Plasmon-Triggered Hot-Spot Excitation on SERS Substrates for

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Biological and Medical Applications of Materials and Interfaces

Plasmon-Triggered Hot-Spot Excitation on SERS Substrates for Bacterial Inactivation and in situ Monitoring Jingwen Xu, Di Wu, Yuzhen Li, Jing Xu, Zhida Gao, and Yan-Yan Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09035 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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ACS Applied Materials & Interfaces

Plasmon-Triggered Hot-Spot Excitation on SERS Substrates for Bacterial Inactivation and in situ Monitoring Jingwen Xu, Di Wu, Yuzhen Li, Jing Xu, Zhida Gao, Yan-Yan Song* Department of Chemistry, Northeastern University, Shenyang 110004, China

ABSTRACT: Bacteria sensing and inactivating is one of the key steps to prevent bacteria propagation and transfer. Here, using Ag nanoparticles-grafted tungsten oxide film (WO3/Ag), we developed a multifunctional platform that may act as a SERS substrate for sensitively capturing and counting bacteria. Moreover, we demonstrated that the use of photon-triggered surface plasmon resonance (SPR) of Ag on the WO3 surface resulted in a significantly improved photocatalytic activity under visible light (638 nm). The photogenerated reactive oxygen species have been shown to be efficient in the inactivation of bacteria and the bacteria inactivation process could be monitored in situ by Raman spectroscopy. Based on the obtained Raman results and fluorescence measurements of green fluorescence protein (GFP) expressing bacteria, the active species triggered by hot spots was demonstrated to account for broken cell walls. The bacteria cell contents subsequently leaked out, leading to cell degradation. Potentially, our work may provide a promising strategy for capturing and monitoring the bactericidal process at low concentration, and specifically, may help in the investigation of related inactivation approaches and mechanisms.

KEYWORDS: multifunctional SERS chip, surface plasmon resonance, bacterial inactivation, in-situ Raman detection, sterilization mechanism, tungsten oxide, reactive oxygen species

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INTRODUCTION Bacteria can be found almost ubiquitously, with various beneficial or damaging effects on the human organism. Every year, a large number of individuals get infected by pathogenic bacteria, potentially leading to serious illnesses, i.e. diarrhea, sepsis and bacteremia, thus posing a threat to public health.1-6 In recent years, a significant amount of interest has focused on the rapid sensing of bacteria, i.e. real-time polymerase chain reactions,7 enzyme-linked immunosorbent assays8 and lateral flow immunoassays.9 Compared to traditional colony counting technologies, these new approaches avoid long-time bacteria cultivation and may therefore help reduce assay time. To date, most of these technologies are based on cytolysis, amplification and quantification of nucleic acids, resulting in complex preprocessing. Surface enhanced Raman spectroscopy (SERS) represents a promising method for biosensing, offering advantages such as concise sample preparation, rapid signal export, great signal enhancement and abundant spectral information.10 Until now, SERS has been applied in the analysis of various biological species, such as lipid,11 saccharides,12 nucleic acids,13,14 proteins15,16 cancer cells17,18 and bacteria.19,20 Generally, the SERS technology in bacteria detection can be divided by labelbased and label-free methods.21 The label-based technology senses bacteria by recognizing the SERS signal from the label of molecules introduced with aptamers or antibodies.22-24 On the contrary, the label-free SERS method utilizes the detection and identification of bacteria directly based on inherent biomolecules of biological species. In this case, the Raman signals usually originate from nucleic acids, lipids and other proteins in bacteria. As a consequence, a substrate exhibiting an excellent SERS effect is generally required.25-27 For example, Wang et al. constructed a Ag nanoparticles-decorated silicon wafer as SERS substrate that has been demonstrated to capture and detect bacteria.28 On the other hand, despite of the rapid and


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sensitive detection of pathogenic bacteria, finding proper approaches to kill bacteria may be the key to prevent further bacteria popagation and transfer.4,5 To date, exploring a multifunctional SERS substrate to gain further information, including in situ sterilization, real-time monitoring and the inactivation mechanisms, still faces a multitude of scientific challenges. Plasmonic metal nanostructures, i.e. noble Au and Ag nanoparticles, are considered to be a fascinating substrate for surface-enhanced Raman scattering (SERS) in ultrasensitive analyses.29 Furthermore, the surface plasmon resonance (SPR) feature of such metal nanostructures attracts increasing attention in the scientific community as it can harvest and convert light to chemical energy via plasmonic excitation.30 In this process, “hot spots”, also reported as energetic electrons, are excited by resonant photons and subsequently transfer to nearby substrates.31 Recently, Au-SPR induced photocatalysis was applied to visible-light triggered surface cleaning,32 drug release,33 water splitting,34 and antibacterial studies.28 In this work, we developed a multifunctional SERS chip based on an Ag nanoparticlesgrafted tungsten oxide film (WO3/Ag) that integrates not only the capture and detection of bacteria, but also the ability of in situ inactivation and investigation of the bactericide mechanism. Owing to the local surface plasmonic resonance (LSPR) of Ag on the WO3-x substrate, the captured bacterial cells were found to be quickly inactived under 638 nm laser irradiation, i.e. the laser wavelength employed in Raman analysis. The related LSPR-triggered sterilization mechanism could be simultaneously studied by Raman spectra. Moreover, this multifunctional chip was also demonstrated to represent a process allowing for a robust antibacterial effect, even under common halogen tungsten lamp irradiation, potentially offering a promising application as a highly efficient antibacterial coating material. EXPERIMENTAL SECTION


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Preparation of SERS chips A glass slide was chosen as substrate for WO3/Ag films. Prior to preparation, the glass slide was ultrasonically cleaned for 10 min in acetone and ethanol, followed by further treatment in a plasma cleaning system for 20 min. WO2.72 nanostructures were grown on the glass slide by solvothermal method. Typically, 4.0 g of WCl6 was added into a solution containing 18 mL of ethanol and 2 mL of ethylene glycol. A transparent yellow solution was obtained and the glass slide was placed at an angle of 30o against the Teflon liner wall with the conducting side facing down. The glass slide together with the solution was sealed in a 30 mL Teflon liner, and the liner was then put into a self-sealing autoclave and heated at 180 oC for 2 h in an electric oven without stirring. After cooling of the autoclave to room temperature, the glass slide coated with dark blue WO2.72 film was taken out and washed with ethanol. Then, the sample was rapidly immersed in a 0.2 mM AgNO3 solution for 10 min and washed with DI water and dried with a stream of nitrogen. In this study, pMBA was used as trapping agent for bacteria. The as-prepared WO3/Ag film was immersed in pMBA aqueous solution at a certain concentration for 12 h to produce the WO3/Ag-pMBA film. The films were cleaned by DI water and dried by N2 and the samples were stored at 4 oC before bacteria capturing experiments and characterization. Capture and detection of Bacteria WO3/Ag-pMBA film was first broken in to pieces with an average size of 0.8 cm × 1.5 cm. Then, the pieces were immersed in 2 mL bacteria solutions of different concentrations in the dark to quantify the capture characteristics of E. coli. When the capture experiment was finished, the film was taken out of the bacteria solution, and was gently rinsed with a 0.1 M PBS buffer three


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times to thoroughly remove non-specific bacteria on the film. The capture efficiency of E. coli was calculated according to the following equation: η (%) = (no-nr) / no×100%

(1)

Where η is the capture efficiency of E. coli, no is the amount of E. coli colonies in the original solution, nr is the remained E. coli colonies after capturing by the WO3/Ag-pMBA film. The amounts of original E. coli colonies and remaining E. coli colonies were calculated by using a standard plate colony counting method. All capturing experiments were repeated three times to obtain averaged values. The number of captured bacteria on the film was determined by SERS mapping measurements. The mapping area was 49.5 µm × 49.5 µm, with a step size of 2 µm. The area was selected randomly and contained 324 random spots to estimate the capture quantity. All measurements were conducted by a 638 nm laser with a 50× objective. The acquisition time was 1 sec and in order to avoid bacteria damages directly caused by high laser power, the laser power was also reduced to 1% of the original laser power (20 mW) using a filter. The confocal hole was 200 µm and the slit aperture was 300 µm. In the traditional SERS method, 10 spectra were randomly collected for each sample within a range of 200~2000 cm−1 and the data were performed by LabSpec6 software. The enhancement factor (EF) value was calculated using the following equation: EF = (ISERS×Nbulk / (Ibulk× NSERS)

(2)

Where, ISERS is the intensity of the peak of pMBA on the SERS substrate, and Ibulk represents the intensity of the peak of pMBA on the glass slide. Nbulk is the number of pMBA molecules


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dispersed on the glass slide, and NSERS represents the number of molecules dispersed on the SERS substrate. Antibacterial tests and monitoring by SERS Antibacterial properties of the WO3/Ag film under xenon lamp source (with a filter of λ > 420 nm) and halogen tungsten lamp were studied. For each antibacterial experiment, the asprepared WO3/Ag film was attached to a quartz cup, which contained 3 mL of an E. coli suspension at a concentration of 105 CFU mL-1. After irradiation for 10 min, the bacteria solution was shaken to provide a uniform solution and 100 µL of the treated bacteria suspension was withdrawn and diluted with sterilized DI water to adjust the amount of survival bacterial colonies in the range of 30~300. The bacterial survival numbers were calculated by counting the bacterial colony numbers on solid LB medium that were incubated at 37 °C for 24 h. For in situ antibacterial monitoring, a 638 nm laser was filtered to 1% of the initial power to avoid bacteria damages directly caused by high laser power. The laser was irradiated on the bacteria captured by the film and Raman spectra of bacteria were recorded at different irradiation times. The capture of reactive oxygen species The generated hydroxyl radicals were detected by fluorescence (FL) spectra with a 3 mM probe TA. Under the irradiation of 635 nm laser (0.51 W/cm2), the WO3 film or WO3/Ag film was immersed in a 2 mL aqueous solution containing terephthalic acid (3 mM), and the fluorescence intensities at 425 nm were recorded with an excitation wavelength of 312 nm. For 1O2 detection, 2 mL 1, 3-diphenylisobenzofuran (1.0 × 10−5 M, DPBF) in ethanol solution containing 100 µM H2O2 of was irradiation by 635 nm laser (0.51 W/cm2) with the


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present of WO3 film or WO3/Ag film. Then absorption intensity of DPBF at 410 nm was recorded via UV-vis spectroscopy every 2 min. The group without laser illumination and sample served as control. RESULTS AND DISCUSSION A layer of WO3−x film was first grown on a glass slide using a solvothermal method (detailed procedure outlined in the Supporting Information section). The scanning electron microscopy (SEM) images obtained show that the as-prepared WO3−x film was a grained nanostructure composed by bunches of nanowires (cf. Figure S1a and S1b). The oxygen deficiencies on WO3−x lead to an in situ redox reaction between metal ions and WO3−x, which resulted in the formation of metal deposition on WO3-x.35 As shown in Figure 1a, Ag nanoparticles were observed on the surface of WO3-x, with a size of approximately 15 nm (cf. Figue 1b) after dippig the WO3-x film in a solution containing Ag+. The mapping images obtained from Energy Dispersive Spectrometry (EDS) indicated a well-dispersed distribution of Ag (cf. Figure S2) by using a glass slide as reference (cf. Figure S3). This Ag formation approach proved to be efficient and did not require any external reduction agents. Furthermore, the as-formed Ag nanoparticles sticked to the WO3 substrate and an excessive accumulation of Ag nanoparticles could be avoided, resulting in a highly reproducible process. X-ray photoelectron spectroscopy (XPS, Figure S4) analysis confirms the formation of Ag metal on WO3, which is evident from the 6.0 eV splitting of the 3d doublet of Ag 3d signals.36 The peak of S 2p is assigned to pMBA. In addition, the peak of Ag−S at 367.8 eV in Ag 3d5/2 also confirms that pMBA molecules are anchored on silver nanoparticles (cf. Figure S5). X-ray diffraction (XRD, cf. Figure S6) patterns further demonstrated the formation of Ag nanoparticles and characteristic peaks of Ag at 2θ = 44.2° and 81.5° could be observed. The bacterial-recognition


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molecule pMBA was grafted onto the WO3/Ag chip via Ag−S bonds between sulfhydryl group in pMBA and Ag nanoparticles. The successful decoration of pMBA on the WO3/Ag films was verified by fourier transform infrared spectroscopy (FT-IR) as shown in Figure S7. Absorption bands appeared at 1390 cm-1 and 2910 cm−1 and could be attributed to the B–O vibrationand C– H stretching vibration from pMBA. Furthermore, owing to the excellent SERS effect of the WO3/Ag substrate, obvious Raman signals originating from the benzene ring of pMBA could be observed (cf. Figure S8). Two strong characteristic peaks appeared at 1071 cm-1 and 1583 cm-1, respectively, corresponding to the benzene-thiol ring37 and boronic acids.38 The peak at 893 cm-1 corresponded to a W-O band. The EF value of the WO3/Ag substrate was 7.0×106, implying that the resulting WO3/Ag film represents a promising substrate for sensitive sensing. The bacterial cell membrane is lined with peptidoglycan which can be identified by boronic acid groups, making pMBA a good trapping agent for bacteia.39,40 To evaluate the bacteria capturing ability of the as-prepared WO3/Ag substrate, we employed Escherichia coli (E. coli) as the model bacterium. Compared to the bare WO3/Ag chip (cf. Figure 1c), a large amount of bacteria could be observed on the pMBA decorated WO3/Ag chip (WO3/Ag-pMBA) after incubation in LB medium containing bacteria at a concentration of 105 CFU mL-1 for 20 min. The latter finding highlights the key role of pMBA in the process of achieving a satisfying bacteria capturing ability. Further studies showed that the capturing ability of bacteria increased upon increasing the pMBA concentration and incubation time. The capturing ability reached saturation at a concentration of 2.0 mg mL-1 (cf. Figure S9a) and the optimal incubation time was shown to be 20 min (cf. Figure S9b). Figure S10 shows that such chips exhibited a satisfying bacteria capturing efficiency as high as ~85%, at a bacteria concentration below 4000 CFU mL-1.


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However, the capturing efficiency was still maintained at ~60% at higher bacterial concentration ranging from 6,000 to 12,000 CFU mL-1. Figure 2a shows the Raman spectra of bare WO3/Ag, WO3/Ag-pMBA before and after E. coli capturing. Due to the large amount of peptidoglycan on the bacteria cell walls, strong interactions between boronic acid groups and the cell walls pulled the bacteria cells towards the SERS substrate. As demonstrated by SEM images (cf. Figures S11a and S11b), the bacterial cells sticked on the Ag nanoparticle surface, with spreading of the cell walls. As a consequence, the cell walls attached onto the SERS substrate, resulting in an enhanced Raman signal as part of the quantitative sensing of bacteria. The characteristic fingerprint band of E. coli cells could be observed in the range between 1128 and 1588 cm-1, a finding that was consistent with previous reports.19,41 The Raman intensity of the peak found at 1276 cm-1 (δ (CH2) amide III from bacteria) was recorded and could be used to quantify the bacteria concentration (the sensing performance was evaluated by recording the number of red points in a mapping area of 49.5×49.5 µm2 at a 2 µm step size). The red points in the SERS mapping images related to the SERS signals of bacteria. The randomly selected area contained 324 spots that were used to estimate the capture situation. As shown in Figure 2b-d, more red points were observed with increasing bacteria concentration. These red points could be used to even distinguish a low bacteria concentration of 10 CFU mL-1, suggesting that the as-prepared chip was sensitive enough for capturing and quantifying bacteria even at a very low concentration. The quantitative evaluation between the number of red points and the bacteria concentration from 10 CFU mL-1 to 1.0×106 CFU mL-1 is shown in Figure S12. The results provided evidence for the promising ability of the WO3/Ag chip to find use as a SERS substrate in the capturing and sensing of bacteria from diluted samples.


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Notably and as shown in Figure 3a, the intensity of the fingerprint band of E. coli was changing in the process of continuously collecting Raman spectra. For example, the intensity of the Raman peak at 1276 cm-1 first increased with irradiation at 638 nm and then reached a maximum value at ~20 min. Then, the intensity of this peak started to decrease with further extension of the irradiation time, and finally disappeared at 60 min. The SEM images revealed that the captured E. coli cells exhibited a complete and intact morphology in the beginning (cf. Figure 3b). However, the cell walls were partially destroyed after irradiation at 638 nm for 20 min, when the inner proteins of bacteria started to leak out (cf. Figure 2c). The released protein molecules then came in direct contact with the WO3/Ag substrate and could be located in the range of “hot spots” generated by WO3/Ag. These generated hot spots are normally due to the electromagnetic enhancement of SERS. As the Raman signal at 1276 cm-1 stemmed from the δ (CH2) amide III of the protein molecules, the Raman signal at 1276 cm-1 was observed to increase within the first 20 min. However, when the irradiation time was further extented to 60 min, the frame of E. coli cells was found to collapse (cf. Figure 2d). For reference, E. coli cells were incubated with WO3/Ag in dark. Owing to the intrinsic antibacterial characteristic of Ag nanoparticles, the E. coli cells presented a rough surface at 90 min (cf. Figure S13). Besides of the well-established antibacterial mechanism that metallic silver and silver ions released by metallic Ag can cause irreversible damages to the functional components of bacteria,28 in this case, the disappearance of Raman signals may be caused by the following additionally two mechanisms. Firstly, with the collapse of cell walls, the bonds between peptidoglycan and boronic acid vanished, resulting in the separation of bacteria from the SERS substrate (cf. Figure S11c-d). Since the hot spots exhibit an effective working distance, the bacteria separated from the SERS substrate may have caused the hot spots in the outer range.19,42 Secondly, considering


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that the SERS chip represents a metal/semiconductor substrate, the SPR-Au-TiO2 coupling induced the formation of free radicals.43-45 These radicals featured a longer living distance than hot spots under visible light irradiation and, potentially, may have attacked protein biomolecules, ultimately leading to the destruction of cell membranes and the degradation of the released active molecules from bacteria. This entire process was well demonstrated by the change of the Raman signal at 1276 cm-1. The LSPR-triggered sterilization mechanism was further verified via the fluorescence signal from green fluorescence protein expressing E. coli (GFP-E. coli), as shown in Figure 4. Using the GFP-E. coli solution stored in dark as reference (curve I), without WO3/Ag decoration, the visible-light irradiation (a 635 nm laser was employed as the light source) presented a negligible influence on the fluorescence signal from GFP-E. coli (curve II). Meanwhile, the fluorescence intensity of GFP-E. coli only exhibited a slight decrease after incubating GFP-E. coli with WO3/Ag films in the dark for 90 minutes (curve III). This discrepancy could be attributed to the well estiblished antibacterial feature of Ag nanoparticles, which is consistent with previous reports.46,47 When GFP-E. coli cells were incubated with WO3/Ag films under irradiation (curve IV), the fluorescence intensity from GFP-E. coli exhibited a slight reduction in the first 20 minutes. However, a sharp fluorescence decrease could be observed upon extending the irradiation period and the fluorescence signal almost disappeared after 60 minutes. As demonstrated by the inset photograph in Figure 4, no fluorescence could be observed in sample IV, indicating the complete degradation of GFP. To further understand the underlying mechanisms, we numerically simulated the LSPR-field of Ag nanoparticles in Figure 5. Compared to the electric fields (hot spots) around one Ag nanoparticle in air (cf. Figure 5a) or on glass slide (cf. Figure 5b), the LSPR mode showed an obvious enhancement at the interface


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between the Ag nanoparticle and the WO3 substrate (cf. Figure 5c). Moreover, the electric fields between two Ag nanoparticles with a distance of 40 nm (according to the SEM image in Figure 1b) on the WO3 substrate also exhibits an intense enhancement (cf. Figure 5d). In addition, the decoration of Ag nanoparticles was found to increase the absorption of the SERS substrate in the visible-light range from 500 to 700 nm (cf. Figure S14). Further experiments demonstrated the formation of a large amount of OH• (cf. Figure 6a) and 1O2 (Figure 6b) on the WO3/Ag film under 635 nm laser irradiation by using the established methods based on terephthalic acid

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and DPBF,49 respectively. These results strongly support

the hypothesis that Ag-SPR could follow a valence-band induced photocatalytic pathway,30 resulting in bacterial inactivation. As illustrated in Figure 6c, under visible-light (i.e. 638 nm) irradiation, “hot spots” were excited by the giant LSPR of Ag on the WO3-x substrate. Here, the energetic electrons remained on Ag nanoparticles and this process lead to an improved photocatalytic efficiency of WO3/Ag due to the hot electrons-induced 1O2. Meanwhile, the photogenerated holes in Ag nanoparticles were transferred to the valence band (VB) of WO3 and further reacted with the absorbed H2O molecules to form OH•. During this process, Ag nanoparticles could trap the photogenerated electrons and thus inhibit the further recombination with holes in WO3. Therefore, the formed Ag/WO3 interfaces can effectively promote charge separation and enhance the photocatalytic activity of the catalyst. These active species (1O2 and OH•) could diffuse several micrometers in the aqueous environment,50 and thus, induce the inactivation of bacteria. To clarify the intrinsic antibacterial activity from Ag nanoparticles and the SPR of WO3/Ag induced bactericidal ability, E. coli cells were incubated with WO3 or WO3/Ag films under xenon lamp source (with a filter of λ>420 nm) or in dark for 10 min. The survival rates were shown in Figure S16. As the SPR-induced photocatalytic activity cannot take


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place without light irradiation, antibacterial effect of WO3/Ag in dark completely comes from the intrinsic antibacterial characteristic of Ag in the WO3/Ag sample. If compare this result with the antibacterial result from WO3 in dark, we can determine the antibacterial effect came from Ag (as illustrated as Part I in Figure S15). If compare the antibacterial results from WO3 and WO3/Ag under light irradiation, the antibacterial effects came from the SPR-induced active species (Part II in Figure S15) can be clear. For potential practical applications of the as-constructed WO3/Ag film, we tested the antibacterial activity under a common halogen tungsten lamp (power density of 0.048 W cm-2). The spectrum of the lamp showed a broad emission peak at the centre of ~630 nm (cf. Figure S16). The bacterial survival rates were only ~14.1 and 13.5% in the presence of WO3/Ag and WO3/Ag-pMBA films, respectively, and after halogen lamp irradiation for 5 min. This finding indicated that the as-prepared films exhibited a satisfying bactericidal effect, even under a homeused light source. Furthermore, the antibacterial efficacy was not attenuated by pMBA grafting. CONCLUSIONS In summary, we developed a multifunctional SERS substrate based on a WO3/Ag film with a satisfying performance demonstrated in the capturing, sensing and counting of bacteria. Importantly, owing to the SPR–Ag-WO3 coupling, the WO3/Ag film was photocatalytically active under visible light, which lead to the inactivation of captured bacteria. Furthermore, the entire processes could be monitored i.e. by following the Raman signal of bacteria. Potentially, this multifunctional chip may offer a novel and alternative strategy for the development of advanced coatings for sensing bacteria and providing efficient solutions for bacteria spreading in public environments.


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ASSOCIATED CONTENT Supporting Information. Experimental procedures and analytical data are provided. This material is available free of charge via the Internet at http://pubs.acs.org.Materials and apparatus, EDS mapping, XPS, XRD, FT-IR UDR of substrate, SEM of bacteria before and after irradiation, capture efficiency and quantitative analysis of E. coli cells, Survival rate of E. coli cells in dark and under visible light irradiation.

AUTHOR INFORMATION Corresponding Author *email: [email protected]; Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21775016), the Fundamental Research Funds for the Central Universities (N160502001, N170502003, N170908001).

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Figure 1. SEM images of as-prepared WO3/Ag films (a) and (b); SEM images of E. coli cells on neat WO3/Ag films (c) and WO3/Ag films with pMBA (d).


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Figure 2. (a) Raman spectra of bare WO3/Ag film, WO3/Ag-pMBA film, and WO3/Ag-pMBA film after E. coli capturing; (b-d) SERS mapping (at 1276 cm-1) of captured E. coli cells with different original concentrations: (b) 1×101,(c) 1×105, (d) 1×107 CFU mL-1. Parameters for the SERS experiments were as follows: laser: 638 nm, acquisition time: 1 s, confocal hole: 200 µm, slit: 300 µm, grating: 1200 g mm-1. scale bars: 2 µm.


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Figure 3. (a) Raman spectra of bacteria on WO3/Ag-pMBA film with different irradiation times; SEM images of bacteria after irradiation with a 638 nm laser for (b) 0 min, (c) 20 min and (d) 90 min (scale bars: 0.5 µm).


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Figure 4. Fluorescence intensityof a GPF-E. coli (107 CFU mL-1) solution: stored in dark (curve I), after incubation with glass slide under irradiation (curve II), incubation with WO3/Ag-pMPA film in dark (curve III), and incubation with WO3/Ag-pMPA film under 635 nm laser irradiation (curve IV). Inset: the optical photograph of the GPF-E. coli solution corresponding to the four curves in Figure 4. The photograph was taken at an incubation time of 60 min and at an excitation wavelength of 365 nm.


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Figure 5. Simulation of the electric field distributions of one Ag nanoparticle (a) in air, (b) on glass surface, and (c) on WO3 surface, and (d) two Ag nanoparticles on WO3 surface.


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Figure 6. (a) Fluorescence intensity at 425 nm, measured from terephthalic acid (2 mM) after incubation with WO3 and WO3/Ag chips under 635 nm laser irradiation; (b) Consumption of DPBF triggered by 1O2 generation; (c) Schematic illustration of the antibacterial mechanisms of the as-prepared WO3/Ag chips.


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For Table of Contents Only


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SPR-triggered antibacterial mechanism is monitored in situ by the WO3/Ag SERS chip. 583x248mm (72 x 72 DPI)


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Plasmon-Triggered Hot-Spot Excitation on SERS Substrates for

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