Aggregation-Induced-Emission Materials with Different Electric

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AIE Materials with Different Electric Charges as Artificial Tongue: Design, Construction, and Assembly with Various Pathogenic Bacteria for Effective Bacterial Imaging and Discrimination Guang-jian Liu, Sheng-nan Tian, Cui-yun Li, Guo-wen Xing, and Lei Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09848 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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ACS Applied Materials & Interfaces

AIE Materials with Different Electric Charges as Artificial Tongue: Design, Construction, and Assembly with Various Pathogenic Bacteria for Effective Bacterial Imaging and Discrimination Guang-jian Liu,║,†

Sheng-nan Tian,║,‡,§ Cui-yun Li,† Guo-wen Xing*,† and Lei Zhou*,‡,§

†College of Chemistry, Beijing Normal University, Beijing 100875, China. ‡National Key Laboratory of Biochemical Engineering, PLA Key Laboratory of Biopharmaceutical Production & Formulation Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. §State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and   Epidemiology, Beijing 100071, China.

ABSTRACT Imaging-based total bacterial count and type identification of bacteria play crucial rules in clinical diagnostics, public health, biological and medical science, environmental protection. Herein, we designed and synthesized a series of tetraphenylethenes (TPEs) functionalized with one or two aldehyde, carboxylic acid and quaternary ammonium groups, which were successfully used as fluorescent materials for rapid and efficient staining of 8 kinds of representative bacterial species, including pathogenic bacteria Vibrio cholera, Klebsiella pneumoniae, Listeria monocytogenes and potential bioterrorism agent Yersinia pestis. By comparing the fluorescence intensity changes of the

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AIE materials before and after bacteria incubation, the sensing mechanisms (electrostatic versus hydrophobic interactions) were simply discussed. Moreover, the designed AIE materials were successfully used as an efficient artificial tongue for bacteria discrimination and all of the bacteria tested were identified via linear discriminant analysis (LDA). Our current work provided a general method for simultaneous broad-spectrum bacterial imaging and species discrimination, which is helpful for bacteria surveillance in many fields.

KEYWORDS: tetraphenylethene; aggregation-induced emission; linear discrimination analysis; bacterial imaging; bacterial discrimination

1. INTRODUCTION Nowadays, ubiquitous bacteria are involved in almost all aspects of human life and are particularly relevant to human welfare and health. The contamination of food, air and water by pathogenic bacteria and their toxic byproducts may elicit foodborne diseases and cause severe public health crises and accidents, also may result in global climate change and environmental pollution.1-2 For example, the constant threats from existing and emerging bacterial infections cause millions of cases of severe illness each year and significant mortality worldwide. Considering that the type and total bacterial count (TBC) are two important indexes for surveilling bacterial contamination, hence, making bacteria visible, countable and especially identifiable is a fundamentally important task, which is necessary for food security and human health.3-6


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To date, various available approaches for bacteria imaging or identification have been established, such as, polymerase chain reaction (PCR),7-9 single-cell and metagenomic DNA sequencing,10 gene microarray,11 plating and culturing,12 target-specific immunoassays13-14 and identification technologies (mass spectrometry,15 Raman spectroscopy,16 diagnostic magnetic resonance17 and fluorescent imaging techniques18). Among them, imaging and identification methods based on fluorescent probes (nanomaterial-based probes, polymer probes and molecule probes) have been widely utilized due to their easy operation and high sensitivity.19-21 For example, Wang and coworkers designed several conjugated polymers for rapid discrimination of fungi, Gram-negative, and Gram-positive bacteria effectively.22-24 In addition, our group developed a self-quenching-resistant carbon nanodot for fast staining of various representative bacteria species within a short time.21 However, on the basis of the reported results, in many cases, broad-spectrum bacterial imaging and specific species discrimination could not be realized simultaneously. Very recently, with the extraordinary development of aggregation-induced emission (AIE) fluorogens, whose fluorescence increasement trigged by restriction of intramolecular motions, a few research groups have designed and applied various AIE materials for bacterial targeting, imaging or identification based on their strong photobleaching resistance and facile functionalization.25-29 For example, Tang designed two positively charged AIE materials for wash-free bacterial imaging, and an uncharged neutral AIE probe for dead bacteria imaging.30-32 Furthermore, some negatively charged AIEgens have also been demonstrated to be available agents for detection of bacteria in real-time.33 Despite previous efforts, the type of pathogens have been studied was limited and the interaction mechanisms between bacteria



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and probes were still controversial. There remains an urgent need for more systematic research to explore the detecting mechanism and more effective method for bacteria discrimination, which encouraged us to construct a series of AIE materials with different electric charges to assemble with various representative bacteria systematically. In this work, a series of AIE materials (Scheme 1) bearing different positive, negative or zero charges with tetraphenylethylene (TPE), one of the representative AIE materials,34-35 as the fluorescent core were designed and synthesized. To our delight, all these novel TPE derivatives (named TPEMN, TPEDN, TPEMC, TPEDC, TPEMA, TPEDA) have capability for broad-spectrum bacterial imaging after optimization of the staining conditions. Fluorescent images under a laser scanning confocal microscope (LSCM) showed that representative bacteria could all be stained, including three species of Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus and Bacillus subtilis) and five species of Gram-negative bacteria (Escherichia coil, Klebsiella pneumoniae, Vibrio cholera, Yersinia pestis and Pseudomonas aeruginosa). Based on the quantitative analysis of the relative fluorescence intensities of TPEs before and after incubation with bacteria (Scheme 2), which may reflect the different enrichment of probes on bacteria for their AIE features, the interaction mechanisms (electrostatic versus hydrophobic interactions) between bacteria and AIEgens were discussed. Furthermore, the

differential fluorescent responses resulting from multivalent

nonspecific interactions between TPEs and bacteria provided an efficient protocol for bacterial discrimination. Though different bacteria are sufficiently similar with each other, through linear discriminant analysis (LDA) which is one of the most common used statistical


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analysis methods for pattern recognition,36-38 all data points collected were well-clustered and divided into different groups corresponding to different kinds of tested bacteria, demonstrating the efficacy of our six-channel sensor array for the pattern recognition of bacteria. Our current work presented an alternative strategy for bacterial imaging and discrimination without the use of radioactive markers and antibodies.

CuCl, TBHP

DMSO,NaHCO 3

CH 3CN, Me3N

CH 3CN, rt

95°C, 65.1%

88.5%

COOH

CHO

Br

N Br

86.4%

1

TPEMN

CH 3CN, Me3N 83.0%

COOH

CHO

Br

N Br

TPEMC

TPEMA

DMSO,NaHCO 3

CuCl, TBHP

95°C, 40.7%

CH 3CN, rt 81.0%

N Br

TPEDN

Br

OHC

2

HOOC

TPEDA

TPEDC

Scheme 1. Synthetic route for AIE materials TPEMN, TPEDN, TPEMC, TPEDC and TPEMA, TPEDA from compound 1 and 2.

Scheme 2. Schematic illustration of TPEs for targeting and imaging bacteria, and the quantitative analysis of the relative fluorescence intensities of TPEs before and after incubation with bacteria.



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2. RESULTS AND DISCUSSION 2.1 Analysis on the fluorescence intensity vs AIEgen concentration The aggregation-induced emission (AIE) effect is an anomalous photophysical phenomenon and different from conventional organic fluorophores showing aggregation-caused quenching (ACQ) effect.39-42 The TPE derivatives are one kind of classic AIEgens which show weak fluorescence in diluted solutions since the dynamic rotations of phenyl rings could nonradiatively deactivated their excited states.27 The six AIEgens (TPEMN, TPEDN, TPEMC, TPEDC, TPEMA and TPEDA) were successfully prepared by a new and facile synthetic route in reasonable yields (Scheme 1). Then, we firstly explored their AIE properties using fluorescence spectrophotometry. As shown in Figure S1a, the fluorescence spectra of TPEMN in physiological saline exhibit a peak maximum at 466 nm with large Stokes shifts, and the emission intensity increased monotonically with the concentration of TPEMN increasing from 4 to 28 µg/mL. The emission intensity of TPEDN also increased progressively with increasing the concentration of TPEDN (Figure S1b). Similarly, the other two kinds of TPEs showed improved fluorescence emission at higher concentrations (Figure S2). Taking TPEMN, TPEMA, TPEMC as examples, the hydrodynamic diameters determined by DLS (dynamic light scattering) indicated the formation of aggregates of TPEs at higher concentrations in physiological saline (Figure S3). These results indicated the typical AIE-active property of the probes used in the study. That is to say, there exist a positive correlation between the fluorescence intensity and the amount of probe bound by bacteria (or


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the binding degree of bacteria with probe), which offered a promising platform for studying the interactions between bacteria and TPE derivatives via fluorescence techniques. 2.2 Photostability of AIE materials

Figure 1. Luminescent decay curves of TPEs (24 µg/mL in aqueous solution) with increasing number of scans. λex(TPEMC) = 337 nm， λem(TPEMC) = 465 nm; λex(TPEDC) = 337 nm， λem(TPEDC) = 465 nm; λex(TPEMA) = 362 nm， λem(TPEMA) = 490 nm; λex(TPEDA) = 369 nm， λem(TPEDA) = 500 nm; λex(TPEMN) = 332 nm， λem(TPEMN) = 466 nm; λex(TPEDN) = 334 nm， λem(TPEDN) = 475 nm. Long-term and real-time bacterial imaging will be more efficient to evaluate bacterial contamination and extend bacterial research, which has drawn increasing attentions.32 In order to further investigate the abilities of our TPEs as well-suited dyes, we characterized their photostabilities by continuous laser irradiation. The fluorescence intensity of each TPE material was normalized and the percentage of fluorescence intensity loss was calculated. As shown in Figure 1, after 50 scans, the signal loss of most TPE materials was less than 25% (for TPEDA ~30%), indicating that these AIEgens exhibit a high photobleaching resistance and long-term tracking ability.



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2.3 Bacteria imaging The above experiments demonstrated that these AIEgens were ideal dyes having potential ability in the sensing field. In an attempt to optimize the working conditions of the AIEgens, the ubiquitous and well-studied Gram-negative E.coli and Gram-positive S.aureus were chosen as model microorganisms for detailed investigations. Following the procedures described in the experimental section below (i.e. LIM, LIM-LSCM observation and LIM-fluorescent spectrum measurement), Gram-negative E.coli was first treated with different concentrations of TPEMN (i.e. 12 µg/mL, 16 µg/mL, 20 µg/mL and 24 µg/mL) for different incubation times. From the images in Figure S4, we found that TPEMN could stain E.coli successfully and give blue fluorescence after incubation only 1 min at the concentration of TPEMN higher than 16 µg/mL. Consistently, the suspensions containing E.coli after incubation with TPEMN for 1 min, 3 min, 5 min, 8 min and 10 min followed by centrifugation (7000 rpm, 5 min) and resuspension emit strongly (Figure S5), and the fluorescence intensity increased gradually with increasing the dye concentrations. Based on the same experimental procedures, we systematically changed the dye concentration and incubation time of TPEMN for staining Gram-positive S.aureus. As shown in Figure S6, fluorescence emission was observed after incubation of TPEMN with S.aureus for a short time and the emission intensity was also enhanced with increasing the staining concentrations of TPEMN (Figure S7). The fluorescence intensity of S.aureus suspensions after incubation with 24 µg/mL of TPEMN for 5 min was more than six times higher than that of TPEMN only in physiological saline (Figure 2). These results unequivocally indicated the intrinsic ability of TPEMN to label bacterial pathogens. The “turn-on” fluorescence of TPEMN after


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incubation with bacteria may result from the restriction of intramolecular rotation of TPE via electrostatic interactions between cationic TPEMN and negatively charged bacterial surface (Scheme 2).43 To obtain good (reproducible) results for bacterial imaging, 24 µg/mL of TPEs were chosen for further experiments.

Figure 2. (a, c) Fluorescence images and (b,d) bright field of (a,b) Gram-negative E. coli and (c,d) Gram-positive S. aureus after incubation with TPEMN (24 µg/mL) for 5 min following described procedures of LIM, LIM-LSCM observation. All images share the same scale bar of 5 µm. (e) Fluorescence spectrum of TPEMN (24 µg/mL) in physiological saline (black line) and fluorescence spectra of bacteria suspensions (Gram-negative E. coli (red line) and Gram-positive S. aureus (blue line)) with a concentration of 108 CFU/mL incubated with 24 µg/mL TPEMN for 5 min followed by centrifugation (7000 rpm, 5 min) and resuspension.

Since TPEMN can be applied for bacterial imaging successfully, we then investigated whether other AIEgens could be applied for bacterial imaging assay. Similarly, after the bacteria with a concentration of 108 CFU/mL were incubated with 24 µg/mL of five other different probes in physiological saline containing 10% DMSO for 1-10 minutes, the cultures were centrifuged (7000 rpm, 5 min) and resuspended in physiological saline. Then we used



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LSCM to record images of Gram-negative E.coli as well as Gram-positive S.aureus, and the fluorescence images were shown in Figure S8 and Figure S9. To our delight, a real-time imaging of

bacteria was achieved. Although these

AIEgens possessed different

positive/negative charges, all of them could bind with the tested strains, which may be due to the hydrophobic interactions between π-conjugated probes and hydrophobic patches prevalent on cells and microbial exteriors (Scheme 2).44 Through side-by-side comparison, the six AIEgens exhibited good staining effects after incubation with bacteria for 3 minutes. To avoid unnecessary signal fluctuation, incubation time of 5 min for all probes was used afterwards for imaging and fluorescence measurements.

Figure 3. LSCM images of Gram-negative bacteria (E. coil, K. pneumoniae, V. cholera, Y. pestis, P. aeruginosa) after incubation with 24 µg/mL of AIEgens (TPEMC, TPEDC, TPEMA, TPEDA, TPEMN and TPEDN) for 5 min following described procedures of LIM and LIM-LSCM


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observation. Scale bar: 5 µm.

To demonstrate the versatility of TPEs for staining representative bacteria, various types of Gram-negative bacterial and Gram-positive bacterial species were separately incubated with 24 µg/mL of six AIEgens for 5 min and subsequently subjected to LSCM for fluorescence imaging. As shown in Figure 3 and Figure S10, the AIEgens were successfully used as fluorescent agents for rapid staining of five representative Gram-negative bacteria, including E. coil, K. pneumoniae, V. cholera, Y. pestis, P. aeruginosa and three Gram-positive bacteria, including L. monocytogenes, S. aureus, B. subtili. Especially, some representative pathogenic bacteria (such as, V. cholera, Y. pestis, L. monocytogens), which can wreak havoc on human beings, could be targeted and the images were bright with clear contours. These high-quality images obtained implied that TPE derivatives should be one kind of promising agents for application in the imaging and enumeration of bacteria in real-time. Furthermore, from the images of the stained bacteria (Figure 3 and Figure S10), we can see that different bacteria with different sensors induced different responses. For example, for the Gram-negative bacteria staining (Figure 3), fluorescence of V. cholera stained by TPEMC and TPEDC and fluorescence of K. pneumoniae stained by TPEDN are relatively weaker than others. However, this phenomenon was suitable for constructing a sensor array to realize specific discrimination of bacteria.33 To further validate our hypothesis and understand the sensing mechanisms for the sensor array, quantitative characterization of fluorescence of eight kinds of bacteria (108 CFU/mL) stained by six AIEgens (24 µg/mL) was performed.



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2.4 Sensing mechanism discussion based on quantitative analysis of relative fluorescence intensity As has mentioned above, the fluorescence intensity measured was closely related to the amount of the probes bound by bacteria (or to the binding degree of bacteria with the probes). After the bacteria were incubated with six AIEgens in physiological saline/DMSO mixture (v/v = 9:1) for 5 min respectively, the mixtures were first centrifuged at 7000 rpm for 5 minutes to remove the unbound AIEgens by bacteria. Then the stained precipitates were re-suspended with isopyknic physiological saline, and the fluorescence of these suspensions were measured using the fluorescence of 24 µg/mL of pure probes alone in physiological saline as a control. The bacteria alone showed no fluorescence in physiological saline and all tests were performed in three replicates. The parameter [(I-I0)/I0] was used to characterize the fluorescence response patterns (Figure 4 and Figure S11) for each TPE-based material against eight kinds of bacteria and to eliminate the influence of TPEs which was not absorbed by bacteria or assembled with themselves.

As can be seen, the three kinds of TPEs with

different substitutions revealed unique fluorescence response patterns against eight bacteria, which may result from the different interactions between them (Scheme 2). Firstly, the TPEs (TPEMN and TPEDN) substituted by quaternary ammonium displayed an intense blue emission with both Gram-positive and Gram-negative bacteria, serving as a “turn-on” fluorescent sensor (Figure S12), which was in line with the observation under microscope. The dramatic enhancement relative to the original value indicated that the binding between TPEMN or TPEDN and bacteria was fairly strong, which restricted the intramolecular rotation of TPE. The results were probably resulted from the Coulombic attraction-induced


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interactions between the synthesized cationic ammonium-functionalized AIEgens and bacteria, verifying that the membranes of most bacteria are highly negatively charged due to the presence of a large amount of anionic phospholipids.26,

43

. Because the electrostatic

attraction is a strong force that brings two oppositely charged species together, the TPEs bearing a positive charged pendant showed light-up characteristics upon binding with the negative surfaces of bacteria. Moreover, as demonstrated by previous literature,33 different bacteria featured different surface electronic properties, which resulted in the different extent of the fluorescence changes in our detecting system (Figure 4).

Figure 4. Fluorescence response patterns ((I − I0)/I0) of eight kinds of bacteria suspensions (108 CFU/mL) stained by six AIEgens (24 µg/mL). Each value was the average of three independent measurements; each error bar shows the standard deviation of these measurements. The excitation wavelengths were 332 nm, 334 nm, 362 nm, 369 nm, 337 nm, and 337 nm for each TPE material, respectively; the emission wavelengths were listed in the figure. I0 is the corresponding fluorescence intensity of each TPE material alone in physiological saline.



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Compared with the cases of positively quaternary ammonium salts (TPEMN and TPEDN), the fluorescence intensity of bacteria treated with neutral TPEMA and TPEDA was quite similar to that of AIEgen itself. The slight fluorescence changes revealed that small amount of probes bound with bacteria tightly or the interaction strengths between bacteria and probe was similar to that between probes themselves. A reasonable explanation was that neutral TPE material could bind with bacteria through hydrophobic interaction, which was generally weaker than electrostatic attraction.44 Due to the hydrophobic effect, hydrophobic AIEgens could enter the small hydrophobic pockets or cavities of patches prevalent on microorganisms, which restricted their intramolecular motions (Scheme 2). Coincidentally, Bunz and coworkers tested both hydrophilic and hydrophobic nanoparticles in their bacterial detection experiments, and found that only hydrophobic ones could produce responses, indicating the hydrophobic interactions between nanoparticles and hydrophobic regions of bacteria.44 In general, lipopolysaccharides are mainly presented within the wall and cell membrane macromolecules of Gram-negative bacteria, whereas lipoteichoic acids and teichoic acids are present on the surface of Gram-positive bacteria.44-48 However, this tiny difference could be reflected in the fluorescence intensity of bacteria treated by our hydrophobic TPEs (TPEMA and TPEDA) decorated with one/two aldehyde groups (Figure 4). Based on the relative fluorescence intensity, we can deduce that the hydrophobic aldehyde-functionalized TPEs had a higher affinity for Gram-positive bacteria than Gram-negative bacteria (except for V. cholera), which was similar to the findings of Xu 49 and Wang 22-24. To note, in the case of TPEMC and TPEDC containing carboxyl groups, upon treating with bacteria at a concentration of 108 CFU/mL, the emission intensity did not lead to a dramatic


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increasement as compared with the initial fluorescence of TPEMC and TPEDC (Figure 4). Conversely, the attenuated emission demonstrated that only small amount of carboxylate derivatives could be absorbed by bacteria or that the interaction strength between bacteria and TPEs was weaker than that of TPEs themselves. The results could be attributed to the lower hydrophobic

interactions

between

TPEMC/TPEDC

and

bacteria

compared

with

TPEMA/TPEDA, and the poorer electrostatic interactions compared with TPEMN/TPEDN. Generally, considering the complexity of bacteria, we consider here both electrostatic interaction and hydrophobic interaction as the major possible sensing mechanisms. Due to the difference between bacterial species and the multivalent interactions toward bacteria, the extents of fluorescence changes of our TPEs were dependent on the types of bacteria, which provided the possibility of discriminating diverse bacteria based on these artificial materials bearing different charges. 2.5 Bacteria discrimination using fluorescent sensor array

Figure 5. Six channels-based 2D canonical score plot for eight kinds of bacteria at 108 CFU/mL analyzed by LDA (three replicates for each kind of bacteria). Cross-validation showed 100% classification accuracy.



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Finally, we expected these different fluorescence responses of the six TPEs towards bacteria to achieve the “fingerprint” discrimination of eight kinds of bacteria. To illustrate the capability of this protocol, the combined fluorescence patterns of the sensors were classified by using linear discriminant analysis (LDA), which is a powerful statistical method to find the linear combination of features that separate two or more classes of objects and to identify multiple analytes simultaneously.36-38, 50-52 As shown in Figure 5, eight kinds of bacteria were well-clustered and discriminated thoroughly from each other in the 2D canonical score plot. The first two canonical factors carried about 72.0% and 20.5% of the total variation respectively. The 100% classification accuracy was confirmed by “leave-one-out” cross-validation, indicating the validity of this six-channel sensor array in discriminative sensing of bacteria analysis. Interestingly, eight bacteria clustered independently in accordance to their categories in the 2D canonical score plot. In this plot, Gram-positive bacteria were at the bottom right, whereas the Gram-negative bacteria were at the middle left and the top (V. cholera), suggesting that there exist a little correlation between bacterial types and bacteria-AIEgen interactions. Furthermore, as discussed above, the interactions between TPEMN, TPEDN, TPEMA, TPEDA and bacteria were much stronger than those of TPEMC and TPEDC, so we simplified the sensor array by getting rid of TPEMC and TPEDC. As expected, satisfactory discrimination between eight kinds of bacteria was achieved based on the four-channel sensor array and the classification matrix with cross-validation reveals 100% accuracy (Figure S13). The first two canonical factors contained 88.8% and 9.4% of the variation, respectively.


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3. CONCLUSION In summary, six simple yet powerful TPE materials were synthesized. These TPEs functionalized by quaternary ammonium, carboxyl or aldehyde groups exhibited typical AIE features, superior resistance to photobleaching and excellent bioimaging activity towards bacteria. Eight representative bacteria, including five species of Gram-negative bacteria (E. coil, K. pneumoniae, V. cholera, Y. pestis and P. aeruginosa) and three species of Gram-positive bacteria (L. monocytogenes, S. aureus and B. subtilis) were efficiently stained using these AIEgens in a short time. Upon quantitative analysis of the fluorescence intensities of each bacteria treated by AIEgens, we deduced both electrostatic and hydrophobic interactions played important roles in the complexation of TPEs with bacteria. More importantly, these sensors can also serve as a novel “artificial tongue” to discriminate different bacteria. Through linear discriminant analysis (LDA), all tested bacteria were well clustered with 100% classification accuracy. Our current work provided an alternative approach to bacterial imaging and discrimination and should have potential applications in the discrimination of a wide variety of microorganisms.

EXPERIMENTAL SECTION Materials All the starting materials and solvents used for the syntheses of TPEs were purchased as reagent grade and used without further purification unless otherwise noted below. Reactions were monitored with analytical thin-layer chromatography (TLC) on silica gel F254 glass



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plates and visualized under UV light (254 nm or 365 nm). Flash column chromatography was performed on silica gel (200 – 300 mesh). NaCl was purchased from Xilong Scientific Co., Ltd. Dimethyl sulfoxide was purchased from Acros Organics. Bacteria used in this study were all preserved in our lab and used by strictly following the related safety operation rules. Yeast extract powder and typtone used for preparation of Luria-Bertani (LB) liquid culture medium were purchased from OXOID Ltd. Brain Heart Infusion was purchased from Becton, Dickinson and Company. The water used in all experiments was purified with a Millipore system. Instrumentation 1

H NMR and

13

C NMR spectra were recorded with a Bruker Avanced III 400 MHz

spectrometer or JEOL's NMR (400MHz or 600 MHz) spectrometers and the chemical shifts (in ppm) were calibrated with deuterated solvent peaks. High resolution electrospray ionization mass spectra (HRMS-ESI) were recorded with Waters LCT Premier XEmass spectrometer. Particle size data were obtained through dynamic light scattering (ZetaPLUS, Brookhaven Instruments Corporation). Fluorescence emission spectra were recorded with a fluorescence spectrophotometer (F-7000) purchased from Hitachi High-Tech Science Corporation with samples contained in 1-cm × 1-cm cells. The optical density (OD) at λ = 600 nm was determined by ultraviolet spectrophotometer from Hitachi High-Tech Science Corporation. Fluorescence imaging experiments were carried out on an Olympus FV1000 LSCM (60 × oil objective; Excitation wavelength: 405 nm). Synthesis


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These AIE materials were synthesized according to the synthetic route different from the previous reports (Scheme 1). Synthesis of 4-(1,2,2-triphenylvinyl)benzaldehyde (TPEMA). To a 25 mL round-bottom flask equipped with a stir bar were added compound 1 (200 mg, 0.47 mmol) and DMSO (1.5 mL). NaHCO3 (80 mg, 0.95 mmol) was added all at once and the mixture was stirred at 90°C for 4h. After cooling to room temperature, the resulting mixture was diluted with ethyl acetate, washed with brine and dried over anhydrous sodium sulfate. Then the solvent was evaporated in vacuo and the residue was purified by column chromatography to give the product TPEMA (110.3 mg, 65.1%, Rf = 0.65, petroleum : EtOAc = 8:1). 1H NMR (400 MHz, CDCl3) δ 9.90 (s, 1H, CHO), 7.62 (d, J = 8.4 Hz, 2H, Ar H), 7.19 (d, J = 8.2 Hz, 2H, Ar H), 7.15 – 7.09 (m, 9H, Ar H), 7.05 – 6.98 (m, 6H, Ar H). HRMS(ESI) calcd for C27H21O [M+H]+: 361.1587, found: 361.1583. The spectroscopic data coincide with that obtained by Suzuki reaction.53 Synthesis

of

4,4'-(1,2-diphenylethene-1,2-diyl)dibenzaldehyde

(TPEDA).

The

main

procedures of the preparation of TPEDA are similar to that of TPEMA. The compound TPEDA was obtained in 40.7% yield. 1H NMR (400 MHz, CDCl3) δ 9.91 (s, 2H, CHO), 7.63 (d, J = 8.2 Hz, 4H, Ar H), 7.20 (d, J = 8.1 Hz, 4H, Ar H), 7.17 – 7.11 (m, 6H, Ar H), 7.04 – 6.96 (m, 4H, Ar H). HRMS(ESI) calcd for C28H21O2 [M+H]+: 389.1536, found: 389.1532. The spectroscopic data coincide with the previous report in which TPEDN was synthesized at -78°C using n-BuLi as a reagent.54 Synthesis of 4-(1,2,2-triphenylvinyl)benzoic acid (TPEMC). To a round-bottom flask were added TPEMA (139 mg, 0.386 mmol), CH3CN (3 mL) and CuCl (11.5 mg). Then an aqueous solution of tert-butyl hydroperoxide (70% in water, 3.0 eq.) was added slowly. The reaction



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mixture was exposed to air and stirred at room temperature for 20 h. After completion of the reaction, the mixture was diluted with saturated NaHSO3(aq.), extracted with ethyl acetate twice and the combined organic layers were dried over anhydrous sodium sulfate, filtered through paper and evaporated to dryness. The desired product TPEMC (125.4 mg, 86.4%) was obtained by column chromatography using petroleum/EtOAc (containing 1‰ acetic acid) as the eluent. 1H NMR (400 MHz, DMSO) δ 12.85 (s, broad, 1H, -COOH), 7.69 (d, J = 7.6 Hz, 2H), 7.21 – 7.05 (m, 11H), 6.97 (dd, J = 5.8, 1.7 Hz, 6H). HRMS(ESI) calcd for C27H19O2 [M-H]-: 375.1380, found: 375.1389. The spectroscopic data coincide with the previous report in which TPEMC was synthesized at -78°C using n-BuLi as the reagent.55 Synthesis of 4,4′-(1,2-diphenyl-1,2-ethenylene)dibenzoic acid (TPEDC). The main procedures were the same as described above for the synthesis of TPEMC. After recrystallization and dried in vacuum, the compound TPEDC was obtained in 81% yield. 1H NMR (400 MHz, DMSO) δ 12.82 (s, broad, -COOH), 7.71 (d, J = 8.3 Hz, 4H), 7.17 (d, J = 6.9 Hz, 6H), 7.10 (d, J = 8.3 Hz, 4H), 7.03 – 6.93 (m, 4H). HRMS(ESI) calcd for C28H19O4 [M-H]-: 419.1278, found: 419.1285. Synthesis of N,N,N-trimethyl-1-(4-(1,2,2-triphenylvinyl)-phenyl)methanaminium bromide (TPEMN). Compound 1 (200 mg, 0.47 mmol) was dissolved in acetonitrile (CH3CN, 5 mL). Then 33% trimethylamine alcoholic solution (0.85 mmol, 1.8 eq.) was added to the mixture, and the contents were stirred at rt for 12 h. Both organic solvent and the excess trimethylamine were evaporated from the crude product mixture under reduced pressure followed by addition of diethyl ether (5 mL). After stirring at rt for another 2 hours, the mixture was filtered and the white solid was rinsed with diethyl ether. The resulting white


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solid was collected and vacuum dried to yield TPEMN (201.6 mg, 88.5%). 1H NMR (600 MHz, DMSO) δ 7.33 – 7.27 (m, 2H), 7.24 – 7.04 (m, 10H), 7.02 – 6.93 (m, 6H), 6.84 (dd, J = 18.0, 8.1 Hz, 1H), 4.42 (d, J = 3.5 Hz, 2H, CH2), 2.94 (s, 9H, N(CH3)3). HRMS (ESI) calcd for [C30H30N]+ :404.2373, found: 404.2378. Synthesis of 1,2-bis[4-(trimethylammoniomethyl)-phenyl]-1,2-diphenylethene dibromide (TPEDN). Using the general procedure described above. Compound 2 (185 mg) gave the title compound TPEDN (188.6 mg, 83.0%). 1H NMR (600 MHz, DMSO) δ 7.36 – 7.27 (m, 4H), 7.19 – 7.13 (m, 6H), 7.11 (t, J = 7.7 Hz, 4H), 7.04 – 6.96 (m, 4H), 4.45 (s, 4H), 2.96 (s, 18H). 13

C NMR (101 MHz, DMSO) δ 144.83, 142.21, 142.16, 140.72, 140.65, 132.20, 131.08,

130.98, 130.65, 130.55, 127.98, 127.92, 126.95, 126.61, 67.45, 51.68. HRMS (ESI) calcd for [C34H40N2]2+: 238.1590, found: 238.1595. Bacteria culture Eight species of bacteria were used in this work. The representative Gram-positive bacteria were L. monocytogenes, S. aureus and B. subtilis (vegetative cells). The representative Gram-negative bacteria were E. coil, K. pneumoniae, V. cholera Y. pestis, and P. aeruginosa. L. monocytogenes was cultured in Brain Heart Infusion liquid medium at 37 °C overnight. Other bacteria were seeded and cultured in LB liquid medium and grown at 37 °C overnight. The bacteria were harvested by centrifugation at 7000 rpm for 5 min. Then the resultant pellet was washed by resuspension and centrifugation using physiological saline (pH = 7.0) for three times. Afterwards, the concentrations of each kind of diluted bacteria suspensions in physiological saline were determined by measuring optical density (OD) at λ = 600 nm. Then the bacteria suspensions were inactivated at 65°C for 30 min.



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Fluorescent staining of bacteria All the representative bacteria were stained with TPEs by the liquid incubation method (LIM) described below. A stock solution of each AIEgen in DMSO at certain concentration was added to the suspensions of bacteria in physiological saline to form aqueous mixtures with 10% DMSO and the final concentrations of bacteria were 1 × 108 colony-forming units (CFU) per milliliter. After incubation for several minutes (1 min, 3 mins, 5 mins, 8 mins or 10 mins), the mixtures were centrifuged at 7000 rpm for 5 minutes, then the supernatants were discarded to remove the unbound AIEgens and the precipitates were re-suspended with isopyknic physiological saline (pH = 7.0, 0.85% NaCl), following vortex shake for 10 seconds. For fluorescent

spectrum

measurement

(i.e.

LIM-fluorescent

spectrum

measurement),

suspensions of samples were recorded with excitation wavelength at 332 nm for TPEMN, 334 nm for TPEDN, 337 nm for TPEMC, 337 nm for TPEDC, 362 nm for TPEMA, 369 nm for TPEDA, respectively. For LSCM observasion (i.e. LIM-LSCM observation), drops of sample suspension were spotted on glass slide and allowed to air-dry. The specimens were imaged immediately under a LSCM with excitation wavelength of 405 nm.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Supplementary fluorescence spectra and LSCM images of bacteria; HRMS, 1H and spectra of AIEgens.


13

C NMR

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AUTHOR INFORMATION Corresponding Author Guo-wen Xing. E-mail: [email protected] Lei Zhou. E-mail: [email protected] Author Contributions ║

Both authors contributed equally to this work.

ACKNOWLEDGMENTS The work was supported by the National Natural Science Foundation of China (21272027), the National High Technology Research and Development Program of China (863 Program) under Grant No. 2013AA032205, and Beijing Nova Program of China (Grant No. Z151100000315086). REFERENCES 1.

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