich.

Characterization:

¹H and ¹³C NMR spectra were measured on Bruker ARX 400 NMR spectrometers using CDCl₃ and methanol-d₄ as the deuterated solvent. High-resolution mass spectra (HRMS) were recorded on a Finnigan MAT TSQ 7000 Mass Spectrometer System operating in a MALDI-TOF mode. UV absorption spectra were taken on a Milton Ray Spectronic 3000 array spectrophotometer. Steady-state fluorescence spectra were recorded on a Perkin Elmer LS 55 spectrometer. Fluorescence images were collected on an Olympus BX 41 fluorescence microscope. Particle sizes were measured on a Zeta potential analyzer (Brookhaven, ZETAPLUS) . The aggregation morphology of TPE-Bacwas investigated using Transmission Electron Microscopy (Japan, JEOL JEM 100CXII) at an accelerating voltage of 100 kV.

Synthesis:

4, 4'- (2- (4-bromophenyl) -2-phenylethene-1, 1-diyl) diphenol (3)

3 was synthesized following the procedure reported in Duan, et al. (Duan, X. -F. ； Zeng, J. ； Lue, J. -W. ； Zhang, Z. -B. A Facile Synthesis of Tetraarylethenes via Cross McMurry Coupling between Diaryl Ketones. Synthesis2007, 5, 713–718) , with some modifications. 4, 4’-dihydroxybenzophenone (3.21 g, 15 mmol) , 4-bromobenzophenone (7.80 g, 30 mmol) , and zinc dust (8.82 g, 135 mmol) were added into a 500 mL two-necked round bottom flask. The flask was vacuumed and purged with nitrogen three times. Afterwards, 200 mL of THF was injected into the flask, followed by cooling down to-78℃ with acetone/dry ice bath. TiCl₄ (6.74 mL, 67.5 mmol) was added into the mixture dropwise. The reaction was then refluxed overnight under nitrogen conditions. After cooling to room temperature, hydrochloric acid (1 M) was added to the reaction mixture to adjust the pH to 2. The organic mixture was extracted with DCM and dried with anhydrous sodium sulfate. The crude product was purified by silica column chromatography, using hexane and ethyl acetate (v/v, 5/2) as elusion solution to give 1 as white solid (4.32 g, 65％) . ¹H NMR (400 MHz, DMSO-d₆, δ) : 9.43 (s, 2H) , 7.30-7.24 (d, 2H) , 7.14-7.02 (m, 4H) , 6.92-6.88 (d, 2H) , 6.85-6.81 (d, 1H) , 6.75-6.68 (m, 4H) ； ¹³C NMR (100 MHz, DMSO-d₆, δ) : 155.869, 155.777, 143.953, 143.467, 143.300, 141.143, 136.233, 133.660, 133.601, 132.701, 131.887, 131.816, 130.564, 130.504, 129.223, 127.694, 127.549, 126.005, 118.871, 114.878, 114.539, 114.376； HRMS (MALDI-TOF) m/z: calcd, 442.0568 [M] ； found, 442.0099 [M] ⁺.

4, 4'- (2- (4-bromophenyl) -2-phenylethene-1, 1-diyl) bis ( (undecyloxy) benzene) (4)

Into a 250 mL two-necked round bottom flask was added potassium carbonate (4.00 g, 28.25 mmol) and 3 (2.5 g, 5.65 mmol) . The flask was vacuumed and purged with dry nitrogen three times. After adding 1-bromoundecane (5.31 mL, 22.6 mmol) and DMF (80 mL) , the reaction was stirred overnight under nitrogen condition at 70℃. After cooling down to the room temperature, the mixture was extracted with DCM, washed with distilled water several times and dried with anhydrous magnesium sulfate. The crude product was purified by running silica column chromatography using hexane and DCM (v/v, 10/1) as elusion solvent to give 4 as light yellow viscous oil (3.30 g, 78％) . ¹H NMR (400 MHz, CDCl₃, δ) : 7.24-7.20 (m, 2H) , 7.14-6.99 (m, 6H) , 6.94-6.87 (m, 5H) , 6.68-6.60 (m, 4H) , 3.92-3.84 (m, 4H) , 1.80-1.69 (m, 4H) , 1.47-1.24 (m, 32H) , 0.92-0.87 (t, 6H) ； ¹³C NMR (100 MHz, CDCl₃, δ) : 158.068, 157.981, 144.092, 143.608, 141.153, 137.843, 136.011, 135.926, 133.270, 132.755, 131.566, 131.033, 127.994, 127.847, 126.433, 120.140, 113.899, 113.717, 68.057, 68.007, 32.127, 29.829, 29.791, 29.639, 29.560, 29.511, 26.273, 22.910, 14.351； HRMS (MALDI-TOF) m/z: calcd, 750.4011 [M] ； found, 750.4021 [M] ⁺.

4'- (1-phenyl-2, 2-bis (4- (undecyloxy) phenyl) vinyl) - [1, 1'-biphenyl] -4-carbaldehyde (6)

Into a 250 mL two-necked round bottom flask was added 4 (2.00 g, 2.60 mmol) , 4-formylphenylboronic acid (5) (0.59 g, 3.96 mmol) , K₂CO₃ (2.28 g, 16.50 mmol) and Pd (PPh₃) ₄ (0.23 g, 0.20 mmol) . The flask was vacuumed and purged with nitrogen three times. Afterwards, THF (80 mL) and water (20 mL) were injected into the flask, followed by refluxing overnight under nitrogen condition. After cooling down to the room temperature, the mixture was extracted with DCM, washed with distilled water several times and dried with anhydrous magnesium sulfate. The crude product was purified by running silica column chromatography using hexane and DCM (v/v, 5/1) as elusion solvent to give6 as yellow viscous oil (1.72 g, 85％) . ¹H NMR (400 MHz, CDCl₃, δ) : 10.03 (s, 1H) , 7.93-7.89 (d, 2H) , 7.74-7.70 (d, 2H) , 7.42-7.39 (d, 2H) , 7.16-7.05 (m, 7H) , 7.00-6.92 (m, 4H) , 6.68-6.62 (t, 4H) , 3.90-3.83 (t, 4H) , 1.78-1.70 (m, 4H) , 1.47-1.24 (m, 32H) , 0.92-0.87 (t, 6H) ； ¹³C NMR (100 MHz, CDCl₃, δ) : 191.240, 157.241, 157.155, 146.129, 144.338, 143.546, 140.432, 137.535, 136.140, 135.351, 134.342, 131.999, 131.967, 131.454, 130.824, 129.582, 127.160, 126.655, 125.880, 125.571, 113.032, 112.903, 67.222, 67.193, 31.280, 28.982, 28.943, 28.803, 28.715, 28.687, 25.439, 22.064, 13.501； HRMS (MALDI-TOF) m/z: calcd, 776.5168 [M] ； found, 776.5176 [M] ⁺.

4-(2- (4'- (1-phenyl-2, 2-bis (4- (undecyloxy) phenyl) vinyl) - [1, 1'-biphenyl] -4-yl) vinyl) -1- (3- (trimethylammonio) propyl) pyridin-1-ium bromide (TPE-Bac)

A solution of 1- (3-trimethylammoniopropyl) -4-methylpyridinium dibromide (7) (0.31 g, 0.40 mmol) and 3 (0.28 g, 0.79 mmol) was refluxed under nitrogen in dry ethanol catalyzed by three drops of piperidine. After cool to ambient temperature, the solvent was evaporated under reduced pressure. The residue was purified by a silica gel column chromatography using dichloromethane and methanol mixture (2: 1 v/v) as eluent to give a red powder TPE-Bac (0.24 g, 53％) . ¹H NMR (400 MHz, CDCl₃, δ) : 9.18 (s, 2H) , 8.01 (s, 2H) , 7.69 (d, 1H) , 7.47 (d, 2H) , 7.35 (d, 2H) , 7.17 (d, 2H) , 7.10-6.83 (m, 12H) , 6.55 (dd, 4H) , 4.90 (s, 2H) , 3.94-3.77 (m, 4H) , 3.77-3.68 (s, 2H) , 3.42 (s, 9H) , 2.77 (s, 2H) , 1.75-1.56 (m, 4H) , 1.45-1.14 (m, 32H) , 0.90-0.77 (m, 6H)； ¹³C NMR (100 MHz, CDCl₃, δ) : 157.160, 157.058, 152.857, 143.839, 143.592, 141.900, 140.753, 140.112, 137.596, 136.028, 135.497, 135.285, 132.764, 131.951, 131.356, 130.795, 128.403, 127.107, 126.416, 125.419, 123.627, 121.471, 112.998, 112.829, 67.096, 62.118, 53.823, 31.267, 31.214, 29.058, 28.980, 28.930, 28.890, 28.829, 28.727, 28.702, 28.648, 28.605, 25.448, 25.378, 22.040, 22.002, 13.487, 13.471； HRMS (MALDI-TOF) m/z: calcd, 1031.6024 [M-Br] ⁺； found, 1031.6024 [M-Br] ⁺.

2-fluorenylphenone (9)

Into a 100 mL two-necked round bottom flask, 8 (3.324 g, 20 mmol) and aluminum (III) chloride (2.667 g, 20 mmol) were dissolved in carbon disulfide (40 mL) under nitrogen atmosphere. The resulting mixture was cooled in an ice bath, after whichbenzoyl chloride (3.093 g, 22 mmol) was added dropwise into the mixture. After completed addition, the mixture was refluxed for 12 h. The mixture was cooled to room temperature and40 mL of water was added to quench the reaction. The solution was then extracted with DCM three times, washed with brine and water, and dried over anhydrous magnesium sulfate. After filtration and solvent evaporation under reduced pressure, the product was purified by silica-gel column chromatography using hexane/DCM as eluent to yield 9 as a white solid in 90％yield (4.86 g) . ¹H NMR (400 MHz, CDCl₃) , δ (TMS, ppm) : 8.01 (s, 1H) , 7.85–7.81 (m, 5H) , 7.61–7.57 (m, 2H) , 7.51–7.47 (m, 2H) , 7.43–7.35 (m, 2H) , 3.95 (s, 2H) . ¹³C NMR (100 MHz, CDCl₃) , δ (TMS, ppm) : 196.7, 145.9, 144.4, 143.0, 140.5, 138.1, 135.8, 132.1, 129.9, 129.6, 128.2, 127.9, 127.0, 126.8, 125.2, 120.8, 119.3, 36.8. HRMS (MALDI-TOF) : m/z 270.1036 (M⁺, calcd. 270.1045) .

9, 9-bis (6-bromohexyl) -2-fluorenylphenone (10)

Into a 100 mL round-bottom flask, with 50 mL of 50％NaOH solution were successively added tetrabutylammomium bromide (0.8 g) , 1, 6-dibromohexane (6 mL, 37.5 mmol) and 9 (2.027 g, 7.5 mmol) . The mixture was then heated at 75℃ for 12 h. After cool to room temperature, the mixture was extracted with DCMseveral times and the organic layers werecombined and washed with dilute hydrogen chloride, saturated brine and water, and dried over anhydrous magnesium sulfate. After filtration and solvent evaporation, the residue was purified by silica-gel column chromatography using hexane/DCM as eluent to yield 10 as yellow oil in 95％yield (4.24 g) . ¹H NMR (400 MHz, CDCl₃) , δ (TMS, ppm) : 7.84-7.81 (m, 3H) , 7.79-7.76 (m, 3H) , 7.62-7.58 (m, 1H) , 7.52-7.49 (m, 2H) , 7.38-7.37 (m, 3H) , 3.28-3.25 (m, 4H) , 2.03-1.98 (m, 4H) , 1.69-1.61 (m, 4H) , 1.23-1.15 (m, 4H) , 1.11-1.04 (m, 4H) , 0.68-0.60 (m, 4H) . ¹³C NMR (100 MHz, CDCl₃) , δ (TMS, ppm) : 196.7, 151.5, 150.3, 145.5, 139.8, 138.2, 136.0, 132.1, 130.1, 129.9, 128.4, 128.2, 127.2, 124.4, 122.9, 120.7, 119.2, 55.1, 39.9, 33.8, 32.5, 29.0, 27.7, 23.6. HRMS (MALDI-TOF) : m/z 597.0724 (M⁺, calcd. 596.1112) .

1, 2-bis [9, 9-bis (6-bromohexyl) -2-fluorenyl] -1, 2-diphenylethene (11)

To a solution of 10 (2.98 g, 5 mmol) , zinc dust (0.82 g, 12.5 mmol) in dry distilled THF was added dropwise of titanium (IV) chloride (1.3 mL, 12.5 mmol) under nitrogen at-78℃. The reaction mixture was warmed to room temperature and then heated to reflux for 12 h. After cooling to room temperature, the reaction was quenched by addition of hydrochloric acid solution. The mixture was then extracted with DCM several times. The organic layers were combined and washed with saturated brine solution and water, and dried over anhydrous magnesium sulfate. After filtration and solvent evaporation, the product was purified by silica-gel column chromatography using hexane/DCM as eluent to yield 11 as green oil in 82％yield (2.38 g) . ¹H NMR (400 MHz, CDCl₃) , δ (TMS, ppm) : 7.61-7.54 (m, 2H) , 7.49-7.40 (m, 2H) , 7.29-7.22 (m, 6H) , 7.13-7.00 (m, 14H) , 3.36-3.22 (m, 8H) , 1.90-1.49 (m, 16H) , 1.26-1.08 (m, 8H) , 1.06-0.85 (m, 8H) , 0.57-0.43 (m, 8H) . ¹³C NMR (100 MHz, CDCl₃) , δ (TMS, ppm) : 150.5, 149.6, 144.1, 143.3, 140.9, 140.8, 139.3, 131.5, 130.5, 127.6, 126.9, 126.8, 126.4, 126.2, 122.6, 119.6, 118.9, 40.0, 34.0, 32.6, 29.0, 27.8, 23.6. HRMS (MALDI-TOF) : m/z 1160.6270 (M⁺, calcd. 1160.2327) .

1, 2-bis {9, 9-bis [6- (N, N, N-trimethylammonium) hexyl] -2-fluorenyl} -1, 2- diphenylethene tetrabromide (TPE-Bac2)

To a solution of 11 (25 mg, 0.02 mmol) in THF (5 mL) was added dropwise trimethylamine (2 mL) at-78℃. The mixture was stirred for 12 h and then allowed to warm to room temperature. The precipitate was redissolved by addition of methanol (5 mL) . After the mixture was cooled to-78℃, additional trimethylamine (2 mL) was addedand the mixture was stirred at room temperature for 24 h. After solvent removal, water was added to redissolve all the precipitates and the aqueous solution was extracted with DCM. The aqueous layer was freeze dried to yield TPE-Bac2 as greenish powders in 83％yield (25 mg) . ¹H NMR (400 MHz, CD₃OD) , δ (TMS, ppm) : 7.69-7.64 (m, 2H) , 7.54-7.50 (m, 2H) , 7.39-7.34 (m, 2H) , 7.33-7.26 (m, 4H) , 7.16-6.95 (m, 14H) , 3.32-3.22 (m, 8H) , 3.11 (s, 36H) , 1.93-1.69 (m, 8H) , 1.63-1.52 (m, 8H) , 1.16-1.03 (m, 16H) , 0.59-0.45 (m, 8H) . ¹³C NMR (100 MHz, CD₃OD) , δ (TMS, ppm) : 152.1, 151.0, 145.5, 144.7, 142.5, 142.3, 140.9, 132.5, 131.6, 128.9, 128.1, 127.7, 124.2, 121.0, 120.4, 67.6, 60.5, 55.9, 53.7, 41.1, 30.4, 26.9, 23.9. HRMS (MALDI-TOF) : m/z1317.6105 [ (M–Br) ⁺, calcd. 1317.6083] .

Sample Preparations:

A stock solution of TPE-Bac in DMSO with a concentration of 10 mM was prepared and stocked in the 4℃ fridge. Phosphate buffer saline (PBS) was prepared by dissolving NaCl (8 g) , KCl (0.2 g) , Na₂HPO₄ (1.44 g) and KH₂PO₄ (0.24 g) in 800 mL distilled water, adjusting pH to 7.4 with HCl, and calibrating to 1 L by adding H₂O. PBS was sterilized by autoclaving for 20 min at 15 Psi (1.05 kg/cm²) on liquid cycle and stored at room temperature.

Bacteria Culturing, Imaging, and Killing:

Bacterial Staining: A single colony of bacteria on solid culture medium [Luria broth (LB) for E. coli and S. epidermidis] was transferred to 5 mL of liquid culture medium and grown at 37℃ for 10 h. The concentrations of bacteria were determined by measuring optical density at 600 nm (OD₆₀₀) and then 10⁹ colony forming unit (CFU) of bacteria was transferred to a 1.5 mL EP tube. Bacteria were harvested by centrifuging at 7000 rpm for 3 min. After removal of supernatant, 1 mL TPE-Bac solution in PBS at the concentration of 10 μM was added into the EP tube. After dispersing with vortex, the bacteria were incubated at room temperature for 10 min.

To take fluorescence images, about 2 μL of stained bacteria solution was transferred to glass slide and then covered by a coverslip. The image was collected using 100× objectives. The bacteria were imaged under an FL microscope (BX41 Microscope) using different combination of excitation and emission filters for each dye: for TPE-Bac, excitation filter ＝ 460-490 nm, dichroic mirror ＝ 505 nm, and emission filter ＝ 515 nm long pass； for PI, excitation filter ＝ 510-550 nm, dichroic mirror ＝ 570 nm, and emission filter ＝ 590 nm long pass.

For PI staining experiment, after 10 min incubation with 10 μM of TPE-Bac, the bacteria were exposed to normal light for 10 min, while the control group was put in the dark. Afterwards, PI was added to both the experiment and control group at a final concentration of 1.5 μM, followed by incubation in the dark for another 10 min. Then the bacteria were imaged under the fluorescent microscope with the following setting: excitation filter ＝ 510-550 nm, dichroic mirror ＝ 570 nm, and emission filter ＝ 590 nm long pass.

For the light-induced toxicity experiment, 10⁸ CFU bacteria were dispersed in 1 mL PBS. After 10 min incubation with the 10 μM of TPE-Bac, the solution was centrifuged at 7000 rpm for 3 min, followed by removal of the supernatant and PBS washing. Then the bacteria were dispersed in PBS and exposed to normal light for designed periods of time, while the control groups were put in dark. Then the viability of bacteria was quantified by the plate-count method.

With the information contained herein, various departures from precise descriptions of the present subject matter will be readily apparent to those skilled in the art to which the present subject matter pertains, without departing from the spirit and the scope of the below claims. The present subject matter is not considered limited in scope to the procedures, properties, or components defined, since the preferred embodiments and other descriptions are intended only to be illustrative of particular aspects of the presently provided subject matter. Indeed, various modifications of the described modes for carrying out the present subject matter which are obvious to those skilled in chemistry, biochemistry, or related fields are intended to be within the scope of the following claims.

Claims

An AIE luminogen comprising fluorescent molecules comprising a backbone structure of:

wherein

at least one of R, R′, R″, R″′, R″″, and R″″′ is independently selected from the group consisting of

R₁, R₂, and R₃ are independently selected from the group consisting of H, C_nH_2n+1, OC_nH_2n+1, and salts thereof；

R, R′, R″, R″′, R″″, and R″″′ are independently selected from the group consisting of H, C_nH_2n+1, OC_nH_2n+1, (OC₂H₄) _n, C₆H₅, C₁₀H₇, C₁₂H₉, OC₆H₅, OC₁₀H₇, and OC₁₂H₉； and

n ＝ 0 to 20.
The AIE luminogen of claim 1, wherein the AIE luminogen is TPE-Bac:
The AIE luminogen of claim 1, wherein the AIE luminogen is TPE-Bac1:
The AIE luminogen of claim 1, wherein the AIE luminogen is TPE-Bac2:
The AIE luminogen of claim 1, wherein the fluorescent molecules generate reactive oxygen species (ROS) upon light irradiation.
The AIE luminogen of claim 5, wherein the AIE luminogen is a photosensitizer.
A method of The AIE luminogen of claim 5, wherein the AIE luminogen is used for photodynamic therapy (PDT) .
The AIE luminogen of claim 7, wherein the AIE luminogen is applied to agar plates for bacteria killing.
The AIE luminogen of claim 1, wherein the AIE luminogen is functionalized with a positive charge and a long hydrophobic tail for cell membrane imaging.
A probe comprising fluorescent molecules exhibiting AIE phenomenon, wherein the fluorescent molecules comprise a backbone structure of:

wherein

at least one of R, R′, R″, R″′, R″″, and R″″′ is independently selected from the group consisting of

R₁, R₂, and R₃ are independently selected from the group consisting of H, C_nH_2n+1, OC_nH_2n+1, and salts thereof；

R, R′, R″, R″′, R″″, and R″″′ are independently selected from the group consisting of H, C_nH_2n+1, OC_nH_2n+1, (OC₂H₄) _n, C₆H₅, C₁₀H₇, C₁₂H₉, OC₆H₅, OC₁₀H₇, and OC₁₂H₉； and

n ＝ 0 to 20.
The probe of claim 10, wherein the probe is used for bacterial study.
The probe of claim 10, wherein the probe is used for biological study.
A method of imaging and quantifying bacteria comprising:

introducing the AIE luminogen of claim 1 to a sample； and

detecting bacteria by observing production of fluorescence from aggregation；

wherein the bacteria is quantified by observing emission intensity.
A method of killing cells, comprising:

introducing the AIE luminogen of claim 1 to a sample containing cells in the presence of normal light；

wherein the AIE luminogen generates ROS upon light irradiation；

wherein exposure to light kills the cells； and

wherein the cells are bacteria or mammalian cells.
The method of claim 14, wherein agar plates containing the AIE luminogen may be reused.
A method of high throughput antibiotics screening and determining bacteria resistance, comprising:

introducing the AIE luminogen of claim 1 to a sample containing an antibiotic；

evaluating the antibiotic based on an emission intensity of the AIE luminogen；

wherein bacteria in the sample turns on emission of the AIE luminogen；

wherein rapid bacteria growth indicates ineffective antibiotics and inhibited bacteria growth indicates effective antibiotics.
A method of photodynamic therapy, comprising:

introducing the AIE luminogen of claim 1 to a sample containing a tumor, wherein the AIE luminogen is a photosensitizer； and

eliminating the tumor by PDT, wherein the photosensitizer produces light-induced toxic reactive species.
A method of determining critical micelle concentration (CMC) , comprising:

introducing the AIE luminogen of claim 1 to a solvent； and

determining the CMC by evaluating fluorescence changes, wherein no emission indicates concentration below CMC and emission is turned on when concentration approaches CMC.