Conjugated Polymer with Aggregation-Directed Intramolecular FÃ¶rster

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Conjugated Polymer with Aggregation-Directed Intramolecular FRET Enables Rapid and Efficient Discrimination and Killing of Microbial Pathogens Shuxian Zhu, Xiaoyu Wang, Yu Yang, Haotian Bai, Qianling Cui, Han Sun, Lidong Li, and Shu Wang Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b00164 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Chemistry of Materials

Shuxian Zhu,#,† Xiaoyu Wang,#,† Yu Yang,† Haotian Bai,‡ Qianling Cui,† Han Sun,‡ Lidong Li,*,†,§ and Shu Wang*,‡ †

State Key Laboratory for Advanced Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China ‡ Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China § State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China ABSTRACT: Rapid and effective differentiation and killing of microbial pathogens are major challenges in the diagnosis and treatment of infectious diseases. Here, we report a novel system based on the conjugated polymer PFDBT-BIMEG, which enables efficient microbial pathogen discrimination and killing. The functional side chains of PFDBT-BIMEG enabled both electrostatic and salt-bridge interactions with microorganisms. Microorganism binding events caused a change in the aggregation structure of PFDBT-BIMEG, which could be recognized by a change of its fluorescence signal by intramolecular fluorescence resonance energy transfer (FRET). This sensing strategy allowed rapid and sensitive distinction of microbial pathogens within 15 min. We performed linear discrimination analysis that featured this advance to confirm that the polymer PFDBT-BIMEG could accurately classify microbial pathogens. Owing to the different adhesion mechanism of PFDBT-BIMEG to the surface of the microorganisms, we applied different sterilization strategies for each kind of microbial pathogen. The microbial pathogens could be efficiently killed by reactive oxygen species produced from PFDBT-BIMEG under irradiation, avoiding the use of any other antibacterial agents. This methodology, which combines pathogen discrimination and killing, represents a promising alternative to current diagnostic platforms.

INTRODUCTION Outbreaks of bacterial infections with high mortalities are a major global threat to public health.1-3 Owing to the variety of bacteria associated with infectious diseases, early bacterial identification and administration of an appropriate antimicrobial treatment are critical for preventing bacterial outbreaks. The current strategy of microbiological identification involves the polymerase chain reaction (PCR), which is a highly sensitive detection tool.4-6 However, quantitative real-time PCR systems are expensive and cannot effectively deliver antimicrobial treatments to kill bacteria. Traditional bacterial culture and analysis of morphological structures is the clinical gold standard; however, this method is greatly restricted by the long procedural times and can only identify certain species. 7,8 These limitations motivate the need for a broad, generic, and accurate system that allows both rapid bacterial identification and efficient sterilization. Fluorescent conjugated polymers (CPs), have drawn great interest for their light-harvesting activity and high quantum efficiency, and have been widely incorporated into optical bioanalysis systems.9-15 The polymer backbone determines the basic optical properties of CPs for optical detection and the functional groups on the side chains enable CPs to interact with targets.16-21 As the outermost part of pathogenic microorganisms, structures of the bacterial cell wall greatly affect their interactions with CPs. Lipopolysaccharides (LPS) present in bacterial outer membranes confer a negative surface charge to

Gram-negative bacteria. Gram-positive bacteria do not have such an outer membrane structure but exhibit a negative surface charge owing to the teichoic acids embedded in the cell wall. Fungi have a cell wall, which consists mainly of mannoproteins and also displays a negative surface charge. 22-24 Therefore, CPs with positively charged pendant groups can attach to the surface of bacteria through electrostatic interactions.25-29 The use of CPs to identify specific types of bacteria from observations of changes in the fluorescent signal is often achieved with complementary materials, such as negatively charged metal nanoparticles or dye-labeled DNAs. These structures can act as emission quenchers or as acceptors in Förster resonance energy transfer (FRET), when complexed with CPs.30-32 In these composite systems, the complementary materials can activate fluorescent signal responses that fundamentally alter the electrostatic interactions between the CPs and bacteria. Hence, such systems show great advantages for distinguishing among different types of bacteria. However, a challenge facing current detection systems is the lack of any inherent antibacterial capacity, which is becoming increasingly desirable for medical applications. Currently, antimicrobial polymeric materials have attracted tremendous interest in antimicrobial applications.33-35 Upon irradiation, CPs can produce reactive oxygen species (ROS) and behave as an antimicrobial agent.36-39 By taking advantage of these properties, novel systems based on CPs could be constructed that correctly identify the type of pathogen and deliver the most effective course of sterilization. Hence, we have developed a sensitive,

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Figure 1. (a) Chemical structure of PFDBT-BIMEG; Emission spectra of PFDBT-BIMEG as a function time in the presence of (b) E. coli, (c) S. aureus, and (d) C. albicans (OD 600 = 0.5). [PFDBT-BIMEG] = 25 μM in RUs. Excitation wavelength was 392 nm. highly efficient method based on one CP poly[(9,9-bis{6ʹ-([N(triethylene glycol methylether)-di-(1H-imidazolium)me thane]hexyl}-2,7-fluorene)-co-4,7-di-2-thienyl-2,1,3benzothiadiazole] tetrabromide (PFDBT-BIMEG, DBT molar ratio is 5%)40 to identify microorganisms through their characteristic cell wall structures. In addition to electrostatic interactions between the microorganisms and PFDBT-BIMEG, saltbridge interactions between the two components were also identified. We studied the aggregation of the CPs on microorganisms, and aggregation-directed FRET of the PFDBTBIMEG enabled the polymer to produce a fluorescence signal response that enabled identification of a diverse range of pathogenic microorganisms. On the basis of a detailed study of the interactions between the microorganisms and PFDBT-BIMEG, appropriate and efficient sterilization strategies were used to kill the identified pathogens through the antibacterial capability of PFDBT-BIMEG. Our system, based on a single CP, represents an efficient approach to microbial pathogen assay and enables subsequent killing of the microbial pathogens. RESULTS AND DISCUSSION The CP PFDBT-BIMEG, shown in Figure 1a, served as the basis for distinguishing among the microorganisms. The backbone of PFDBT-BIMEG consists of energy donor poly(fluorene) (PF) and energy acceptor 1,4dithienylbenzothiadiazole (DBT) units at 5 mol%. These units produce two fluorescent signals based on FRET. The bisimidazolium (BIM) groups, linked to the side chains of the fluorene units, enable electrostatic interactions through their positive charges and can also form ionic hydrogen bonds through the C–H group at the 2-position to interact with anions in the detection system. The ethylene glycol (EG) side chain

increases the water-solubility of PFDBT-BIMEG. As shown in Figure S1, the UV-Vis absorption spectrum of PFDBTBIMEG showed a strong absorbance from the PF component at 392 nm and a weak absorbance from the DBT units at 530 nm. Thus, PFDBT-BIMEG exhibits a maximum emission at 423 nm from the PF components and an emission at 655 nm from DBT units. Hence, the FRET effect from PF to DBT was weak owing to the good water-solubility of PFDBT-BIMEG and the extension of the polymer chains maintained in pure PFDBT-BIMEG solutions. To explore the discrimination ability of the synthesized PFDBT-BIMEG for microorganisms, we selected Gramnegative (E. coli) and Gram-positive (S. aureus) bacteria and fungi (C. albicans) as analytes. As shown in Figure 1b-d, after addition of PFDBT-BIMEG to the microorganism solutions, the emission spectra of PFDBT-BIMEG changed. In general, the PF emission decreased and the DBT emission increased. For the blank group without any microorganisms, no discernable differences in the PFDBT-BIMEG emission appeared over 15 min (Figure S2). Hence, the change suggests that PFDBTBIMEG aggregation resulted from nonspecific interactions between the CP and the microbial surface. However, the trend of the PFDBT-BIMEG fluorescence change showed differences among the microorganisms. For E. coli, after mixing with PFDBT-BIMEG, the emission of PF decreased sharply and the emission of DBT increased simultaneously within 1 min. No further notable changes of the PFDBT-BIMEG emission occurred over a prolonged time period. The PFDBTBIMEG emission when mixed with S. aureus exhibited a gradual change over approximately 5 min, whereas the change for C. albicans required a longer time of approximately 15 min.


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Figure 3. Fluorescence images of PFDBT-BIMEG at 1, 3, 5 and 15 min in the presence of E. coli, S. aureus, and C. albicans, respectively. Excitation wavelength was 365 nm.

Figure 2. FRET efficiency (I655 nm/I423 nm) of PFDBT-BIMEG as a function of time in the absence and presence of E. coli, S. aureus, and C. albicans (OD600 = 0.5), respectively. [PFDBTBIMEG] = 25 μM in RUs. Excitation wavelength was 392 nm. The FRET efficiency (I655 nm/I423 nm) of PFDBT-BIMEG as a function of time after interacting with different microorganisms is shown in Figure 2. The FRET efficiency increased much more rapidly for E. coli than for the other two microorganisms and reached its maximum value at 1 min. The degree of FRET efficiency reached maximum values after 5 min for S. aureus and 15 min for C. albicans. Furthermore, the maximum FRET efficiency of PFDBT-BIMEG decreased for the different microorganisms in the order 2.15 for E. coli > 1.33 for S. aureus > 0.96 for C. albicans. The blank group maintained a relatively low FRET efficiency after 15 min. The decrease in the sensitized PF emission and the increase in DBT emission suggest a decrease in the average distance between the FRET donor-acceptor pair40. We believe that the change in the FRET efficiency corresponds to the different aggregation extents of the PFDBT-BIMEG on the surface of the different microorganisms. The different fluorescence signal changes could be directly visualized under UV light (Figure 3 and Figure S3). After mixing the PFDBT-BIMEG with E. coli, the mixture appeared red at 1, 3, 5 and 15 min. For S. aureus, the color of

the mixture changed from purple to pink and then light red. For C. albicans, the color of the mixture changed more gradually than the others, from light purple, to purple, pink, and then light red. In the absence of any microorganisms, the PFDBT-BIMEG solution maintained its blue fluorescence. These differences in the change of the signals might enable us to distinguish among the different microorganisms with this CP. To gain a deeper insight into how the different fluorescence signals corresponded to different species, we first visualized the binding of PFDBT-BIMEG on the three microorganisms using confocal laser scanning microscopy (CLSM). As shown in Figure S4, for all three microorganisms, each cell was stained with PFDBT-BIMEG and showed blue and red fluorescence. We used ζ-potential measurements to analyze the interactions of PFDBT-BIMEG and the different microorganisms. As shown in Figure 4a-4c, the average ζ-potential values of E. coli, S. aureus, and C. albicans were –43, –24 and –22 mV, respectively. The microorganisms displayed net negative charge as expected. Because PFDBT-BIMEG is positively charged, the microorganisms stained with PFDBT-BIMEG showed a shift of their ζ-potentials to more positive values as incubation time increased. These results confirmed the binding of PFDBT-BIMEG to the different microorganisms, which is consistent with our CLSM results. In addition, the ζ-potential of E. coli increased faster and to a greater degree after incubation with PFDBT-BIMEG than for other microorganisms (Figure 4d). The ζ-potential of E. coli increased by 20 mV and reached a plateau within 3 min. The ζ-potential of S. aureus increased by 8 mV and reached a plateau at approximately 5 min. A similar trend was observed for fungi C. albicans; however, the ζ-potential increase for C. albicans was almost linear over time. These results reveal that the faster and greater increase in the ζ-potential of E. coli can be attributed to it having the greatest amount of negative surface charge among the three microorganisms. This surface charge provides a stronger electrostatic interaction and attracts the positive CP molecules. Thus, PFDBT-BIMEG rapidly accumulated on the surface of the E. coli and the FRET efficiency increased. The S. aureus



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Figure 4. ζ-potential measurements of (a) E. coli, (b) S. aureus and (c) C. albicans (OD600 = 0.5) as a function of time after incubation with PFDBT-BIMEG. (d) Increased ζ-potential values of E. coli, S. aureus and C. albicans as a function of time after incubation with PFDBT-BIMEG.

Figure 5. Fitting curves of observed enthalpy changes ΔHobs against the PFDBT-BIMEG/microorganism molar ratio by titrating PFDBT-BIMEG into (a) E. coli, (b) S. aureus, and (c) C. albicans. Dilution enthalpy of the polymer was deducted. ΔH values are expressed in kcal/mol of polymer. OD600 = 0.5. and C. albicans both showed similar initial surface potentials and the changes of ζ-potential were slower and similar.It is difficult to explain the trends observed in Figure 2. The electrostatic interaction between the PFDBT-BIMEG and microorganisms plays an important role in the binding effect but is not the only factor affecting binding. To further understand the interaction mechanism between the PFDBT-BIMEG and microorganisms, we conducted isothermal titration microcalorimetry (ITC) measurements. As shown in Figure 5, different titration plots were obtained for the microorganisms. By fitting the ITC curves with one set of sites binding model, the binding thermodynamic parameters were calculated and summarized in Table 1. Both the enthalpy changes (∆H) and entropy changes (∆S) were negative for all the microorganisms. In biological systems, there are numerous ionic hydrogen bonds. The salt-bridge is a novel form of an ionic hydrogen bond in which oppositely-charged groups are sufficiently close for electrostatic attraction.41 Microbial surfaces are rich in oxyanionic groups, such as phosphate groups in the cell well and carboxylate groups in protein.42 Considering the structure of PFDBT-BIMEG, the imidazolium groups

can form ionic hydrogen bonds with anions, and are more likely to form (C–H)+---O=P bonds with phosphate groups.43 Thus, the only contributions to the negative entropy and enthalpy changes arise from the salt-bridge between the BIM groups of PFDBT-BIMEG and oxyanionic groups on the microbial surface. The large areas of contact and interaction between microorganism and PFDBT-BIMEG may lead to the large enthalpy changes and entropy changes.44 E. coli contains high levels of LPS in the outer membrane; however, S. aureus has only a single lipid membrane covered by peptidoglycan and teichoic acid.45 The ∆H and T∆S values for E. coli were higher than those for S. aureus. Furthermore, the binding constant (K) for E. coli was larger than that for S. aureus. These results indicate that PFDBT-BIMEG was more tightly bound to the E. coli surface than to S. aureus owing to the higher content of LPS in the former. The cell wall of C. albicans is mainly composed of mannoproteins.46 Thus, ∆H and T∆S were lowest for C. albicans, and the K value was smallest among these microorganisms. As a result, the ability of PFDBT-BIMEG to bind C. albicans was weakest and PFDBTBIMEG was loosely packed on the surface of C. albicans.


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Scheme 1. Schematic illustration of PFDBT-BIMEG for discrimination of different microorganisms. Electrostatic interactions induced positive PFDBT-BIMEG to aggregate on the microbial surfaces and salt-bridges induced PFDBT-BIMEG to bind with the surface of the microorganisms.

Figure 6. (a) Partial fluorescence response patterns of PFDBT-BIMEG in the presence of various microorganism (OD600 = 0.5). [PFDBT-BIMEG] = 25 μM in RUs. Error bars show standard deviations calculated from six parallel measurements. (b) Canonical score plot for the fluorescence patterns, as derived by LDA. The 95% confidence regions for the individual microorganism are depicted by dashed lines. Table 1. Thermodynamic parameters for the binding of PFDBT-BIMEG to microorganisms at 298 K in water. Microorganisms

Binding constant (K) (×106 M-1)

△H

T△S

(kcal/mol)

(kcal/mol)

E. coli

46.1

-436.6

-426.3

S. aureus

11.6

-302.2

-292.5

C. albicans

7.58

-24.62

-15.23

Together with the results shown in Figure 2, the variation of FRET efficacy changes among the three microorganisms can likely be attributed to the aggregation speed and tightness of binding of PFDBT-BIMEG to the surfaces of the different microorganisms. The ζ-potential and ITC analyses demonstrate the existence of both electrostatic and salt-bridge interactions between microorganism and PFDBT-BIMEG. They can reflect the association of the observed fluorescence signal for PFDBT-BIMEG

in the presence of the different microorganisms. On the basis of the above results, we can infer the binding mechanism of PFDBT-BIMEG to the different microorganisms. As shown in Scheme 1, the aggregation of PFDBT-BIMEG on the surface is dominated by both electrostatic attractions and salt-bridge interactions. After addition of PFDBT-BIMEG to the microorganism solutions, electrostatic interactions induced positive PFDBT-BIMEG to aggregate on the microbial surface. The binding of PFDBT-BIMEG on the microbe surfaces was dominated by the formation of salt-bridges. Owing to a more negative ζ-potential and greater amounts of phosphate groups, E. coli exhibited faster and stronger binding with PFDBTBIMEG than that of S. aureus. For C. albicans, the composition of the membrane is different from that of the bacteria, such that its binding was the slowest and weakest. Over all, the binding ability between the PFDBT-BIMEG and microorganisms showed a positive correlation with the fluorescence signal change, that is, E. coli exhibited the fastest FRET effect and highest FRET efficiency, followed by S. aureus and C. albicans.



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Figure 7. Bactericidal activity of PFDBT-BIMEG toward (a) E. coli, (b) S. aureus and (c) C. albicans in the dark and under white light irradiation (25 mW/cm2, 5 min) after incubation of the microorganisms with PFDBT-BIMEG for various times. Notably, the interaction mechanism of the PFDBT-BIMEG with different microorganisms was determined, and the resulting fluorescence changes were reproducible, depending upon the type of microorganism. Differentiation in the FRET efficiency explicitly demonstrated the ability of PFDBT-BIMEG to discriminate among the three microorganisms. Changes in the PFDBT-BIMEG emission in the presence of the different microorganisms could be quantified by the parameter δ, as defined by the following equation: 𝑅𝑡 𝐴𝑡 ⁄𝐷𝑡 𝐴𝑡 𝐷0 𝛿= = = · 𝑅0 𝐴0 ⁄𝐷0 𝐴0 𝐷𝑡 where t is the time after microorganism addition; R0 and Rt are the intensity ratio of the emission at 655 nm and 423 nm, before and after addition of the microorganisms, respectively; A0 and At are the corresponding emission intensities of the acceptor DBT at 655 nm and D0 and Dt are those of the donor PF at 423 nm. Figure 6a summarizes part of the δ responses for the different microorganisms. Linear discrimination analysis (LDA) was used to explore the process parameter δ and to identify the microorganisms. LDA is a statistical method, which is based on the shortest Mahalanobis distances strategy.47 Unknown samples can be classified by calculating the Mahalanobis distances between sample points and the class center in canonical coordinates. For this study, we processed the fluorescence responses per min over 15 min, six times for each microorganism (that is, 15 constructs × 3 species × 6 replicates). Two canonical factors were generated that collated 100% (88.9% for factor 1 and 11.1% for factor 2) of the variance. As shown in Figure 6b, LDA transformed the series of δ to canonical scores, which were clustered into three groups according to the individual microorganism. Distribution areas of E. coli, S. aureus and C. albicans were completely separated and no overlap was observed with a 95% confidence interval. These results revealed that the different features of the microorganisms contributed to significant changes in their response. Hence, the microorganism classification by PFDBT-BIMEG shows excellent accuracy. Moreover, we distinguished two gram-positive bacteria (S. aureus and B. subtilis) and tow gram-negative bacteria (E. coli and P. aeruginosa) with this methodology. As shown in Figure S5a and b, similar PFDBT-BIMEG fluorescence changes were observed for S. aureus and B. subtilis; and the changes of PFDBT-BIMEG fluorescence emission showed almost same between E. coli and P. aeruginosa. Then, we quantified these changes in the PFDBT-BIMEG emission by the parameter δ (Figure S5c) and transformed the series of δ to canonical

scores with LDA (Figure S5d). Each of the bacteria formed their own distribution area without overlap. Significantly, different strains of gram-positive bacteria or gram-negative bacteria can be easily distinguished by using PFDBT-BIMEG. Comparing the classical staining assays that require trained operators, the advantage of microorganism classification by PFDBT-BIMEG is its easy operation. Furthermore, introducing LDA improves the accuracy and reliability of microorganism classification.

Figure 8. SEM images of E. coli, S. aureus and C. albicans incubated with and without PFDBT-BIMEG under the same irradiation conditions (25 mW/cm2, 5 min). Scale bar is 5 μm for C. albicans and 1 μm for the others. Incubation times were 1 min for E. coli, 5 min for S. aureus and 15 min for C. albicans. On the basis of the good binding ability of PFDBT-BIMEG to the microorganisms and its ability to produce ROS 48, we subsequently studied the bacterial killing efficiency of PFDBT-BIMEG through a plate-counting technique. First, we used 2,7-dichlorofluorescein (DCFH) as the ROS probe to demonstrate the ROS production of PFDBT-BIMEG. ROS oxidize nonemissive DCFH to fluorescent 2ʹ,7ʹdichlorofluorescein (DCF).49 The increase of DCF fluorescence intensity at 524 nm can be monitored to prove the presence of ROS. As shown in Figure S6, upon continuous irradia-


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tion DCFH with PFDBT-BIMEG for 10 min, the DCF fluorescence intensity gradually increased and became much higher than that of a blank group containing no PFDBT-BIMEG. It should be noted that PFDBT-BIMEG itself could not be excited at 488 nm. These results demonstrate the significant ROS generation from PFDBT-BIMEG. Then, we incubated the PFDBT-BIMEG with three microorganisms. As shown in Figure 7, PFDBT-BIMEG exhibited a highest killing efficiency of 95% toward all microorganisms after irradiation at 25 mW/cm2 for only 5 min. By comparison, the killing efficiency was less than 20% in a dark control experiment. These results confirm that the bactericidal activity of PFDBT-BIMEG derived from the ROS produced upon irradiation. However, the killing efficiency was strongly dependent on the incubation time. After E. coli was incubated with PFDBT-BIMEG for 1 min, a 99%-reduction of the bacteria was obtained upon irradiation (Figure 7a and Figure S7). Increasing the incubation time to 5 min, the killing efficiency towards S. aureus increased from 57% to 97% (Figure 7b and Figure S8). As shown in Figure 7c and Figure S9), by further extending the incubation time to 15 min, the killing efficiency towards C. albicans reached 96%. These results further confirmed our discussion of the range of interactions of PFDBT-BIMEG with different microorganisms, as illustrated in Scheme 1. The combination of PFDBT-BIMEG with microorganisms is beneficial for efficiently killing microbial pathogens. Furthermore, a suitable sterilization strategy could be selected for the different microorganisms. Direct visualization of morphological changes of the microorganisms was observed by SEM imaging (Figure 8). Compared with the control groups, the microorganisms maintained their appearance after interacting with PFDBT-BIMEG upon irradiation and no fractured cells were observed. These results indicate that anchoring of PFDBT-BIMEG to the surface of the microorganisms induced PFDBT-BIMEG to form a polymer network on the microorganisms, which helped to maintain their morphologies. Flat cells were detected for E. coli, and shriveled cells was observed for S. aureus, and C. albicans. Considering the low dark toxicity of PFDBT-BIMEG, as illustrated in Figure 7, the photoexcited PFDBT-BIMEG produced a substantial quantity of ROS, which formed defects on the cell walls and caused death of the microorganisms through leakage of intracellular components50,51. This insight into the interactions between PFDBT-BIMEG and the microorganisms is helpful for understanding the antibacterial mechanism of PFDBT-BIMEG. CONCLUSIONS In summary, we have developed a new system based on the CP PFDBT-BIMEG for robust and simple differentiation and killing of microbial pathogens. The PFDBT-BIMEG features an intrinsic intra-FRET backbone and functional BIMEG side chains. The side chains of PFDBT-BIMEG induced both electrostatic attractions and salt-bridge interactions enabling binding with the microorganisms. Depending on the cell surface characteristics of the microorganisms, differences in their interactions with PFDBT-BIMEG were reflected by different aggregation degrees of PFDBT-BIMEG. The aggregatingdirected FRET change of PFDBT-BIMEG over 15 min could be used as a fluorescence signal for distinguishing the type of bacteria. The quantized fluorescence signal was analyzed by LDA to enable accurate classification of the pathogens. Fur-

thermore, on the basis of the interaction mechanism between PFDBT-BIMEG and the microorganisms, suitable strategies could be used to kill the different microbial pathogens rapidly and efficiently through the excellent antibacterial capability of PFDBT-BIMEG. Ultimately, our system based on a single CP will permit both differentiation and killing of microbial pathogens. Such systems could have far-reaching benefits in the diagnosis and treatment of infectious diseases. EXPERIMENTAL SECTION Materials. Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis) and Candida albicans (C. albicans) were purchased from Beijing Bio-Med Technology Development Co., Ltd. Phosphate buffered saline (PBS, pH 7.4) was purchased from HyClone. PFDBT-BIMEG was synthesized according to a published procedure. 52 All other chemicals were purchased from Sigma-Aldrich and used as received without further purification. Sterile water was used throughout the experiments. Characterization. Ultraviolet-visible (UV-vis) absorption spectra were measured with a Hitachi U-3900H spectrophotometer. Fluorescence spectra of the samples were measured with a Hitachi F-7000 spectrometer. Photographs were captured with a Nikon D-7000 camera. Fluorescence microscopy images were acquired with an Olympus FV1000-IX81 confocal fluorescence microscope with 405-nm excitation. The ζpotentials were measured with a Nano ZS90 Zetasizer (Malvern Instruments Ltd., UK) based on laser doppler microelectrophoresis. Calorimetric measurements were conducted on a MicroCal ITC200. A CXE-350 xenon lamp (Beijing OPT Photoelectric Technology Co., Ltd.) was used as a white light source with emission from 400 to 800 nm. Scanning electron microscopy (SEM) images were recorded with a SUPRA 55 SAPPHIRE (Carl Zeiss Jena GmbH). Preparation of Microorganism Solutions. A single colony of E. coli on a solid Luria-Bertani (LB) agar plate was transferred to 10 mL of liquid LB culture medium and grown at 37 °C for 12 h. Following the same method, P. aeruginosa on LB agar plate, S. aureus on a solid nutrient broth (NB) agar plate, B. subtilis on Beef-extract Pepton Yeast-extract (BPY) agar plate and C. albicans on a solid yeast-extract tryptone dextrose (YTD) agar plate were transferred to liquid LB, NB, BPY and YTD culture media, respectively, and incubated under the same conditions. Bacteria were harvested by centrifuging (4500 ×g for 2 min) and washing with PBS three times. The bacteria were resuspended in PBS and diluted to a specified optical density at 600 nm (OD600). These solutions contained ~108 bacteria or ~106 fungi, to give an OD600 of 1.0. Confocal Fluorescence Microscopy Measurements. Portions of 100 μL of the three microorganism solutions (OD 600 = 0.5) were mixed with 10 μL 2.5×10−4 M PFDBT-BIMEG. After incubation at room temperature for 15 min, the mixture was centrifuged (4500 ×g for 2 min) and washed with PBS three times, and the bacteria were resuspended in 100 μL of sterile water. Similarly, 10 μL of sterile water was mixed with 100 μL of the diluted microorganism solutions in control experiments. Portions of the suspensions (3 μL) from each sample were dropped onto microscope slides for fluorescence microscopy measurements.



ζ-Potential Measurements. The three 1-mL microorganism solutions (OD600 = 0.5) were mixed with PFDBT-BIMEG. The final concentration of the conjugated polymer was 2.5×10−5 M. The mixtures were incubated for 1, 3, 5, 10 and 15 min at room temperature. The microorganism solutions were centrifuged 4500 ×g for 2 min) and washed with PBS three times, followed by resuspension in 1 mL of sterile water for ζpotential measurements. Assay by Isothermal Titration Microcalorimetry (ITC). The calorimetric measurements were conducted at 25 °C. To monitor the binding interactions of the PFDBT-BIMEG with E. coli, S. aureus, and C. albicans, the sample cells were initially loaded with 200 μL of the different microorganism solutions (OD600 = 0.5) or PBS, and the reference cell was loaded with PBS. Then a certain concentration of PFDBT-BIMEG solution was injected into the sample cell as a 0.2-μL portion with continuous stirring for 650 rpm, until the interaction process was completed. The concentrations of PFDBT-BIMEG solution (in repeat units, RUs) were 5 μM for E. coli, 10 μM for S. aureus and 30 μM for C. albicans. The heat of dilution was excluded by subtracting the heat collected when PFDBT-BIMEG was injected into the PBS. The detailed ITC analysis process is provided in the supporting information. ROS measurements. To obtain DCFH solution, 2’-7’dichlorofluorescin diacetate (DCFH-DA) solution (0.5 mL, 1 mM in ethanol) was added to the NaOH solution (2 mL, 10 mM). The PFDBT-BIMEG (final concentration is 10 µM) we added to 1.0 mL of the DCFH solution (40 µM). The fluorescence spectra were measured at 1 min intervals while the solution was irradiated with white light (1 mW/cm2). Assessment of Antibacterial Activity. The E. coli, S. aureus and C. albicans solutions were diluted to OD600 = 1.0. A 10-μL portion of 5×10−4 M PFDBT-BIMEG was added into the 100-μL microorganism solutions. The samples were incubated for 1, 5, 10 and 15 min at room temperature. One group was exposed to 25 mW/cm2 white light for 5 min and another group was left in the dark for the same time. The E. coli and S. aureus solutions were further diluted 100,000-fold with PBS. The C. albicans solutions were further diluted 3,000-fold with PBS. Each 100-μL portion of the diluted bacteria solution was spread on a corresponding solid agar plate. After incubation at 37 °C, the number of colony-forming units (CFUs) was recorded. A 100-μL portion of microorganism solution, which was treated by under the same light/dark conditions, was used as a blank sample, followed by dilution of 100,000 of E. coli and S. aureus and 3,000 of C. albicans with PBS. Each 100μL portion of the diluted bacteria solution was spread on the same solid agar plate and incubated as described above. Preparation of SEM Samples. The E. coli, S. aureus and C. albicans solutions were diluted to OD600 = 1.0. 10 μL 5×10−4 M PFDBT-BIMEG solutions were mixed with 100 μL of these microorganism solutions. After incubation times of 1 min for E. coli, 5 min for S. aureus and 15 min for C. albicans at room temperature, the mixtures were exposed to 25 mW/cm2 white light for 5 min. The bacteria were washed with sterile water three times and resuspended in 100 μL of 0.1 vol.% glutaraldehyde for 30 min to fix the morphology of bacteria. Next the bacteria were dropped onto clean silicon slices and dried naturally. Finally, the specimens were washed with sterile water and gradient dehydration was performed with ethanol (15%, 30%, 50%, 70%, 90%, and 100%, each for 10 min). Another group of bacteria without any treatment was used as a blank

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control. All samples were dried in a freeze dryer and coated with platinum before SEM observations.

Supporting Information. ITC analysis process for the model of single set of binding sites. Normalized UV−vis absorption and emission spectrum of PFDBT-BIMEG. Emission spectra of PFDBT-BIMEG as a function of time. Fluorescence images of PFDBT-BIMEG at 1, 3, 5 and 15 min in the water. CLSM images of E. coli, S. aureus, and C. albicans stained with PF-DBTBIMEG. Emission spectra of PFDBT-BIMEG as a function time in the presence of B.subtilis and P.aeruginosa. Partial fluorescence response patterns of PFDBT-BIMEG in the presence of B.subtilis and P.aeruginosa. Canonical score plot for the fluorescence patterns, as derived by LDA. The 95% confidence regions for the individual gram-positive bacteria and gram-negative bacteria are depicted by dashed lines. Fluorescence intensity of DCF in PBS with PFDBT-BIMEG under irradiation. Plate photographs of E. coli, S. aureus and C. albicans incubated with PFDBT-BIMEG in the dark and under continuous white light irradiation. This material is available free of charge via the Internet at http://pubs.acs.org.

*[email protected] *[email protected]

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / #These authors contributed equally.

The National Natural Science Foundation of China, the Fundamental Research Funds for the Central Universities and the State Key Laboratory of Fine Chemicals. The authors declare no competing financial interest.

We thank for the support from the National Natural Science Foundation of China (51673022, 51703009), the Fundamental Research Funds for the Central Universities (FRF-TP-16-026A1) and the State Key Laboratory of Fine Chemicals (KF1613).

PFDBT-BIMEG, poly[(9,9-bis{6ʹ-([N-(trieth ylene glycol methylether)-di-(1H-imidazolium)me thane]hexyl}-2,7-fluorene)-co4,7-di-2-thienyl-2,1,3-benzothiadiazole] tetrabromide; FRET, Förster resonance energy transfer; CPs, conjugated polymers; PF, poly(fluorene); DBT, 1,4-dithienylbenzothiadiazole; EG, ethylene glycol; ITC, isothermal titration microcalorimetry; LDA, linear discrimination analysis.

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Chemistry of Materials SYNOPSIS TOC


Conjugated Polymer with Aggregation-Directed Intramolecular FÃ¶rster

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