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Supramolecular Conjugated Polymer Materials for In-Situ Pathogen Detection Haotian Bai, Hui Chen, Rong Hu, Meng Li, Fengting Lv, Libing Liu, and Shu Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09807 • Publication Date (Web): 27 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016

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ACS Applied Materials & Interfaces

Supramolecular Conjugated Polymer Materials for In-Situ Pathogen Detection Haotian Bai, Hui Chen, Rong Hu, Meng Li, Fengting Lv*, Libing Liu and Shu Wang* Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China. E-mail: [email protected]; [email protected]

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ABSTRACT: Cationic poly (fluorene-co-phenylene) derivative (PFP-NMe3+) forms a supramolecular complex with cucurbit[7]uril (CB[7]), which could be reversibly disassembled by amantadine (AD) to release PFP-NMe3+ due to the formation of more stable CB[7]/AD complex. The cationic PFP-NMe3+ is an amphiphilic structure and could bind to negatively charged membrane of pathogen by multivalent interactions. Upon the formation of PFP-NMe3+/CB[7] complex, the CB[7] could bury the side-chain alkyl groups and decreases the hydrophobic interactions of PFP-NMe3+ on the surface of pathogens, thus PFP-NMe3+ exhibits different interaction modes with pathogens before and after assembly with CB[7]. The PFP-NMe3+/CB[7] supramolecular complex could be explored as optical sensor for simple, rapid and in-situ detection and discrimination of multiple pathogens taking advantage of optical signal changes of PFP-NMe3+/CB[7] complex before and after disassembly by AD on the pathogen surfaces. The new sensor can realize in-situ detection and identification of Gram-negative bacteria (E. coli, P. aeruginosa), Gram-positive bacteria (B. subtilis, S. aureus, E. faecalis) and the fungi (C. albicans, S. cerecisiae), and can also discriminate different strains of the same species. Blend samples of these pathogens could be identified successfully as well. In comparison with conventional blood culture-based pathogen assay methods requiring at least for 24 hours, the PFP-NMe3+/CB[7] complex only needs 2 hours (including pathogen culture, pathogen harvest by centrifuging and optical assay procedures) to stratify diverse pathogen types, and also does not require specific biomarkers or cell labeling. KEYWORDS: conjugated polymer, supramolecular materials, microorganisms, sensor, detection


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1. INTRODUCTION In the clinical diagnosis and food industry, fast and reliable detection and identification of microorganisms is crucial for treating infectious disease and nutriment contamination.1,2 Previous studies indicated that S. aureus and E. coli caused half of all bacterial infections and C. albicans caused more than half of fungal pathogen infections.3-5 Foodborne and waterborne pathogens have caused numerous epidemic diseases in recent years.6 Multiple pathogen infection caused by spreading among patients makes the early diagnosis more complicated.5,7 The expensive BacT/Alert blood culture system, as a widely used identification method in clinical diagnosis, usually takes more than 24 h to give the result of samples.8,9 In recent years, considerable attention has been paid to exploring facile materials and systems to identify and discriminate pathogens, including nanoparticles conjugated polymers, PCR, etc.1-3,10-17 But few systems could guarantee fast in-situ assay, good precision of each sample, accuracy and reproducibility of data with multiplex steps. Thus it is necessary to develop new identification and discrimination methods for pathogens continually to meet the above requirements.18 Nowadays, supramolecular chemistry has been made an important effect on the interdisciplinary scientific research in chemistry, biology and material sciences.19-20 During the past two decades, supramolecular analytical chemistry as a vital and new field has received much attention.21-26 Our previous work has reported that the supramolecular complex of cationic conjugated polymers with cucurbit[7]uril (CB[7]) before and after assembly exhibits different interaction manners with pathogen surfaces.25 Thus supramolecular system based on cationic conjugated polymers inspires us to explore a rapid and facile method for discrimination and identification of pathogens. In this work, the cationic polyfluorene/CB[7] supramolecular complex is explored as optical sensor for simple, rapid and in-situ detection and discrimination of multiple pathogens taking advantage of optical signal changes of the complex before and after disassembly on the pathogen surfaces. The new sensor can in-situ realize the identification of Gram-negative bacteria (E. coli, P. aeruginosa), Gram-positive bacteria (B. subtilis, S. aureus, E.



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faecalis) and the fungi (C. albicans, S. cerecisiae), and can also discriminate different strains of the same species. Blend samples of the three pathogens could be identified successfully as well.

2. RESULTS AND DISCUSSION In this study, three different kinds of microorganism (fungi, Gram-positive and Gram-negative bacteria) are used as targeted pathogens. The cell walls of fungi and bacteria are both negatively charged, but possessing different surface structures (Scheme 1a).27-29 The mechanism of supramolecular strategy for identification and discrimination of pathogens is shown in Scheme 1b-c. The cationic poly (fluorene-co-phenylene) derivative (PFP-NMe3+) is an amphiphilic structure and could bind to negatively charged membrane of pathogen by multivalent (electrostatic and hydrophobic) interactions.3 Upon the formation of PFP-NMe3+/CB[7] complex, the CB[7] could bury the side-chain alkyl groups and decreases the hydrophobic interactions of PFP-NMe3+ on the surface of pathogen. Upon adding amantadine (AD) into the PFP-NMe3+/CB[7] complex, the PFP-NMe3+ is released through competitive replacement due to the formation of more stable CB[7]/AD complex. Thus PFP-NMe3+/CB[7] complex exhibits different interaction modes with pathogens before and after disassembly by amantadine (AD).30,31. Simple, rapid and in-situ detection and discrimination of pathogens is realized by probing the fluorescence intensity changes of PFP-NMe3+/CB[7] complex before and after adding AD. Since PFPNMe3+ exhibits different interactions with the cell surfaces of fungi and bacteria, multiple pathogens in one sample could be identified successfully as well.


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Scheme 1. (a) Illustration of the different envelope structures of the Gram-negative bacteria, Grampositive bacteria and fungi. (b) Schematic supramolecular complex of PFP- NMe3+/CB[7] and disassembly by AD reversibly. (c) Schematic representation of supramolecular strategy for in-situ identification and discrimination of pathogens.

Variable temperature 1H NMR spectra were measured to study the binding of CB[7] to PFP-NMe3+. As shown in Figure S1, with the addition of CB[7] into PFP-NMe3+ solution in D2O, the quaternary 5 Environment ACS Paragon Plus


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ammonium protons in PFP-NMe3+ displayed significant shift toward higher field (from 3.9 - 3.6 ppm to 2.8 - 2.5 ppm) and the broad peaks of protons in the range of 0.7 - 2.0 ppm are more distinct and clear. It should be noted that the broad aromatic protons (from 8.3 – 8.6 ppm) of PFP-NMe3+ backbone show about 0.5 ppm shift to lower field and the proton peaks become much narrower upon adding CB[7]. These results indicate the formation of PFP-NMe3+/CB[7] complex by the noncovalent host-guest interactions. As PFP-NMe3+ is a classical amphiphilic conjugated polymer possessing hydrophobic backbone and hydrophilic pendants, CB[7] that owns a hydrophobic cavity and hydrophilic exterior could wrap PFP-NMe3+ through encapsulating pedent alkyl and QA moieties as shown in Scheme 1b . The isothermal titration calorimetry (ITC) experiments were carried out to obtain the thermodynamic information and elucidate the binding ability of PFP with CB[7], as shown in Figure S2. The photophysical characterizations of PFP-NMe3+ and PFP-NMe3+/CB[7] were conducted by UV/Vis absorption and fluorescence methods. As shown in Figure 1a-b, compared with PFP-NMe3+ alone, the maximum absorption of PFP-NMe3+/CB[7] complex red shift from 385 nm to 397 nm, while the fluorescence intensity significantly increases. The fluorescence quantum yield (QY) of PFPNMe3+/CB[7] complex in water is 33 % that is higher than that of PFP-NMe3+ itself (29 %). These results demonstrate that the assembly of CB[7] and PFP-NMe3+ reduces the aggregation and decreases the self-quenching of conjugated polymers in aqueous. Dynamic light scattering (DLS), zeta potential and scanning electron microscope (SEM) were further carried out to display the aggregate size and morphology of PFP-NMe3+ and PFP-NMe3+/CB[7] complex in aqueous solution. As shown in Figure 1c-f, the average diameter of PFP-NMe3+ is enlarged from 167 ± 5 nm to 246 ± 1 nm, while the zeta potential decreases from 50 ± 3 mV to 18 ± 2 mV upon adding CB[7] to disperse the aggregation of PFP-NMe3+. SEM also objectively demonstrates that aggregate size and morphology of PFPNMe3+/CB[7] complex have more loose structure than PFP-NMe3+ itself. These experimental results exhibit that CB[7] encapsulates pedent alkyl and QA moieties and changes the aggregated state of PFPNMe3+, which is consistent with that of 1H NMR experiments.


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Figure 1. (a) UV/Vis absorption spectra of PFP-NMe3+ before and after adding CB[7]. (b) Fluorescence spectra of PFP-NMe3+ before and after adding CB[7]. (c) Size distribution histograms and zeta potential of PFP-NMe3+. (d) SEM images of PFP-NMe3+. (e) Size distribution histograms and zeta potential of PFP-NMe3+/CB[7] complex. (f) SEM images of PFP-NMe3+/CB[7] complex. For a,b,c,d,e,f: PFPNMe3+/CB[7] = 1:20, [PFP-NMe3+] = 15 µΜ in repeat units (RUs). Scale bar is 100 nm.

To explore different interaction modes of PFP-NMe3+/CB[7] complex with pathogens before and after disassembly by amantadine (AD), zeta potential and scanning electron microscope (SEM) were carried out. The eight pathogens used in this work include fungi (C. albicans (CA10231) and S. 7 Environment ACS Paragon Plus


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cerecisiae (P11)), Gram-negative bacteria (E. coli (TOP10) and P. aeruginosa (JCM5962)) and Grampositive bacteria (B. subtilis (DSM2109 and NCIB3610), S. aureus (ATCC6538), E. faecalis (JCM5803)). The different interaction modes of PFP-NMe3+/CB[7] complex with pathogens lead to different zeta potential shifts. For PFP-NMe3+/CB[7] complex, the cationic QA groups are inhibited to insert into pathogen membrane, thus enhances the electrostatic interactions of PFP-NMe3+/CB[7] on the surface of pathogen. In addition, inserting into the pathogen membrane by hydrophobic interactions does not change its zeta potential, but the electrostatic binding results in obvious positive shift of potential. The released QA group side chains of PFP-NMe3+/CB[7] by AD could interact and intercalate into pathogen membrane by electrostatic and hydrophobic interactions. On the basis of the positive tendency of zeta potential shifts as shown in Figure 2a, the PFP-NMe3+/CB[7] complex has more affinity with both Gram-negative bacteria (E. coli (TOP10) and P. aeruginosa (JCM5962)) and fungi (C. albicans (CA10231) and S. cerecisiae (P11)), which are mainly driven by electrostatic interactions. While the disassembled PFP-NMe3+/CB[7] complex by adding AD has a better inserting ability into Gram-positive bacteria (B. subtilis (DSM2109 and NCIB3610), S. aureus (ATCC6538), E. faecalis (JCM5803)), which is facilitated by hydrophobic interactions. It is noted that after adding AD aqueous solution, the zeta potentials of E. coli treated with the disassembled PFP-NMe3+/CB[7] are almost same as those of E. coli treated with PFP-NMe3+ alone. To acquire further evidence for the interactions between pathogen and PFP-NMe3+ or PFP-NMe3+/CB[7] complex directly, SEM was used to observe the morphology change of pathogen membrane. Gram-negative E.coli, Gram-positive S. aureus and fungi C. albicans are chosen as the representative analysis samples. As shown in Figure 2b, the surfaces of all pathogens treated with PFP-NMe3+ alone and PFP-NMe3+/CB[7] complex become more rough than those without any treatment (blank). Also Gram-negative (E. coli) and fungi (C. albicans) show more rough surfaces than Gram-positive (S. aureus) possibly because Gram-negative bacteria and fungi are much easier to contact with fluffy PFP-NMe3+/CB[7] by electrostatic interactions. The most important fact is that, by adding AD to release the cationic QA groups, the surface morphology of pathogens treated with PFP-


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NMe3+/CB[7] complex is similar to that treated with PFP-NMe3+ alone due to the release of PFP-NMe3+ from the complex.

a

b

Figure 2. (a) Zeta potential of eight species of pathogens upon the addition of PFP-NMe3+ and PFPNMe3+/CB[7] complex, also the zeta potential of them after adding AD. (b) SEM images of E. coli (Top



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10), S. aureus (ATCC6538) and C. albicans (CA10231) treated with PFP- PFP-NMe3+/CB[7] before and after adding AD. PFP-NMe3+/CB[7] = 1:20, CB[7]:AD = 1:5, [PFP-NMe3+] = 15 µΜ in RUs.

Actually, the pathogens with different size and shape are easily distinguished by fluorescence microscope in bright field (Figure 3a), but not for those with similar size and shape. For example, the Gram-negative bacteria P. aeruginosa and Gram-positive bacteria E. faecalis look the same, so microscope in bright field could not discriminate them. In order to visualize and identify pathogens directly, the fluorescence images were employed to qualitatively analyze and discriminate different pathogens. Here the software DVCView and Microsoft Excel were taken to calculate quantitative fluorescence intensity. As shown in Figure 3b, the fluorescence intensity of species stained by PFPNMe3+/CB[7] enhances or decreases to different degree before and after the addition of AD. The fluorescence intensity of Gram-positive bacteria (B. subtilis, S. aureus, E. faecalis) significantly increases after adding AD, while both Gram-negative bacteria (E. coli and P. aeruginosa) and fungi (C. albicans and S. cerecisiae) decreases distinctly. In addition, more intensity decrease was observed for Gram-negative bacteria than fungi. It should be noted that the fluorescence change degree is still different between strains of the same pathogen species. As shown in Figure 3b, the fluorescence intensity change degree of Gram-negative bacteria is obviously negative (E. coli is 14% and P. aeruginosa is 30%) and the fungi is almost unchanged, while the Gram-positive bacteria is positive distinctly. The fluorescence intensity of E. faecalis has increased nearly twice and that of S. aureus increased 33% after adding AD, meanwhile different strains of B. subtilis (DSM2109 and NCIB3610) are different significantly. Therefore, as shown in Figure 3c, the supramolecular complex PFPNMe3+/CB[7] can realize the discrimination of these eight species of pathogen in-situ by calculating the fluorescence intensity change degree before and after disassembly by AD. Similarly, PFP-NMe3+/CB[7] and AD can also image and discriminate the mixed samples of two species of microbes through quantitative data. As displayed in Figure S2 and Figure 4, three representative blended samples were


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performed to interact with PFP-NMe3+/CB[7] and corresponding fluorescence images were obtained to quantitatively analyze and discriminate different pathogens with the help of bright field images. The sensitivity of PFP-NMe3+/CB[7] and AD system for pathogen detection was carried out using S. aureus with different concentrations (OD600 = 0.25 - 2.0) as representative samples. All the samples were imaged by fluorescence microscope followed by the quantitative calculation of fluorescence intensity changes before and after adding AD (Figure S3). The degree of fluorescence intensity change almost keeps unchanged even when pathogen concentration is lower (OD600 = 0.25), which means that our insitu supramolecular sensor has good sensitivity for pathogen detection. Also for supramolecular sensor, it is not necessary to fix the specific concentration of pathogens in different batches of detection since the evaluation criterion is the change of fluorescence intensity. From the practical point of view, this point is extremely important since the supramolecular sensor could provide good precision of each sample, accuracy and reproducibility of data. Also our in-situ supramolecular PFP-NMe3+/CB[7] sensor only needs 2 hours to complete detection task, including pathogen culture and harvest by centrifuging and optical assay procedure.



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Figure 3. (a) The bright field images of eight species of pathogens treated with PFP-NMe3+/CB[7] for 20 min. (b) The fluorescence images and intensity changes of PFP-NMe3+/CB[7] to the surface of eight pathogens before and after adding AD (scale bar is 20 µm). All the microbes species were stained in blue fluorescence treated with PFP-NMe3+/CB[7] for 10 min, after adding AD and interact for another 10 min. The exposure time is 5 ms. The fluorescence intensity changes in parallel experimental groups are obtained from DVC View and Microsoft Excel softwares. PFP-NMe3+/CB[7] = 1 : 20, CB[7] : AD = 1 : 5, [PFP-NMe3+] = 15 µM in RUs. (c) The histogram of fluorescence intensity changes according to the data in (b).

Figure 4. The bright field images of the three mixed pathogens treated with PFP-NMe3+/CB[7] for 20 min and the histogram of fluorescence intensity change according to the data in Figure S2.


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3. CONCLUSION In summary, we have designed and constructed a simple, rapid supramolecular sensor for in-situ detection and discrimination of pathogens based on the disassembly of cationic conjugated polymer complex (PFP-NMe3+/CB[7]) on the surface of pathogens. Scanning electron microscope, dynamic light scattering and zeta potential measurements were employed to certify that the PFP-NMe3+/CB[7] complex has different degree of electrostatic and hydrophobic interactions with various pathogens before and after disassembly. By taking advantage of optical signal changes of PFP-NMe3+/CB[7] complex before and after disassembly on the pathogen surfaces, in-situ detection and discrimination of multiple pathogens could be realized. More importantly, it is not necessary to fix the specific concentration of pathogens in different batches of detection since the evaluation criterion is the change of fluorescence intensity before and after disassembly of PFP-NMe3+/CB[7] complex. Thus the supramolecular sensor could guarantee good precision of each sample, accuracy and reproducibility of data. Furthermore, in comparison with conventional blood culture-based pathogen assay methods requiring at least for 24 hours, PFP-NMe3+/CB[7] complex is sensitive and only needs 2 hours to complete the whole analysis, including pathogen culture and harvest by centrifuging and optical assay procedure. The supramolecular strategy based on cationic conjugated polymer materials provides a proof-of-concept to identify and discriminate pathogens successfully in this work, and it is also expected to be used for other biological analysis, such as mammalian cells, virus, protein, DNA and environmental samples

4. EXPERIMENTAL SECTION Materials and measurements In the experiments, all chemicals and organic solvents were bought from Sigma-aldrich Chemical Company, Alfa-Aesar or Beijing Chemical Works and they were used as received. The variable temperature 1H NMR spectrum was obtained using a Bruker AVIII500WB apparatus. UV-Vis absorption measurements and the concentration of microscope were measured on a JASCO V-550 13 Environment ACS Paragon Plus


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spectrophotometer. Fluorescence spectrua were conducted with a Hitachi F-4500 fluorometer equipped with a xenon lamp excitation lamp excitation source. The fluorescence quantum yield (QY) was conducted with a Hamamatusu absolute PL quantum yield spectrometer C11347. The size of polymer was measured on a Nano ZS90 (Malvern, UK). Zeta potentials were measured on a Nano ZS (ZEN3600) system. The morphology of microbial pathogens was determined using scanning electron microscopy (SEM, JEOL JSM-7401F). The concentration of the microbial pathogens was determined by a JASCO V-550 spectrophotometer. Phase contrast bright-field and fluorescence images were taken with a fluorescence microscope (Olympus 1×71) with a mercury lamp (100 W) as a light source.

Prepare of bacteria and fungi solution A single colony of Ampr E. coli (Top 10) on a solid Luria-Bertani (LB) agar plate was transferred to 10 mL of liquid LB culture medium (supplemented with 50 µg/mL ampicillin) and grown at 37°C for 6 hours. Bacteria were harvested by centrifuging (7100 rpm for 2 min) and they were washed with phosphate buffer saline (PBS, 10 mM, pH = 7.4) twice. The supernatant was discarded and the remaining Ampr E. coli were resuspended in PBS, and diluted to an optical density of 1.0 at 600 nm (OD600=1.0). A single colony of P. aeruginosa (JCM5962) on a solid LB agar plate was transferred to 10 mL of liquid LB culture medium (without ampicillin) and grown at 37°C for 6 hours. The following operations were totally as the same as those of Ampr E. coli. A single colony of S. aureus (ATCC6538) on a solid Nutrient Broth (NB) agar plate was transferred to 10 mL of liquid YPD culture medium and grown at 37 °C for 8 h. The following operations were identical to those of Ampr E. coli. As for B. subtilis (DSM2109 and NCIB3610), except that the culture medium was replaced by Beef-extract Pepton Yeast-extract (BPY); while E. faecalis (JCM5803), expect that the culture was replaced by Enterococcus faecalis liquid culture medium, other experimental conditions and operations were identical to that of Ampr E. coli. A single colony of C. albicans (CA10231) and S. cerecisiae (P11) on solid Yeast-extract Peptone Dextrose (YPD) agar plate were respectively transferred to 10 mL of liquid


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YPD culture medium and grown at 30 °C for 12 h. The following operations were totally the same as those of E. coli expect the bacterial fluid of them was 2.0.

Zeta potential measurement Suspensions of C. albicans and S. cerecisiaw (100 µL, OD 600 = 2.0), E. coli and P. aeruginosa (100 µL, OD 600 = 1.0), B. subtilis, E. faecalis (100 µL, OD 600 = 1.0) and S. aureus (100 µL, OD 600 = 1.0) in final volume of 500 µL PBS were incubated with PFP-NMe3+ or PFP-NMe3+/CB[7] complex at 37 °C for 20 min. While in the disaasembly groups, the AD was added into the bacteria treated with PFP-NMe3+/CB[7], and then were incubated for 10 min. The bacteria were harvested by centrifuging (7100 rpm for 5 min) floowed by removal of the supernatant, and the resulted eight species of microbes were then resuspended in 10 µL of PBS, respectively. The microbes were washed once and suspended in 1 mL of H2O for zeta potential measurements.

SEM measurements After the treatment described in Zeta potential measurement, microbe samples were immediately fixed with glutaraldehyde (0.5%) in PBS at room temperature for 30 min. The bacteria were centrifuged (10000 g for 5 min) and the supernatant was removed, and then the pellets were suspended in sterile water. 2-3 µL of microorganism suspension was dropped onto clean silicon slices and it was dried naturally in the air. Once the specimens became dry, 0.1% glutaraldehyde was added to fix it for 1 h and then 0.5% glutaraldehyde for another 2 h. Next, the specimens were washed twice with sterile water and then were dehydrated by adding ethanol in a graded series (70% for 6 min, 90% for 6 min, and 100% for 6 min), and dried after that. Finally, the specimens were platinum-coated before being put into the experiment of SEM.

Fluorescence Microscopy Measurements The suspensions of C. albicans and S. cerecisiaw (100 µL, OD 600 = 2.0), E. coli and P. aeruginosa 15 Environment ACS Paragon Plus


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(100 µL, OD 600 = 1.0), E. faecalis (100 µL, OD 600 = 1.0), B. subtilis (300 µL, OD aureus (100 µL, OD

600

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= 1.0), S.

= 1.0) in final volume of 500 µL PBS were treated and incubated through the

same steps as the zeta potential experiment. And then bacteria solution (3 µL each) was added to clean glass slides for imaging. The fluorescence images were taken by fluorescence microscopy with the exposure time of 5 ms. The false color was blue for PFP-NMe3+. The type of light filter was D455/70 nm exciter, 500 nm beamsplitter, D455/70 nm emitter. Magnification of the object lens was 100×. The intensity value of fluorescence images was obtained from the software DVCView.

ASSOCIATED CONTENT Supporting Information. Additional Figures S1- S3. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (S.W.); [email protected] (L.L.).

ACKNOWLEDGEMENTS The authors are grateful to the National Natural Science Foundation of China (Nos. 21533012, 91527306, 21373243), the Major Research Plan of China (No. 2013CB932800), and Strategic Priority Research Program of the Chinese Academy of Sciences (XDA09030306).

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SYNOPSIS TOC


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