On-demand Modulation of Bacterial Cell Fates on Multifunctional

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On-demand Modulation of Bacterial Cell Fates on Multifunctional Dynamic Substrates Inseong Choi, Jinhwan Lee, Wontae Kim, Hyunook Kang, Se Won Bae, Rakwoo Chang, Sunghyun Kim, and Woon-Seok Yeo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18132 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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ACS Applied Materials & Interfaces

On-demand Modulation of Bacterial Cell Fates on Multifunctional Dynamic Substrates Inseong Choi†, Jinhwan Lee||, Wontae Kim‡, Hyunook Kang†, Se Won Bae§, Rakwoo Chang‡, Sunghyun Kim||, and Woon-Seok Yeo†,*

†Department

of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk

University, Seoul, 143-701, Republic of Korea ||

Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, Republic

of Korea

‡Department

§Green

of Chemistry, Kwangwoon University, Seoul 139-741, Republic of Korea

Materials and Process Group, Research Institute of Sustainable Manufacturing

System, Korea Institute of Industrial Technology, Cheonan 31056, Korea KEYWORDS antibacterial surface, dynamic surface, electroactive, low density selfassembled monolayers, mycoplasma

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ABSTRACT This paper reports unprecedented dynamic surfaces based on zwitterionic low density self-assembled monolayers (LDSAMs) of alkanethiolates on gold, which integrate three interconvertible states – bacteria-adherable, bactericidal, and nonfouling states – through electrical modulations. The conformations of alkanethiolates were electrically modulated to generate zwitterionic, anionic, and cationic surfaces, which responded differently to bacteria and determined the fate of bacteria. Furthermore, the reversible switching of multi-functions of the surface was realized for killing bacteria and subsequently releasing dead bacteria from the surface. For practical application of our strategy, we examined the selective antibacterial effect of our surface for eradication of mycoplasma contaminant in contaminated mammalian cells cultures.

INTRODUCTION Dynamic surfaces on which surface properties can be modulated by external stimuli, including temperature, organic reagents, enzymes, pH, and electrical potentials, leading to chemical or conformational conversions of surface molecules.1-3 Chemical conversions, which are mostly involved in chemical reactions, allow accurate and predictable modulation of surface properties; however, in some cases, they are not practical for the use under physiological conditions and for repeatable modulations because of their invasiveness to biological systems and irreversible changes in covalent bonds of surface molecules. In contrast, conformational conversions and subsequent swelling, shrinking, and bending of surface molecules can be reversed with lower energy compared with chemical reactions, and therefore enables changes in surface properties under mild conditions, ensuring noninvasiveness and repeatability.4 Self-assembled monolayers (SAMs) of alkanethiolates on gold have been actively used to construct dynamic surfaces, particularly electrical potential responsive surfaces, because of their convenience in use, synthetic flexibility for introducing 2 ACS Paragon Plus Environment

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various biological/chemical groups, and biocompatibility with many types of biological systems, as well as the intrinsic electrical conductivity of gold.3,5 Therefore, a dynamicity stemming from the conformational conversion of alkanethiolates in SAMs triggered by low electrical potentials can provide unprecedented opportunities in numerous practical applications.6-10 Following the pioneering work by Lahann et al. introducing electrical potential-responsible low density SAMs (LDSAMs) of thiolates with sufficient spaces for the conformational change of thiolates between bent and straight forms,6 a few LDSAMs with charged head groups have been reported for the dynamic control of surface properties such wettability,7 protein absorption,8 and cell adhesion.9-10 To extent the conformational modulations of LDSAMs using electrical potentials, we for the first time developed a multi-functional LDSAM system equipped with both negatively charged carboxylate (CB) head group and positively charged quaternary ammonium (QA) head group to generate three electrically interconvertible surfaces: zwitterionic, anionic, and cationic surfaces. This approach is highly significant because the three charged surfaces with distinct properties can be combined into a single unit. Particularly, we focused on the different effects of the charged surfaces on bacteria and recent themes in biomedical and tissue engineering – preventing microbial infection, microbial colonization, and biofilm formation. Our dynamic surface integrates three interconvertible states – bacteria-adherable, bactericidal, and non-fouling states – through in-situ electrical modulations. The conformations of thiolates in the zwitterionic LDSAMs are electrically modulated to present cationic or anionic charges on the surface, which respond differently to bacteria determining their fate in physiological conditions (Figure 1a). Upon a negative potential treatment, the positive ion-containing alkanethiolates are attracted to the gold electrode and the resulting anionic surface mediates the adhesion of live bacteria.11-12 The cationic surface resulting from 3 ACS Paragon Plus Environment


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a positive potential treatment effectively kills the attached bacteria by inducing the membrane leakage.13 The zwitterionic surface at open circuit (OC) releases dead bacteria from the surface, and at the same time, prevents the adhesion of bacteria in the media.14-15 Figure 1b shows the chemical structure of LDSAMs prepared from protected carboxylate-quaternary ammonium (PCB-QA) disulfide and the conformational changes by electrical potential treatments. Figure 1c shows the structures of PCB-QA disulfide and control disulfides used in this study. MATERIALS AND METHODS Materials. Gold-coated slides were prepared by vacuum deposition of titanium (5 nm for cell culture, 10 nm for surface characterizations) followed by gold (20 nm for cell culture, 50 nm for surface characterizations) onto #2 glass coverslips. Gold nanoparticles (AuNPs) were synthesized as reported previously. 2-Mercaptoethanol (MET), 11-mercaptoundecanoic acid (MUA),

(11-mercaptoundecyl)-N,N,N-trimethylammonium

(MUTA)

bromide,

acetylthiocholine chloride, 1,4-benzoquinone, potassium ferricyanide (III) [K3Fe(CN)6], formaldehyde solution, ampicillin, 2, 4, 6-trihydroxyacetophenone monohydrate (THAP), sinapinic acid (SA), propidium iodide (PI), β-casein, and lysozyme were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Media (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and phosphate buffered saline (PBS) were purchased from WelGENE Inc. (Seoul, Korea). Molecular probe SYTO9, an amine-modified microsphere (0.2 µm, ex. 580/em. 605) and carboxylate-modified microsphere (0.2 µm, ex. 505/em. 515) were purchased from Invitrogen (Carlsbad, CA, USA). Luria-Bertani (LB) medium, BactoTM agar broth, and BBLTM mycoplasma broth base were obtained from BD Difco (Franklin Lakes, NJ, USA). Heat inactivated horse serum was purchased from Thermo Fisher Scientific (San Jose, CA, USA). Acholeplasma laidlawii (A. laidlawii, ATCC 23206) 4 ACS Paragon Plus Environment

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was obtained from ATCC (Koram Biotech, Korea). Bacillus subtilis (B. subtilis, KCTC 1021) was obtained from the Korean Collection for Type Cultures (KCTC). All other chemicals and regents for the organic synthesis were purchased from Sigma-Aldrich unless otherwise specified. Preparation of CB-QA-presenting LDSAMs and MALDI TOF MS analysis. Gold chips were cleaned in piranha solution (sulfuric acid:hydrogen peroxide (30%) = 7:3. Caution! extremely hot and corrosive) before use. A gold chip was immersed in a solution of PCB-QA (0.5 mM in ethanol) for 12 h, rinsed with ethanol, and dried under a stream of nitrogen. The (trifluoromethyl)benzyl protecting group was then removed using 0.5 M KOH solution in the presence of 1 mM MET as a backfilling molecule for 6 h. The resulting CBQA-presenting LDSAM was washed with water and ethanol and dried under a stream of nitrogen. SAM formation and deprotection reactions were verified by MALDI-TOF MS analysis. Mass analysis was performed with an Autoflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) using a Smartbeam laser as an ionization source. All spectra were acquired with an accelerating voltage of -19 kV, a 50 Hz repetition rate, and in positive mode with an average of 1000 shots using THAP (5 mg/mL in acetonitrile) as a matrix. Preparation of CB-QA-, CB-, and QA-presenting HDSAMs. As controls, three types of high-density monolayers were prepared. A gold chip was immersed in a solution of CB-QA disulfide (0.5 mM in ethanol) consisting of CB and QA groups without the bulky protecting group for 12 h. The resulting monolayer was rinsed with ethanol, backfilled with 1 mM MET for 6 h, washed with water and ethanol, and dried under a stream of nitrogen. For CB- and QA-presenting HDSAMs, the chips were immersed in a mixed solution (1 mM in ethanol) of MUA and MET or in a mixed solution (1 mM in ethanol) of MUTA and MET in a ratio of



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80:20 for 12 h, respectively. The resulting CB- and QA-presenting HDSAMs were washed with water, ethanol, and dried under a stream of nitrogen. Contact angle measurement. Contact angle measurements were performed at room temperature by sessile drop method using an Attention goniometer (Biolin Scientific, Sweden) with a liquid droplet of water. Sessile droplet was dropped by lowering 1 µL of distilled water from a syringe needle onto the monolayer. Photographs and data acquisition were performed using analysis software (Attention theta software ver. 4.1.0). Cyclic voltammetry (CV). Electrochemical measurements were performed with an electrochemical analyzer CHI 1000A series multi-potentiostat (CH Instruments, Austin, TX, USA). CV was performed with degassed 0.1 M KOH solution as an electrolyte, monolayer as a working electrode, Pt wire as a counter electrode, and Ag/AgCl as a reference electrode, with a scan range between –0.6 and –1.1 V at a scan rate of 0.2 V/s. The amount of alkanethiolates on gold was determined by integrating the area of the reduction peaks in the voltammograms. Atomistic molecular dynamics simulation. To estimate the area per molecule for selfassembled monolayer (SAM) systems, we performed atomistic molecular dynamics (MD) simulations. SAM prepared from 1-undecanethiol was tested as a model system, and the CBQA-presenting alkanethiolates for HDSAM and PCB-QA-presenting alkanethiolates for LDSAM were used for quantitative comparison with the experimental results. Force fields for surfactant molecules and the ethanol solvent were taken from CHARMM generalized force fields16 and atomic charges were obtained by ab initio quantum calculation with the B3LYP/6-31++G(2d,2p) method using Gaussian 09 software.17 For the initial configuration of each SAM system, 36 surfactant molecules were first placed on square lattice points with spacing of 3 Å to form a monolayer and two monolayers were then aligned in a face-to-face manner to form a symmetric SAM system. To mimic SAMs adsorbed on the Au surface, 6 ACS Paragon Plus Environment

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constraints were applied to sulfur atoms in the surfactant molecules so that their z positions were fixed to z = 1.5 Å in the upper SAM and z = –1.5 Å in the lower SAM. In case of the PCB-QA-presenting LDSAM system, 36 Cl- ions were added to neutralize the simulation system. After the monolayers were set up, ethanol molecules were added in the bulk region. Restraints were also applied to heavy atoms in both surfactant and ethanol molecules so that they could not enter the intermediate region (|z| < 1.5 Å) between the sulfur atoms in the upper and lower SAM. Figure S3 shows initial snapshots of the three SAM systems. The CHARMM software package (ver. c38a1)18 were used for MD simulations for the three SAM systems with a time step of 2 fs. After system equilibration for the initial 1 ns with the NVT ensemble, the production run with the NPT ensemble was conducted at a temperature of 298.15 K and pressure of 1 bar. At least 15 ns trajectories for each SAM system were used to calculate the area per molecule data. Error bars were obtained by block averaging. Impedance measurement. To characterize the conformational changes of CB or QA groups on the surfaces, impedance measurement was performed using negatively charged Fe(CN)63- or positively charged QA-conjugated benzoquinone as an electroactive probe. Electrochemical impedance spectroscopy data were acquired in an electrolyte solution of K3[Fe(CN)6] (1 mM in PBS, pH 7.4) to characterize bending of the CB group or QAconjugated benzoquinone (1 mM in PBS, pH 7.4) to characterize bending of the QA group on the monolayers upon treatment with +0.24 V or –0.3 V, respectively. The monolayers were scanned between 104 and 10-1 Hz, and all impedance spectra were taken at an open circuit potential and AC modulation of 10 mV. As a control CB-QA-presenting HDSAM was prepared and impedance measurement was performed under same experimental conditions as above. Fluorescent microspheres absorption. CB-QA-presenting LDSAM was treated with an electrical potential of OC, +0.2 V, or –0.2 V for 1 min in PBS. The carboxylate-modified 7 ACS Paragon Plus Environment


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microspheres with green fluorescence and amine-modified microspheres with red fluorescence were washed three times with distilled water and centrifuged (16,000 ×g, 1 min), and then diluted to 1 % (v/v) in PBS, pH 7.4. The two diluted fluorescent microspheres were mixed in an equal volume ratio, and 2 mL of the mixture was used to treat the monolayers while maintaining a potential of OC, +0.2 V, or –0.2 V for 10 min. The monolayers were gently rinsed with distilled water and dried under a stream of nitrogen. The selective absorption of fluorescent microspheres was verified using a fluorescence array scanner. Protein and AuNPs absorptions and MALDI-TOF MS analysis. The CB-QApresenting LDSAM was treated with a protein mixture of β-casein (pI 4.6–5.1, 25 µM) and lysozyme (pI 11.0, 12.5 µM) in PBS, pH 7.4, while maintaining a potential of OC, +0.2 V, or –0.2 V for 10 min. The monolayer was gently rinsed with distilled water, dried under a stream of nitrogen, and analyzed by MALDI-TOF MS with SA (5 mg/mL in 50% aqueous acetonitrile) as a matrix. For the AuNP absorption experiment, carboxylate-modified AuNPs and quaternary ammonium-modified AuNPs were prepared as follows. AuNPs (1 mL, 3.3 nM) were washed three times with distilled and centrifuged (16,000 ×g, 3 min), and then incubated with a mixed solution (100 µM in ethanol) of 6-mercapto-1-hexanol and MUA in a ratio of 8:2 for 12 h. The resulting CB-presenting AuNPs with negative surface charges were washed with ethanol and PBS, pH 7.4, by centrifugation (16,000 ×g, 3 min), and suspended in 1 mL of PBS at pH 7.4. Positively charged QA-presenting AuNPs were prepared using 6methoxyhexane-1-thiol and MUTA in a ratio of 8:2 for 12 h as described above. The mixture of CB-presenting AuNPs (1.65 nM) and QA-presenting AuNPs (0.83 nM) in 2 mL of PBS, pH 7.4, was used under the same experimental condition as the protein absorption experiment above, and then analyzed by MALDI-TOF MS without using an organic matrix.


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Bacteria culture. E. coli DH5α and B. subtilis were used as model bacteria to represent gram-negative and gram-positive bacteria. The E. coli DH5α or B. subtilis were first cultured in separate pure cultures overnight at 37°C on LB agar plates. One colony was inoculated into 5 mL of LB medium (20 g/L) and then incubated at 37°C with shaking at 150 rpm (LSI1005R, LabTech, New Delhi, India) for 12 h. This culture was then used to inoculate a second culture in 100 mL of LB medium. When the second suspended culture reached an optical density of 0.8 at 600 nm, bacteria were collected by centrifugation at 3000 ×g for 5 min at 4°C. From the centrifuged sub-cultures, the pellet was washed twice with PBS, pH 7.4, and then pooled. The bacteria pellets of E. coli or B. subtilis were diluted with PBS, pH 7.4, to obtain samples of E. coli of 5 × 108 cells/mL and B. subtilis of 1 × 108 cells/mL. The fresh bacteria suspensions were then used immediately for the bacteria viability assay. Viability assays of E. coli and B. subtilis. The simultaneous treatment of electrical potentials and incubations of bacteria on CB-QA-presenting LDSAMs were achieved by using a custom-designed Teflon cell containing a reservoir with a 28 mm2 circular hole for culture media. The CB-QA-presenting LDSAMs were treated with electrical potentials of OC, +0.2 V, or –0.2 V for 10, 30, or 60 min with E. coli (2 mL of 5 × 108 cells/mL in PBS, pH 7.4) or B. subtilis (2 mL of 1 × 108 cells/mL in PBS, pH 7.4), and gently washed with PBS to remove loosely bound and unbound bacteria cells. Bacteria on the monolayers were stained for 15 min with live/dead assay fluorescent probes containing 3.34 µM SYTO9 and 20 µM PI in water. The monolayers were rinsed with water to remove excess staining fluorescent probes, and live and dead cells on the monolayers were analyzed by fluorescent microscopy through FITC and Texas Red filters. The numbers of live and dead bacteria cell were calculated using ImageJ software (NIH) to determine cell viability and bacterial density. CB-QA-, CB-, and QA-presenting HDSAMs were prepared and then used as controls under



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the same experimental conditions, except for treatment with electrical potentials as described above. E. coli growth on agar plates. The CB-QA-presenting LDSAMs were treated with electrical potentials of +0.2 V or –0.2 V for 60 min with E. coli (2 mL of 5 × 108 cells/mL in PBS, pH 7.4) and gently washed with PBS. The monolayer was dried at room temperature for 20 min and then incubated on an agar plate for 1 h at 37°C. LB media (50 µL) was added and spread after removal of the monolayer from the agar plate, and then the agar plate was incubated for 24 and 48 h. Bacteria cell growth on agar plates was visualized using a Gel-Doc image analysis system. Scanning electron microscopy (SEM). To study morphological differences of E. coli on monolayers depending on electrical potential treatment, SEM was utilized. CB-QApresenting LDSAMs were treated with electrical potentials of +0.2 V, or –0.2 V for 60 min with E. coli (2 mL of 5 × 108 cells/mL in PBS, pH 7.4), and gently washed with PBS. The E. coli on the monolayers was immediately fixed with 4% formaldehyde solution in PBS, pH 7.4, for 30 min, and then washed with water. The samples were coated with platinum and analyzed by SEM. As control experiments, CB- and QA-presenting HDSAMs on gold were prepared and analyzed by SEM under the same experimental conditions except for the treatment with electrical potentials as described above. Cyclic treatment of electrical potentials. CB-QA-presenting LDSAMs were treated with an electrical potential of –0.2 V for 30 min with E. coli (2 mL of 5 × 108 cells/mL in PBS, pH 7.4). The monolayers were washed three times with PBS, pH 7.4, and then treated with electrical potential at +0.2 V for 30 min with E. coli (2 mL of 5 × 108 cells/mL in PBS, pH 7.4). The monolayers were gently washed with PBS and then stained with fluorescent probes for live/dead assay. Bacterial behaviors were examined according to cyclic treatment with


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electrical potentials, including from +0.2 V to –0.2 V, –0.2 V to OC, and +0.2 V to OC, under the same experimental conditions described above. Mammalian cell culture and viability assay. NIH 3T3 cells were cultured in DMEM containing 4.5 g/L D-glucose supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. CB-QA-presenting LDSAMs were treated with electrical potentials of OC, +0.2 V, or –0.2 V for 10, 30, and 60 min with NIH 3T3 (2 mL of 2.5 × 104/mL in 10% DMEM in PBS, pH 7.4), and then gently washed with PBS. NIH 3T3 cells on the monolayers were stained for 15 min with live/dead assay fluorescent probes containing 3.34 µM SYTO9 and 20 µM PI in PBS. The monolayers were rinsed with PBS to remove excess staining fluorescent probes and live and dead cells on the monolayers were analyzed by fluorescent microscopy through FITC and Texas Red filters. Viability assay of mixture of E. coli and NIH 3T3 cells. Bacteria cells and mammalian cells were mixed in 10% DMEM in PBS, pH 7.4 without antibiotics. The E. coli (1 mL of 1 × 109/mL in 10% DMEM in PBS, pH 7.4) and NIH 3T3 cells (1 mL of 5 × 104/mL in 10% DMEM in PBS, pH 7.4) were mixed and treated on CB-QA-presenting LDSAMs with electrical potentials of OC, +0.2 V, or –0.2 V for 1 h. The monolayers were gently washed with PBS and then analyzed by fluorescent microscopy after staining with live/dead assay fluorescent probes containing 3.34 µM SYTO9 and 20 µM PI in PBS. Viability assays of mycoplasma only and mixture of mycoplasma and NIH 3T3 cells. Acholeplasma laidlawii, a type of mycoplasma, was first cultured on an agar plate (20 g/L of BBLTM mycoplasma broth, 20 mL/L of heat-inactivated horse serum, and 15 g/L of BactoTM agar broth) for 5 days at 37°C in a 5% CO2 incubator. One colony was inoculated into 5 mL of liquid medium (20 g/L of BBLTM mycoplasma broth and 20 mL/L of heatinactivated horse serum) and incubated for 5 days at 37°C in 5% a CO2 incubator. This culture (100 µL of liquid media) was then used to inoculate a second culture in 80 mL of 11 ACS Paragon Plus Environment


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liquid medium, which was incubated for 15 h at 37°C in a 5% CO2 incubator. The mycoplasma was collected by centrifugation at 3000 ×g for 5 min at 4°C after incubation. From the centrifuged sub-cultures, the pellet was washed twice with PBS, pH 7.4 and then pooled. The pellets were re-suspended in 20 mL of PBS, pH 7.4, and then diluted by 100-fold into PBS, pH 7.4. The fresh bacteria suspensions were then used immediately under the same experimental conditions as the E. coli viability assay described above. For the viability assay of a mixture of mycoplasma and NIH 3T3 cells, mycoplasma was cultured under the same culture conditions described above, and then diluted by 50-fold into 10% DMEM (in PBS, pH 7.4). This diluted mycoplasma (1 mL of 50-fold diluted cells in 10% DMEM in PBS, pH 7.4) and NIH 3T3 cells (1 mL of 5 × 104/mL in 10% DMEM in PBS, pH 7.4) were mixed and then used under the same experimental conditions as the viability assay described above. RESULTS AND DISCUSSION To generate LDSAMs, we utilized bulky protecting groups to the terminal of alkanethiols which form SAMs on gold via sulfur-gold coordination bonds, and subsequent removal of the bulky protecting group creates a space between alkanethiolates. In this study, we used a (trifluoromethyl)benzyl protecting group reported by Pranzetti et al.10 and designed a heterodisulfide molecule PCB-QA, consisting of a CB with a bulky protecting group and QA (for the synthetic scheme, see the Supporting Information Figure S1). The monolayer formation and subsequent removal of the protecting group was verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contact angle measurement. MALDI-TOF MS has been effectively used to analyze SAMs on gold, a process termed SAMDI MS, which provided information regarding the molecular weights of the alkanethiolates in SAMs.19-21 MALDI analysis of PCB-QA-presenting SAMs showed a major peak at m/z 900.8 [M]+ corresponding to the PCB-QA heterodisulfide (Figure S2a, top left panel). After treatment with 0.5 M KOH solution, MS analysis of the monolayer showed 12 ACS Paragon Plus Environment

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that the peak at m/z 900.8 was absent and new peaks at m/z 462.3 [M]+ and at m/z 490.4 [M]+ arose, corresponding to CB-QA heterodisulfide and QA-terminated homodisulfide (Figure S2a, bottom left panel). In addition, sessile contact angles of water on the monolayer were measured before and after KOH treatment. The contact angle of the initial monolayer was 80° (Figure S2a, top right panel), which dramatically decreased to 47° after KOH treatment (Figure S2a, bottom right panel), indicating the removal of the hydrophobic fluorinecontaining protecting group. Taken together, MS analysis and contact angle measurement indicated that the PCB-QA-presenting monolayer was successfully formed on gold, and subsequent removal of the bulky protecting group yielded CB-QA-presenting SAMs. To verify that PCB-QA-presenting SAMs were in low density of alkanethiolates, we performed quantitative analysis using cyclic voltammetry and molecular dynamics simulation. Figure S2b shows the cyclic voltammograms recorded for the CB-QA-presenting high density SAMs (HDSAMs, solid line) prepared using the CB-QA control molecule and PCB-QA-presenting LDSAMs (dot line) prepared using PCB-QA disulfide. A cathodic peak at –950 mV for the CB-QA-presenting SAMs stemming from reductive desorption of alkanethiolates from gold significantly decreased for PCB-QA-presenting SAMs, indicating a lower packing density of alkanethiolates on PCB-QA-presenting SAMs compared to on CBQA-presenting SAMs. The amount of alkanethiolates on gold was determined by integrating the area of the reduction peaks in the voltammogram, which was summarized as the area per molecule (Am) and loading density in Figure S2c. The simulation results of the model systems were in good agreements with corresponding experiments, indicating that the essential mechanism of the SAM structure was captured in the model systems. For details regarding molecular dynamics simulation, see Figure S3 and the discussion in the supporting information. The loading density of 3.0 × 1013 cm-2 for QA groups in CB-QA-presenting



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LDSAMs was also well comparable with the suggested data for effective antibacterial effect.22 The ability of zwitterionic CB-QA-presenting LDSAMs to switch surface states by conformational changes of thiolates in response to applied potentials was investigated by impedance measurements, fluorescence, and mass analysis. A mixture of two materials with a positively or negatively charged surface was incubated with zwitterionic LDSAMs and treated with electrical potentials of OC, +0.2 V, and –0.2 V at pH 7.4 in PBS. The initial zwitterionic monolayer at OC prevented absorption of both negatively and positively charged materials via its non-fouling property, whereas a positively or negatively charged material was selectively absorbed on the CB- or QA-exposed monolayer via electrostatic interactions (Figure 2a). Electrochemical impedance measurement using negatively charged Fe(CN)63- or positively charged QA-conjugated benzoquinone as an electroactive probe revealed electrontransfer resistance by a redox process indicating the proximity of the electroactive probe to electrodes.23 Figure 2b shows the electrochemical impedance spectra obtained for CB-QApresenting HDSAM (black line) or LDSAM (red line). The electron-transfer resistance on HDSAM was higher than that on LDSAM at +0.24 V in the presence of K3Fe(CN)6 (1 mM, in PBS, pH 7.4), denoting that negatively charged Fe(CN)63- was accumulated on the LDSAM (Figure 2b, left panel). This indicates that the positive electrical potential treatment exposed positively charged QA groups on the LDSAM via selective bending of the CBcontaining alkanethiolates and subsequent preferential absorption of the negatively charged probe, contributing to active electron transfer between the probe and gold electrode. Similarly, electron-transfer resistance on HDSAM was higher than that on LDSAM when positively charged QA-conjugated quinone (1 mM, in PBS, pH 7.4) was used at –0.30 V, indicating accumulation of the positively charged probe on the gold electrode (Figure 2b, right panel). For the synthetic scheme of QA-conjugated benzoquinone, see Figure S1. We 14 ACS Paragon Plus Environment

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further verified the conformational change of LDSAM and selective absorption of charged materials using equal concentration mixtures of fluorescent microspheres (CB-modified microspheres with green fluorescence and amine-modified microspheres with red fluorescence), proteins (β-casein with pI 4.6–5.1 and lysozyme with pI 11.0), and gold nanoparticles (AuNPs) (CB-modified AuNPs and QA-modified AuNPs). As expected, green fluorescence was only observed on the cationic surface, which resulted from positive potential treatment (Figure 2c, top, middle), whereas red fluorescence was only observed on the anionic surface stemming from negative potential treatment (Figure 2c, bottom, right). In addition, β-casein and lysozyme which have a negative net charge and a positive net charge at pH 7.4, respectively, preferentially bound on CB- or QA-exposed LDSAM depending on the electrical potential treatments evidenced by MALDI-TOF MS analysis (Figure 2d). We also observed similar results as above using a mixture of CB-modified AuNPs and QAmodified AuNPs (Figure 2e). Notably, no detectable fluorescences or mass peaks were observed at OC indicating the non-fouling properties of zwitterionic surfaces. The results obtained above clearly imply that CB-QA-presenting LDSAMs were successfully formed via hydrolytic removal of bulky protecting groups, and conformation changes was induced according to applied electrical potentials. In addition, the surfaces showed selective absorptions and non-fouling properties depending on their surface charges for various materials. We next demonstrated the antibacterial effect of the LDSAM with zwitterionic, cationic, and anionic states following electrical adjustments by observing bacteria growth on agar plates (Figure S4). Gram-negative bacteria Escherichia coli (E. Coli, 5 × 108 cells/mL in PBS, pH 7.4) were incubated on CB-QA-presenting LDSAM with electrical potential treatments for 1 h, released, and spread on agar plates. The images of colonies on the agar plates were obtained after incubation for 24 h (Figure S4, first row) and 48 h (Figure S4, 15 ACS Paragon Plus Environment


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second row) at 37°C. As a control, LB media was spread on an agar plate. Because of the non-fouling properties of the zwitterionic surface and antibacterial effect of the QA group, no colonies of E. coli were observed on agar plates for the monolayers treated with OC and +0.2 V, whereas live E. coli from the CB monolayer at –0.2 V grew on the agar, and thus numerous colonies were observed. Next, we modulated bacteria cell fates on the CB-QA-presenting LDSAM by electrical potential treatment. As controls, CB-QA-, CB-, and QA-presenting HDSAMs were prepared using CB-QA disulfide, (11-mercaptoundecyl)-N,N,N-trimethylammonium (MUTA), and 11-mercaptoundecanoic acid (MUA), respectively (for the structures, see Figure 1c). The SAMs were treated with E. coli (5 × 108 cells/mL in PBS, pH 7.4) and incubated for various times from 10 to 60 min. After brief rinsing, bacteria on the SAMs were stained with the live/dead assay fluorescent probes SYTO9 (green, for live cells) and propidium iodide (PI, red, for dead cells), which have been widely used to characterize live and dead bacteria according to their membrane integrity. On CB-QA-presenting LDSAM and CB-QApresenting HDSAM, only a few E. coli were observed, demonstrating the inertness of the zwitterionic surfaces (Figure 3a and Figure S5a, left panel). While QA-exposed LDSAM following treatment with an electrical potential of +0.2 V and QA-presenting HDSAM prepared from MUTA showed efficient bactericidal properties (Figure 3a and Figure S5a, middle panel), numerous live bacteria were observed on CB-exposed LDSAM following treatment with an electrical potential at –0.2 V and CB-presenting HDSAM prepared from MUA indicating the bacteria-adherable properties (Figure 3a and Figure S5a, right panel). Quantitative analysis indicated that antibacterial ability of absorbed E. coli on both QApresenting SAMs increased with incubation time showing more than 99.5% killing efficiency after 1 h (Figure 3b and Figure S5b), and bacterial cell density gradually increased over time on both the QA- and CB-presenting SAMs (Figure 3c and Figure S5c). To determine whether 16 ACS Paragon Plus Environment

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the cationic surface readily induced damage of the cell wall, scanning electron microscopy (SEM) was used to visualize morphological differences of the bacteria (Figure 3d). The E. coli were incubated on MUTA and MUA control monolayers and CB-QA-presenting LDSAMs with electrical potential treatment of +0.2 V and –0.2 V for 1 h. Bacteria cells in contact with cationic surfaces of the MUTA monolayer (Figure 3d(i)) and the +0.2 V-treated QA-presenting LDSAM (Figure 3d(iii)) lost their cellular integrity by leakage of intracellular components. However, the morphology of E. coli adhered to the anionic surfaces of the MUA monolayer (Figure 3d(ii)) and the –0.2 V-treated CB-presenting LDSAM (Figure 3d(iv)) showed a clear edge and intact shape without any disruption of the cell wall. Overall, this series of experiments and the results clearly demonstrate that bacterial cell fates can be modulated on-demand on CB-QA-presenting LDSAMs via electrical controls, as evidenced by the different bacterial responses to zwitterionic, cationic, and anionic states as shown in Figure 1. The antibacterial activity and bacterial density on LDSAM and the control HDSAM toward gram-positive bacteria Bacillus subtilis (B. subtilis) were also evaluated (Figure S6). We observed that antibacterial efficiency against B. subtilis was significantly lower than that against E. coli on the QA-presenting surfaces. Although the antibacterial mechanism of QApresenting surfaces remains unclear, differences in the cell wall structure between gramnegative and gram-positive bacteria may affect the mechanism. Various studies have reported the antibacterial effect of materials with positively charged groups against gram-negative bacteria.24-25 A suggested bactericidal mechanism involves exchange of divalent cations on outer membrane of gram-negative bacteria to hold the negatively charged lipopolysaccharide network to positively charged groups, leading to destabilization of the outer membrane and bacterial death. Another possible mechanism involves damage to the gram-negative bacteria cell membrane by penetration of positively charged groups, leading to leakage of cytoplasmic 17 ACS Paragon Plus Environment


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components and eventual bacterial death.24,26 On the contrary, gram-positive bacteria have a thicker layer of peptidoglycan compared to gram-negative bacteria which prevents the penetration of charged groups, and therefore, may account for lower antibacterial efficiency against B. subtilis compared to that against E. coli on the QA-presenting surfaces.24 Next, the reversible change of surface ionic states of CB-QA-presenting LDSAMs in consecutive electrical treatments was evaluated to determine the recyclable dual-functions to kill bacteria and release dead bacteria from the surface. CB-QA-presenting LDSAMs with E. coli were treated with an electrical potential of –0.2 V followed by +0.2 V. A live/dead assay revealed an efficient antibacterial effect with high cell density, clearly demonstrating that the cells initially adhered to the CB-presenting monolayer at –0.2 V were killed by the exposed QA groups induced by treatment with +0.2 V (Figure 4a(i)). However, electrical potential treatment of +0.2 V to –0.2 V enabled adhesion of both live and dead cells with high cell density (Figure 4a(ii)). This observation corresponded well with the results showing that the CB-presenting monolayer leads to cell adhesion and ensures cell viability, while the QApresenting monolayer kills the bacteria after cell adhesion. When CB-QA-presenting LDSAMs were treated from –0.2 V to OC (Figure 4a(iii)) or from +0.2 V to OC (Figure 4a(iv)), only a few live or dead cells were adsorbed, respectively, indicating the non-fouling effect of the zwitterionic surface at OC. Quantitative analyses of live and dead cells (circle, solid line) and their densities (square, dot line) on CB-QA-presenting LDSAMs with cyclic treatment of electrical potentials are shown in Figure 4b. These results are highly significant because they validate that our surface can not only actively kill bacteria, but also release absorbed dead bacteria via electrical potential adjustments in the same batch without a decrease in activity. Recently, various antibacterial materials containing QA groups such as hydrogels27 and surface-modified nanomaterials have been developed.28-30 However, the practical applications 18 ACS Paragon Plus Environment

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of these materials have been limited because of their cytotoxicity against mammalian cells.29 In this regard, we confirmed the biocompatibility toward mammalian cells and selective killing effect toward bacteria without compromising the viability of mammalian cells of our system (Figure S7). As a model mammalian cell line, NIH 3T3 fibroblasts were treated on the CB-QA-presenting LDSAM with electrical potential treatments for various times from 10 to 60 min. Figure S7a shows representative fluorescence microscopy images of live (green) and dead (red) cells of NIH 3T3 on LDSAMs. Only a few adhered cells were observed on the CB-QA-presenting LDSAM because of the non-fouling property (Figure S7a, left panel). However, a large number of live cells were observed both on the QA- (Figure S7a, middle panel) and CB-exposed surfaces (Figure S7a, right panel) following treatment with an electrical potential of +0.2 V or –0.2 V, respectively. Next, the mixture of E. coli and NIH 3T3 cells were treated on the LDSAMs, and a dead/live cell assay was performed. E. coli and NIH 3T3 cells did not adhere to inert CB-QA-presenting LDSAM (Figure S7b, left panel), while a number of E. coli and NIH 3T3 cells adhered to both QA- (Figure S7b, middle panel) and CB-exposed surfaces (Figure S7b, right panel). Furthermore, the QA-exposed surface showed selective cytotoxicity against bacteria and the CB-exposed surface showed negligible cytotoxic effects on both E. coli and NIH 3T3 cells, validating that our system has selective antibacterial activity without compromising the viability of mammalian cells. The selective killing of bacteria on the surfaces can be explained by the differences in structural lipid compositions as well as in the cell surface charge between mammalian cells and bacteria cells.31-32 Negatively charged lipids such as phosphatidylglycerols and lipopolysaccharides are

abundant

in

the

bacterial

membrane,

while

zwitterionic

lipids

such

as

phosphatidylcholines and sphingomyelins are major components of the mammalian cell membrane. Therefore, the negatively charged bacteria cells preferentially bound to positively charged QA-exposed monolayers. Furthermore, cholesterols in the mammalian cell 19 ACS Paragon Plus Environment


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membrane play a role as a stabilizer to reduce membrane disruption, preventing destruction of the membrane by the QA group. As a practical application of our system, we utilized our surface for mycoplasma eradication from mammalian cell culture. Mycoplasma, the smallest and simplest selfreplicating bacteria microorganism, are well-known major contaminants of cell culture with contamination rates of 5–35% and as high as 65–80% depending on the location.33-35 Mycoplasma infection causes various cytopathic effects on cultured cells, such as changes in cell function and metabolism, limiting accurate biological analysis.34 In addition, mycoplasma infection for an extended period induces genetic abnormalities in infected cells, and many types of cancers are associated with mycoplasma, such as colon, gastric, lung, prostate, and renal cancer.36-37 Because mycoplasma lacks a peptidoglycan-based cell wall, and thus, theoretically does not respond to commercial antibiotics, various methods for eliminating mycoplasma from infected cell cultures have been developed, including treatment with specialized chemical molecules,38 in vivo passage of cells in nude mice,39 co-culture of infected cells with macrophages,40 and repeated treatments with guinea pig or rabbit serum.41 However, some of these methods are detrimental to mammalian cells and require complicated equipment and protocols. In this study, we used Acholeplasma laidlawii, one of the five most common cell culture contaminants, to evaluate the mycoplasma killing ability of our system. As shown in Figure 5a, CB-QA-presenting LDSAM showed mycoplasma bacteria-adherable, bactericidal, and non-fouling properties controlled by electrical modulation. Quantitative comparisons of antibacterial efficiency and attached cell density on LDSAM (Figure 5b and 5c) indicated that more than 99.8% of mycoplasma were dead on QA-presenting LDSAM, and cell density gradually increased over time on both QA- and CB-presenting LDSAM. Based on the excellent antibacterial effect against mycoplasma, we utilized our system to selectively eradicate mycoplasma contaminants from mammalian cell culture. A non-fouling 20 ACS Paragon Plus Environment

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effect towards mycoplasma and mammalian cells was observed on CB-QA-presenting LDSAM (Figure 5d, left panel), and numerous cells were adhered to CB-exposed LDSAM (Figure 5d, right panel). Notably, QA-exposed LDSAM showed a significant antibacterial effect against mycoplasma, while mammalian cells remained intact (Figure 5d, middle panel). Note that the yellow fluorescence was due to the superposition of dead mycoplasma (red) and live mammalian cells (green). These results indicate that mycoplasma cells, which are not susceptible to commercial antibiotics, can be selectively and efficiently removed from mammalian cell cultures. CONCLUSION In conclusion, we prepared zwitterionic LDSAMs of anionic CB-containing and cationic QA-containing alkanethiolates by utilizing bulky protecting groups. The conformations of alkanethiolates in LDSAMs were electrically modulated to result in zwitterionic, anionic, and cationic surface states. These three interconvertible states showed bacteria-adherable, bactericidal, and non-fouling properties, respectively, and were used for on-demand, in situ determination of bacteria cell fates under physiological conditions. Furthermore, we demonstrated selective antibacterial activity in a contaminated culture of mammalian cells for selective eradication of mycoplasma contaminant. We believe our multi-functional dynamic surfaces can be practically used for various research applications including biomedical/tissue engineering, healthcare devices, and basic cell studies.



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Figure 1. (a) Schematic representation of our strategy for on-demand modulation of bacteria cell fates on electro-active substrates and (b) the chemical structures on the surface. (c) Structures of disulfides and alkanethiols used in this study.


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Figure 2. Verification of conformational changes depending on electrical potentials. (a) Schematic representation of three surface states by treatment of different electrical potentials and selective absorption of surface-charged materials. (b) Electrochemical impedance spectra obtained on HDSAM (black line) or LDSAM (red line) of CB-QA at +0.24 V in the presence of 1 mM K3Fe(CN)6 (left) or at –0.30 V in the presence of 1 mM QA-conjugated quinone (right). (c) A mixture of CB-modified green fluorescent microspheres and amine-modified red fluorescent microspheres was incubated with zwitterionic LDSAMs under the same experimental conditions as described in (a). (d) A mixture of β-casein (pI 4.6–5.1) and lysozyme (pI 11.0) in PBS (pH 7.4) was incubated with zwitterionic LDSAMs under the same experimental conditions as described in (a). (e) A mixture of CB-modified AuNPs and QA-modified AuNPs was also tested and showed similar results as above.



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Figure 3. (a) Fluorescence microscopy images of live (green) and dead (red) cells of E. coli on CB-QA-presenting LDSAM with electrical potential treatments. (b, c) Quantitative analysis of antibacterial efficiency and bacterial density of E. coli in Figure 3(a). (d) SEM images of E. coli on the monolayers of MUTA (i) and MUA (ii) as control monolayers, and CB-QA-presenting LDSAMs with electrical potential treatments of +0.2 V (iii) and –0.2 V (iv) for 1 h. Highly magnified SEM images of E. coli are shown in the right panel.


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Figure 4. (a) Fluorescence microscopy images of live (green) and dead (red) cells of E. coli on CB-QA-presenting LDSAMs with cyclic treatment of electrical potentials from (i) –0.2 V to +0.2 V, (ii) +0.2 V to –0.2 V, (iii) –0.2 V to OC, and (iv) +0.2 V to OC. (b) Quantitative analysis of antibacterial efficiency (circle, solid line) and bacterial density (square, dot line) for Figure 4(a). The error bars show the standard deviation determined from three independent assays.



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Figure 5. Mycoplasma (a) or mycoplasma/NIH 3T3 co-culture (d) were treated on CB-QApresenting LDSAMs with electrical potential treatments of OC (left panel), +0.2 V (middle panel), and – 0.2 V (right panel). Note that the yellow fluorescence was due to the superposition of dead mycoplasma (red) and live mammalian cells (green). (b, c) Quantitative analysis of antibacterial efficiency and bacterial density for mycoplasma in Figure 5(a).


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthesis of functional molecules, characterization of monolayers, dynamic simulation and extended evaluation of antibacterial activity AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (W.-S.Y) Author Contributions I.C. and W.-S.Y. conceived the project, analyzed the data, and wrote the paper. J.L. performed the impedance measurement experiment. R.C. and W.K. performed molecular dynamics simulation. I.C. performed all other experiments including synthesis, MS analysis, electrical analysis, fluorescence measurements, and cell culture. H.K., S.W.B., R.C., and S.K. analyzed and discussed the data. W.-S.Y. oversaw the project. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported by the Priority Research Centers Program (2009-0093824) through the National Research Foundation (NRF) of Korea (NRF-2016R1D1A1A09918111) funded by the Ministry of Education and by the Agri-Bio Industry Technology Development



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On-demand Modulation of Bacterial Cell Fates on Multifunctional

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