Performance of NIR-Mediated Antibacterial Continuous Flow

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Performance of NIR-Mediated Antibacterial Continuous Flow Microreactors Prepared by Mussel-Inspired Immobilization of Cs0.33WO3 Photothermal Agents Young Kwang Kim,† Eun Bi Kang,‡ Sung Min Kim,‡ Chan Pil Park,§ Insik In,*,†,∥ and Sung Young Park*,†,‡ †

Department of IT Convergence, ‡Department of Chemical & Biological Engineering, and ∥Department of Polymer Science & Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea § Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea S Supporting Information *

ABSTRACT: An antibacterial continuous flow microreactor was successfully prepared by sequential mussel-inspired surface engineering of microchannels by using catechol-grafted poly(N-vinylpyrrolidone) and immobilization of near-infrared active Cs0.33WO3 nanoparticles inside the polydimethylsiloxane(PDMS)-based microreactors. Excellent phothothermal antibacterial acitivity over 99.9% was accomplished toward Gram-positive and -negative bacteria upon near-infrared irradiation during continuous operation up to 30 days. This was achieved without releasing Cs0.33WO3 nanoparticles from the surface of the microchannels, confirming the robust immobilization of photothermal agents through the mussel-inspired chemistry. The cleaning of used microreactors was easily attainable by simple acid treatment to release immobilized photothermal agents from the surface of the microchannels, enabling efficient recycling of used microreactors. KEYWORDS: microreactor, antibacterial, photothermal, near-infrared irradiation, mussel-inspired chemistry

1. INTRODUCTION Intensive research interest has been focused on the formulation of a new generation of nanomaterial-based antimicrobials because the increasing antimicrobial resistance of pathogenic bacteria has rendered the traditional antimicrobials ineffective.1 Antimicrobials such as silver nanoparticles (AgNPs) and polyelectrolytes such as chitosan that intrinsically show antibacterial activity have been widely utilized as antibacterial nanomaterials.2,3 However, other types of organic or inorganic nanomaterials such as metal nanostructures (Au and Pd), semiconductor nanomaterials (TiO2 and CuS), carbon nanomaterials (carbon nanotube and graphene oxide), and conjugated polymers (polyaniline and poly(vinylpyrrolidone)) that extrinsically show photothermal antibacterial performance through the absorption of near-infrared (NIR) light have been recently developed as alternative antimicrobials with bactericidal properties potentially applicable to water disinfection.4−11 Ideal photothermal antibacterial agents should display strong optical absorbance in the NIR region, which is hardly attainable for most biological tissues.12 Therefore, many research efforts have been focused on providing nanomaterials or their hybridized nanostructures to accomplish strong NIR absorption, potential long-term stability, and efficient transfer of absorbed NIR energy into heat.13 However, little attention has been paid to the practical application of photothermal © XXXX American Chemical Society

antibacterial agents to water disinfection from the view-points of both recyclability and continuous operation. Generally, the complete separation or recovery of antibacterial nanomaterials after each bactericidal event from aqueous environments is highly discouraging because the small sizes of the nanomaterials hamper their effective isolation from water. Few studies have reported the preparation of recyclable and reusable antibacterial agents through either the hybridization of photothermal antibacterial agents with magnetic NPs such as magnetite (Fe3O4) and maghemite (γ-Fe2O3), or the direct synthesis of coherently antibacterial and magnetically responsive nanomaterials such as Hägg iron carbide (Fe5C2) NPs.14−16 However, the demonstration of a continuously operating antibacterial platform has hardly been attempted until now. Very recently, an easily recyclable and continuously operating photocatalytic membrane was successfully fabricated through the wrapping of photocatalytic akaganéite (β-FeOOH) nanorods on polydopamine (PDA)-coated porous substrates.17 The β-FeOOH nanorods-immobilized membrane reactor was very effective for the continuous photodegradation of organic dyes under the exposure of direct sunlight, which enables the design of a light-driven wastewater treatment system. But, the Received: December 26, 2016 Accepted: January 3, 2017 Published: January 3, 2017 A

DOI: 10.1021/acsami.6b16634 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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microreactors were prepared according to the previous report.28 In details, the channel shape is rectangular and 85 μm (height) × 300 μm (width). The total channel length in a single microreactor is 120 cm. In the initial part of a microwave, a waved channel with a length of 12 cm was constructed for the mixing and dispersion of CA-PVP or Cs0.33WO3 NPs. A silicon wafer was spin-coated with a UV-curable SU-8-50 to get the 85 μm layer. After a softbake at 110 °C, the silicon wafer was exposed to UV-light through a specially designed photomask, and postbaked at 110 °C. Subsequently, the wafer was developed using SU-8 developer and cleaned. A mixture containing the silicon elastomer and the curing agent (10:1 weight ratio) was poured onto the silicon master and baked for 4 h at 60 °C. After the layer was peeled off from the silicon master, the inlets and outlet of the microfluidic device were punched. The PDMS was bonded on a glass substrate after both surfaces were treated by oxygen plasma for 1 min. After bonding, the PDMS microreactor was heated up in an oven for 12 h at 110 °C to achieve higher bonding strength. Figure S2 was provided to visualize the photo image of typical PDMS-based microreactor in this study. Poly(vinylpyrrolidone) (PVP, Mw ∼ 40 000), 2-chloro-3′,4′dihydroxyacetophenone (CA), cesium tungsten oxide (Cs0.33WO3), ethanol, trizma base (99%), trizma HCl, (99%), deuterium oxide (D2O), S. aureus, E.coil, MRS, LB, and agar were purchased from Sigma-Aldrich Reagent Company and used without further purification. SYTO 9 green was purchased from Thermo Fisher Scientfic Company and used without further purification. Fourier transform infrared (FT-IR) spectra were obtained from a Nicolet IR 200 (USA). Field emission scanning electron microscopy (FE-SEM) analysis was performed with a JSM-7610F ultra high resolution field emission scanning electron microscope from JEOL Ltd. Transmission electron microscopy (TEM, JEM-2100F, JEOL, Akishima, Tokyo, Japan) was performed at an operating voltage of 200 kV. X-ray diffraction was performed using a Bruker Instrument with Cu Kα radiation (λ = 1.5405 Å). Attenuated total reflectance infrared (ATR-IR) spectra were obtained with a Nicolet IR 200 (USA). NIR laser irradiation (808 nm) (PSU-III-LRD, CNI Optoelectronics Tech. Co. LTD, China) was used as a method to evaluate the photothermal activity. Confocal microscopy image analysis was performed with a LSM510 confocal laser scan microscope (Carl Zeiss, Germany). 2.2. Synthesis of CA-PVPs. A 15.0 g sample of PVP and a 9.16 g sample of CA were dissolved in 100 mL of ethanol in a 250 mL flask. The solution was stirred under nitrogen purging and heated at 70 to 80 °C for 24 h. After cooling to room temperature, most of the solvent was evaporated by rotary evaporator under reduced pressure, resulting in crude CA-PVP powder. After dissolving the crude CA-PVP powder into ethanol, a bright brown CA-PVP powder was obtained from the precipitation into diethyl ether. After vacuum drying for 12 h, the yield was 86.1%. 2.3. Decoration of CA-PVP on the Microchannels of the PDMS Microreactor. For decoration of the PDMS-based microchannels, fresh CA-PVP solution in Tris buffer was prepared by dissolving 50 mg of CA-PVP into 1 mL of Tris buffer (100 mM, pH 8.5) just before use. Then, the inner volumes of the microchannels were fully charged with CA-PVP solution. Both the inlet and outlet of the microreactor were tightly sealed to minimize the leakage of CAPVP solution. After 24 h at room temperature, the inner volumes of the microchannels were repeatedly cleaned with a continuous flow of deionized water and dried under reduced pressure at room temperature, resulting in CA-PVP decorated microchannels. 2.4. Immobilization of Cs0.33WO3 Nanoparticles on CA-PVP Decorated Microchannels. The dispersion of Cs0.33WO3 nanoparticles with different concentrations was prepared by adding 0.5, 1.0, and 5.0 mg of Cs0.33WO3 nanoparticles into 1 mL of Tris buffer (100 mM, pH 8.5). After brief sonication in a bath sonicator, 1 mL of Cs0.33WO3 nanoparticle dispersion was injected into the CA-PVP decorated microchannel. Both the inlet and outlet of the microreactor were tightly sealed to minimize the leakage of the Cs0.33WO3 nanoparticle dispersion. After 24 h at room temperature, the microchannels were repeatedly cleaned with a continuous flow of

incorporation of photocatalysts into the surface of the membrane might show certain limitations due to the restricted catalyst immobilization on the flat membrane. This spatial limitation can be overcome by adopting the design of a continuously operating microreactor system because microreactors have much larger surface areas due to the presence of microchannels inside the microreactor.18 In our previous study, mussel-inspired immobilization of photocatalytic TiO2 NPs inside polydimethylsiloxane (PDMS)-based microreactors was successfully attempted. TiO2-immobilized microreactors accomplished continuous photocatalytic degradation of organic dyes for up to 30 days without any noticeable release of TiO2 NPs.19 In this study, a continuously operating antibacterial microreactor was first constructed through the mussel-inspired immobilization of Cs0.33WO3 NPs on PDMS-based microreactors. Tungsten bronze-type compounds (MxWO3, where M = Cs, K, Na, etc.) are well-known transition metal oxides showing NIR shielding ability due to the strong NIR absorption originating from localized surface plasmon resonances (LSPRs), which qualifies Cs0.33WO3 as excellent candidates for photothermal wiping of bacteria.20−22 Tungsten suboxide (WO3-x) similarly possesses mixed-valence W ions (W6+ and W5+) and has been successfully utilized as a rapid and effective NIRdriven photothermal agent for the ablation of cancer cells in vivo.23−25 In addition to the feasible continuous antibacterial operation up to 30 days, the construction of an antibacterial microreactor through the immobilization of Cs0.33WO3 into the microchannels of PDMS-based microreactors provides extra benefits for enhanced photothermal antibacterial performance compared with the batch-type system. To maximize NIR absorption of photothermal agents incorporated in batch reactors, homogeneous dispersion or external agitation is a prerequisite because any aggregation or settlement of photothermal agents in batch reactors could result in diminished generation of photothermal heat due to possible NIR shielding by the agents themselves or less efficient heat transfer to the bacteria culturing media. This behavior is less expected in microreactor systems because of the uniform and ultrathin decoration of photothermal agents through the microchannels inside the microreactor together with enhanced mixing performance of continuous flow microreactors by chaotic advection or turbulence.26 Because of the above benefits of antibacterial microreactors, Cs0.33WO3-immobilized continuous flow microreactors were designed for effective photo killing of more than 99.9% Gram-positive and -negative bacteria upon NIR irradiation (808 nm) in a bacteria concentration of 1 × 105/mL at a flow rate of 50 μL/min. This antibacterial efficiency of photothermal sterilizing agentimmobilized microreactors was substantially preserved without any noticeable release of photothermal agents into aquatic media during microreactor operation lasting 30 days. The easily achievable scale-up processes for microreactors compared with batch-type reactors might bring photothermal agent-immobilized antibacterial microreactors closer to practical applications in water disinfection on a mass scale.

2. EXPERIMENTAL SECTION 2.1. Materials and Characterization. Cs0.33WO3 NPs with lateral dimension, synthesized from ammonium tungstate as previously reported, was confirmed by high resolution transmission electron microscopy (HR-TEM), showing the average size in the range of 20.27 ± 3.7 nm (Supporting Information, Figure S1).27 PDMS-based B


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Scheme 1. Schematic Drawings for (a,b) the Preparation of Cs0.33WO3-Immobilized Microreactor and (c) Antibacterial Activity of Cs0.33WO3-Immobilized Microreactor through the Photothermal Effect upon NIR Irradiation

distilled water. Finally, vacuum drying under reduced pressure at room temperature produced Cs0.33WO3-immobilized microchannels. 2.5. SYTO and Propidium Iodide Costained Live/Dead Bacteria Staining Assay. Imaging of the photothermal cytotoxicity was carried out using the SYTO and propidium iodide (PI) costaining method. In detail, the samples containing S.aureus and E. coli were flowed into the Cs0.33WO3-immobilized microchannels. In one typical sample, 5 mL of bacteria at a density of 1 × 105, 1 × 106, and 1 × 107 bacterial/mL was flowed into the Cs0.33WO3-immobilized microchannels at a flow rate of 0.05, 0.1, and 0.2 mL/min, respectively. The microchannels were then irradiated with an 808 nm laser at a power density of 80 W/cm2. The bacteria were costained with SYTO and PI for another 30 min. Finally, confocal microscopy was used for imaging the stained (live/dead) bacteria. 2.6. Bacteria MTT Assay. The S. aureus and E. coli solutions were flowed onto Cs0.33WO3-immobilized microchannels. Typically, 5 mL of bacteria at a density of 1 × 105, 1 × 106, and 1 × 107 bacteria/mL was added to Cs0.33WO3-immobilized microchannels at a flow rate of 0.05, 0.1, and 0.2 mL/min, respectively. The microchannels were then irradiated with an 808 nm laser at a power density of 80 W/cm2. The treated bacteria were harvested by centrifugation, rinsed once with PBS solution and recentrifugated. The bacteria were then stained with 20 μL of SYTO solution. The stained bacteria were incubated in the dark at 37 °C with shaking for 4 h followed by centrifugation and washing. Finally, 200 μL of MTT solution was added to the precipated bacteria and absorbance was observed at 500 nm wavelength using a microplate reader (Varioskan flash, Thermo Fisher Scientific). The control was CA-PVP with NIR irradiation.

Si−O−Si, C−H, and C−Si linkages are not reactive in typical organic synthesis, mussel-inspired immobilization of preformed Cs0.33WO3 nanoparticles into the microchannels of PDMSbased microreactors was attempted by mimicking strongly adherent catechol agents.31−33 Several catecholic small compounds such as dopamine have been successfully utilized for the generation of “polymeric glue”-like polydopamine on several types of substrates including polymers, metals, and metal oxides possibly through the formation of a suprastucture of catecholic compounds either by covalent oxidative polymerization or physical self-assembly pathway in basic media.32 In this study, catechol-grafted poly(N-vinylpyrrolidone) (CAPVP) was employed instead of dopamine or its analogues because precise control of the number density of “glued” nanomaterials on the specific substrate is easily attainable through control of the number of CA moieties in CA-PVP.34 CA-PVP was synthesized by quaternization reactions between PVP (Mw of 40 000 Da) and CA similarly to previous reports.19 The intense peaks at 255 and 360 nm in UV−vis spectra represent the existence of a catechol moiety, which indicates π−π* electron transitions in phenolic compounds as shown in Figure S3a. The number of catechol moieties in PVP was 140 units estimated by NMR spectra in the area 6.5−7.5 ppm, in which the efficiency is 93% compared to the initial number (150) (Figure S3b). The decoration of CA-PVP inside the PDMS-based microreactor was attempted by incorporating a Tris-buffered silane (TBS) solution (pH 8.5) of CA-PVP (concentration of 50 mg/mL) through the full internal volume (11.4 μL, 0.3 mm (height) × 0.1 mm (width) × 380 mm (length)) of the microchannels. After 24 h at room temperature, the internal microchannels of the PDMS-based microreactors were repeatedly washed with a continuous flow of

3. RESULTS AND DISCUSSION PDMS-based microreactors were selected as the platform for photothermal agent-immobilized antibacterial microreactors due to the feasibility of polymer-based microreactors compared with their glass-based counterparts.29,30 Because PDMS is one of the hydrophobic and water-repelling polymers in which only C


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Figure 1. (a) FE-SEM images of Cs0.33WO3-immobilizaed microchannels depending on the concentration of dispersed Cs0.33WO3 and (b) ATR-IR spectra of the microchannels after CA-PVP decoration and Cs0.33WO3 immobilization.

increase of the inclusion of W element from 0.59 to 3.58 atomic % was observed in the EDX analysis during SEM analysis (Figure S4), showing that the mussel-inspired chemistry based on CA-PVP in this study can precisely control the number of immobilized Cs0.33WO3 NPs photocatalysts. For comparison, polydopamine and other polycatechol-decorated substrates typically do not reveal a specific morphology after musselinspired reactions. The presence of Cs0.33WO3 in the microchannels after the mussel-inspired immobilization of Cs0.33WO3 NPs on the polycatechol-decorated microchannels was also examined by ATR-IR spectroscopy (Figure 1b). CAPVP decorated microchannels showed only two strong peaks at 1273 (Si-CH3) and 1142 cm−1 (Si−O−Si), which originated from PDMS.35 The characteristic peaks for CA-PVP or its polymerized form were not clearly detected probably because the “organic glue” layer on the microchannels was much thinner (often less than 50 nm) than the depth of IR penetration of typical ATR crystals (often more than 0.5 μm). After immobilization of the Cs0.33WO3 NPs, the characteristic W−O−W stretching peaks for WO3 were clearly increased at 820 cm−1.36 Along with the increased lateral density of Cs0.33WO3 in the microchannels, significantly enhanced WO3 peak intensity was observed. The XRD spectrum of the photothermal agent-immobilized microchannels evidently revealed the characteristic hexagonal scattering pattern of Cs0.33WO3 NPs together with a broad diffraction halo at 11.8°, indicating the amorphous morphology of PDMS (Figure S5).35 Also, XPS analysis of both Cs0.33WO3 NPs and Cs0.33WO3immobilized microreactors clearly demonstrate the characteristic binding peaks of both Cs (Cs 3d3/Cs 3d5) and W (W 4d3/W 4d5/W 4f) (Figure S6). From all the above characterizations, it is clear that the utilization of CA-PVP facilitates the immobilization of photothermal agents such as Cs0.33WO3 NPs into the microchannels of microreactors through the buildup of “organic glue”, which is very effective for attaching or hybridizing two (nano)materials that are dissimilar in composition and morphology. The facile immobilization of Cs0.33WO3 NPs prompted us to investigate the photothermal antibacterial performance of

deionized water for another 24 h. Completion of the washing step was confirmed by observing the disappearance of strong absorption at 360 nm where extended transition of CA groups was monitored in the ultraviolet−visible spectrum of CA-PVP. Next, immobilization of Cs0.33WO3 NPs on the CA-PVP decorated microchannels was attempted by a similar process using TBS dispersion (pH 8.5) of Cs0.33WO3 NPs (concentrations of 0.5, 1, and 5 mg/mL, respectively), producing Cs0.33WO3-immobilized microreactors, WO3-MR-0.5, WO3MR-1, and WO3-MR-5, respectively (Scheme 1). The control of Cs0.33WO3 concentration over the microchannels with “polymeric glue” modulates the lateral density of photothermal agents in the microchannels. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the microreactors showed a lateral density of tungsten as 3.95, 6.07, and 9.66 μg/cm2, respectively, for the Cs0.33WO3 photothermal agents. Definitely, increasing the concentration of Cs0.33WO3 NPs dispersion resulted in the immobilization of photothermal agents with higher lateral density on the microchannels (Table S1). The immobilization of Cs0.33WO3 NPs on CA-PVP decorated microchannels in neutral condition was not successful possibly because mussel-inspired chemistry is not effective under neutral conditions. Instead, absorption of catechol groups on Cs0.33WO3 NPs mainly through ligand to metal charge transer (LMCT) process is feasible. However, this absorption is reversible and significant release of immobilization of Cs0.33WO3 NPs from the microchannels was observed under steady flow of even neutral water.34 The mussel-inspired immobilization of Cs0.33WO3 photothermal agents was confirmed by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy with attenuated total reflection attachment (ATR-IR), and X-ray diffraction (XRD) analysis. SEM images of Cs0.33WO3-immobilized microchannels showed highly aggregated morphology for the Cs0.33WO3 NPs embedded into the organic matrix (Figure 1a). The higher was the initial concentration of Cs0.33WO3 NPs, the more crowded was the morphology of Cs0.33WO3, which coincides with the above-mentioned ICP−MS analysis. As the concentration of Cs0.33WO3 NPs dispersion increased, the D


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ACS Applied Materials & Interfaces Cs0.33WO3-immobilized continuous flow microreactors.37,38 We’ve measured UV−visible spectra of Cs0.33WO3 NPimmobilized microchannels, aligning along with the light pass to increase the optical signal. In the case of Cs0.33WO3 NPimmobilized microchannels, strong optical absorption between 700−1100 nm was observed in all Cs0.33WO3 NP-immobilized microchannels. This optical absorption was increased along with the increase of NP immobilization (Figure S7). At first, the NIR-driven heat generation ability of Cs0.33WO3-immobilized microreactors was compared with the pristine PDMS-based microreactor without photothermal agents under NIR irradiation using an 808 nm laser with a power density of 2 W/cm2. Then, the internal temperature of the microchannels was monitored with a thermal imaging camera. Apparently, the Cs0.33WO3-immobilized microreactors showed significantly increased temperature ranging from 55.2, 63.9, to 72.9 °C along with the increase of Cs0.33WO3 loading amount after NIR irradiation for 5 min (Figure 2). The bare PDMS-based

control group (CA-PVP), which demostrated almost 100% cell viability for all flow rate tests, indicating that CA-PVP is nontoxic and has no antibacterial activity even after NIR exposure. At a flow rate of 100 and 200 μL/min, antibacterial efficiency was significantly retarded with high bacteria concentrations of 1 × 106 and 1 × 107 for the microreactors with low amounts of Cs0.33WO3 immobilization, WO3-MR-0.5, and WO3-MR-1. However, the photothermal performance of WO3-MR-5 remained above 95% regardless of bacteria concentration. With a bacteria concentration of 1 × 105, the antibacterial efficiency of WO3-MR-5 approached 99.9% for both types of bacteria, revealing the excellent photothermal performance of Cs0.33WO3-immobilized microreactors. The effect of photothermal treatment on the morphology of the bacterial cells was investigated by examining SEM images of E. coli with a bacteria concentration of 1 × 107 before and after passing through the Cs0.33WO3-immobilized microreactors under NIR irradiation. Following photothermal treatment, E. coli demonstrated a significant change in its bacterial morphology (Figure S8). The clear bacterial membrane of E. coli was significantly collapsed after photothermal treatment. WO3-MR-1 and WO3-MR-5 with higher loading amounts of photothermal agents caused more disruption of cell morphology compared to WO3-MR-0.5. Next, the photothermal antibacterial activity of Cs0.33WO3immobilized microreactors was evaluated by using the commercially available LIVE/DEAD BacLight bacterial viability kit.39 Both S. aureus and E. coli with a bacteria concentration 1 × 105 was passed through WO3-MR-0.5, WO3-MR-1, and WO3-MR-5 under NIR irradiation with varying flow rates of 50, 100, and 200 μL/min. Before the photothermal treatment, both untreated bacterial cells showed green fluorescence in confocal microscopy images, revealing that both bacterial cells were alive. After photothermal treatment through WO3-MR-5, only red fluorescence was observed in the confocal microscopy images regardless of the flow rate, revealing the high bacteria killing efficiency of WO3-MR-5 (Figure 4). With both WO3MR-0.5 and WO3-MR-1, only red fluorescence was observed at a flow rate of 50 μL/min but few spots showing green fluorescence appeared at higher flow rates of 100 and 200 μL/ min (Figures S9 and S10). Therefore, it is clear that Cs0.33WO3immobilized microreactors with higher loading amounts of photothermal agents preserve their photothermal antibacterial activity at high flow rates. Decreasing Cs0.33WO3 loading into the microreactors results in a moderate decrease of photothermal antibacterial activity but the decrease of the flow rate into the microreactors enables the preservation of excellent antibacterial activity through the increase of retention time for bacterial cells inside the Cs0.33WO3-immobilized microchannels of the microreactors compared with control that does not have the Cs0.33WO3-immobilized microchannels. Finally, continuous antibacterial operation of the Cs0.33WO3immobilized microreactors was attempted for both S. aureus and E. coli bacterial cells for 30 days. A bacteria concentration of 1 × 105 was used with a flow rate of 50 μL/min in this test. The photothermal antibacterial performance of over 99.9% for both types of bacteria was maintained even after 30 days of continuous operation regardless of the extent of Cs0.33WO3immobilization in the microreactors (Figure 5a). This result clearly shows that the Cs0.33WO3 NPs immobilized on the surface of the microchannels remain chemically and physically robust even after 30 days of continuous NIR irradiation and the subsequent photothermal event. The robustness of mussel-

Figure 2. Temperature monitoring of Cs0.33WO3-immobilized microchannels after NIR irradiation (the colored images are the corresponding pictures of microreactors upon NIR irradiation for 300 s captured from thermal imaging camera).

microreactor also showed a slightly increased temperature of 32.2 °C compared with the initial temperature of 19.6 °C, which is not as dramatic as in the Cs0.33WO3-immobilized microreactors. Therefore, the above significant temperature increase in the microchannels of the Cs0.33WO3-immobilized microreactors is regarded to originate from the photothermal modulation of immobilized Cs0.33WO3 NPs along the surface of the microchannels due to the strong NIR absorption, which is not hampered by PDMS possessing significant NIR transparency. The photothermal antibacterial performance of Cs0.33WO3immobilized microreactors was evaluated using a 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) based cell viability assay after applying a continuous flow of S. aureus (Gram-positive) and E. coli (Gram-negative) bacteria into CA-PVP as control, WO3-MR-0.5, WO3-MR-1, and WO3-MR-5 upon irradiation with an NIR laser with varying bacteria concentrations (1 × 105, 1 × 106, and 1 × 107) and flow rates (50, 100, and 200 μL/min) (Figure 3). At a flow rate of 50 μL/min, antibacterial activity above 99.9% was accomplished for both types of bacteria regardless of the amount of immobilized photothermal agents and the bacteria concentration. With WO3-MR-5, antibacterial performance was highest and approached 99.9% with a bacteria concentration of 1 × 105, revealing that higher immobilization of photothermal agents provides better photothermal antibacterial activity through enhanced photothermal effects compared with the E


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Figure 3. Photothermal antibacterial performance of Cs0.33WO3-immobilized microreactors after applying continuous flow of S. aureus and E. coli bacteria into Cs0.33WO3-immobilized microreactors upon irradiation of the NIR laser with varying flow rates of (a) 50, (b) 100, and (c) 200 μL/min.

Figure 4. Confocal images of S. aureus and E. coli bacteria treated with NIR irradiation through (a,e) bare microreactor, (b,f) WO3-MR-0.5, (c,g) WO3-MR-1, and (d,h) WO3-MR-5 with the flow rate of 50 μL/min (green and red bacteria images are regarded as live and dead bacteria, respectively). All the scale bars are 1.0 μm.

agent was 3.91, 5.99, and 9.60 μg/cm2, for WO3-MR-0.5, WO3MR-1, WO3-MR-5, respectively. The decrease in the lateral density of Cs0.33WO3 NPs was just 1.1%, 1.2%, and 0.9% for WO3-MR-0.5, WO3-MR-1, WO3-MR-5, respectively (Table S1). Therefore, the mussel-inspired immobilization of photothermal agents results in robust incorporation of Cs0.33WO3 NPs, which is one of the essential features for the practical

inspired immobilization of photothermal agents into the microreactors was verified by SEM analysis. After 30 days of continuous operation, there was no noticeable change in the surface morphology of the Cs0.33WO3-immobilized microchannels (Figure 5b).40 ICP−MS analysis further supports the stability of mussel-inspired immobilization. After 30 days of operation, the lateral density of the Cs0.33WO3 photothermal F


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Figure 5. (a) Monitoring of antibacterial activity of Cs0.33WO3-immobilized microreactors against E. coli and S. aureus bacteria for 30 days, and (b) FE-SEM images of microchannels after first microreactor operation, 30 days of operation, and regeneration with acidic buffer solution. All the scale bars are 1.0 μm.

washing with strong acid. Therefore, the antibacterial microreactors with immobilized photothermal agents are easily cleanable and subsequently recyclable through exploitation of the stability of mussel-inspired immobilization that bridges photothermal nanomaterials with PDSM-based microreactors in different circumstances.

application of antibacterial agents. Another merit of the microreactors whose surface engineering was attempted by mussel-inspired immobilization is the easy regeneration of the microreactors. While the above Cs0.33WO3-immobilized microreactors preserve excellent photothermal antibacterial performance with prolonged operation of up to 30 days, further continuous operation may diminish the antibacterial activity probably due to the partial release or poisoning of the photothermal agents. The cleaning and recycling of used microreactors can reduce the preparation time and labor required for the preparation of new microreactors.41 Bacteria killing ability comes from the photothermal effect of immobilized Cs0.33WO3 photocatalysts. CA-PVP plays as a kind of double-side adhesive that can bind on PDMS substrate and hold Cs0.33WO3 photocatalysts on it. The stability of this mussel-inspired chemistry has been demonstrated for the continuous operation for 30 days. As demonstrated with the cleaning and recycling of photocatalytic microreactors in the previous study, “organic glue” composed of a polymerized form of CA-PVP can be easily hydrolyzed in strongly acidic media, resulting in the nearly complete release of immobilized nanomaterials from the surface of the microchannels. Cs0.33WO3-immobilized microreactors operated continuously for 30 days were treated with acidic TBS solution (pH 1). SEM images of the acid-treated microchannels using TBS (pH 1) for 3 days showed that most of the Cs0.33WO3 NPs were completely released from the microchannel surface regardless of the loading amount of photothermal agents (Figure 5b). While the surface morphology of the cleaned microreactors is not very clean compared with that of CA-PVP decorated microchannels, there was no traceable photothermal agent after

4. CONCLUSIONS Immobilization of photothermal antibacterial Cs0.33WO3 nanoparticles inside PDMS-based microreactors was successfully attempted by sequential mussel-inspired surface engineering of the microchannels using CA-PVP and Cs0.33WO3 nanoparticles as photothermal agents. Excellent phothothermal antibacterial activity above 99.9% was accomplished during continuous operation for up to 30 days without releasing Cs0.33WO3 nanoparticles from the surface of the microchannels, confirming the robust immobilization of photothermal agents through the mussel-inspired chemistry. Cleaning of the used microreactors was easily attainable by simple acid treatment to release immobilized photothermal agents from the surface of the microchannels, enabling efficient recycling of used microreactors. Our approach can be widely extendable for the construction of various nanocatalyst- or photocatalyst-immobilized microreactors which are important in many applications such as water disinfection, photodegradation of organic pollutants from water, etc.

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ICP-mass of Cs0.33WO3 immobilized microchannel and 30 day recycle microchannel in acid condition for 3 days; images and spectra from analyses; image of the PDMSbased microreactor in this study (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Sung Young Park: 0000-0002-0358-6946 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by Grant Nos. 10062079, R0005303 and R0005237 from the Ministry of Trade, Industry & Energy (MOTIE), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2014055946)

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Performance of NIR-Mediated Antibacterial Continuous Flow

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