Journal of Colloid and Interface Science 505 (2017) 910–918

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Regular Article

Urea-derived graphitic carbon nitride (u-g-C3N4) films with highly enhanced antimicrobial and sporicidal activity John H. Thurston a,⇑, Necia M. Hunter a, Lacey J. Wayment a, Kenneth A. Cornell b a b

Department of Chemistry, The College of Idaho, 2112 Cleveland Blvd, Caldwell, ID 83605, USA Department of Chemistry and Biochemistry, Boise State University, Boise, ID 83725, USA

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 10 May 2017 Revised 22 June 2017 Accepted 25 June 2017 Available online 27 June 2017 Keywords: g-C3N4 Photocatalysis ROS Antibacterial Biocidal Sporicidal

a b s t r a c t In this manuscript, we describe the fabrication of photoactive biocidal or sporicidal films from ureaderived graphitic carbon nitride (u-g-C3N4). Co-deposited films of u-g-C3N4 and Escherichia coli O157: H7 (IC50 = 14.1 ± 0.2 mJ) or Staphylococcus aureus (methicillin resistant IC50 = 33.5 ± 0.2 mJ, methicillin sensitive IC50 = 42.7 ± 0.5 mJ) demonstrated significantly enhanced bactericidal behavior upon administration of visible radiation (400 nm
k
426 nm). In all cases, complete eradication of the microbial sample was realized upon administration of 100 mJ of visible radiation, while no antimicrobial activity was observed for non-irradiated samples. In contrast, Bacillus anthracis endospores were more resistant to u-g-C3N4 mediated killing with only a ca. 25% reduction in spore viability when treated with a 200 mJ dose of visible radiation. Characterization of u-g-C3N4 reveals that the improved activity results from enhancements of both the surface area and reduction potential of the material’s conduction band edge, coupled with fast injection of charge carriers into localized states and a decline in radiative recombination events. The results of this study demonstrate that g-C3N4-based materials offer a viable scaffold for the development of new, visible light driven technologies for controlling potentially pathogenic microorganisms. Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction

⇑ Corresponding author. E-mail address: [email protected] (J.H. Thurston). http://dx.doi.org/10.1016/j.jcis.2017.06.089 0021-9797/Ó 2017 Elsevier Inc. All rights reserved.

It was recently estimated that there were 722,000 nosocomial infections in the United States, of which approximately 75,000 of these cases were fatal [1]. To this end, nosocomial infections are

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directly correlated with a variety of negative healthcare outcomes including increased patient mortality and morbidity, increased length of hospitalization and an increase in total cost of care [1]. While a variety of mechanisms contribute to microbial transfer and infection in the hospital environment, it is widely accepted that bacteria can persist on solid surfaces for extended periods of time [2]. Consequently, contaminated surfaces can serve as environmental reservoirs for a variety of pathogenic microorganisms, including vancomycin-resistant Enterococcus (VRE), methicillinresistant Staphylococcus aureus (MRSA), E. coli, Klebsiella spp. and Candida albicans [2,3]. Indeed, contact with a VRE-contaminated surface has been reported to result in microbial transfer with approximately the same frequency as direct contact with a colonized patient [4]. More generally, contact with a contaminated surfaces in a health care facility has been correlated with an average nosocomial infection rate of 10% [1]. This infection rate can increase to as high as 30% in specific areas of the hospital, such as the intensive care unit [1]. Importantly, environmental decontamination measures have been reported to successfully suppress outbreaks of both MRSA and VRE in healthcare facilities, highlighting the importance of environmental reservoirs of these organisms in sustaining the patient infection cycle [4]. Consequently, developing new technologies that prevent or combat the colonization of surfaces in healthcare facilities remains fundamental to the goal of reducing the incidence rate of nosocomial infections. Thin films and coatings have the potential to impart desired reactivity without adversely affecting or modifying the bulk structural or mechanical properties of a material. The development of antimicrobial coatings remains an active field of research and a variety of materials, including semiconductors [5,6], silver and silver salts [7], synthetic and biopolymers [8–11], carbon nanotubes [12,13] and functionalized clays [5,14] have been used to produce antimicrobial coatings. Among these technologies, narrow or intermediate band gap semiconductors that possess the ability to generate cytotoxic reactive oxygen species (ROS) from molecular oxygen show particular promise for the reduction of bacterial populations in interior environments [15]. Graphitic carbon nitride (g-C3N4) is a metal-free, intermediate band gap semiconductor that is readily produced via direct thermal polymerization of a variety nitrogen-rich molecular precursors [16,17]. The potential photocatalytic applications of this material have been described in several recent review articles [17–21]. This material is particularly attractive for environmental remediation and biocidal applications due to its’ metal free composition, which could limit incidental toxicity, and the fact that it is photoresponsive to visible wavelengths, allowing it to be easily employed in interior environments without the need for specialized light sources. Unfortunately, the potential utility of g-C3N4 is frequently compromised by relatively high recombination rates of photogenerated charge carrier species and correspondingly low observed photocatalytic activities. Despite this potential shortcoming, several recent studies have elegantly demonstrated that g-C3N4–based materials can achieve the disinfection of various microorganisms in solution [22–27]. In contrast to the studies outlined above, the ability of g-C3N4 to exhibit antibiotic activity in the solid state, an application for which this material appears to be uniquely suited, is almost completely unexplored [15]. As such, further study of the utility of g-C3N4-based films and coatings for biocidal applications is both needed and justified. The chemical and electronic properties of g-C3N4 are known to vary as a function of precursor identity. Specifically, samples of g-C3N4 produced from oxygen-containing precursors such as urea (u-g-C3N4) and cyanuric acid exhibit improved reactivity for a variety of photochemical applications [28–33]. These results clearly suggest that it may be possible to further promote the antimicrobial activity of g-C3N4 via judicious selection of the catalyst precur-

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sor. To test this hypothesis, we have explored the fabrication of photoactive, antimicrobial films from u-g-C3N4. The materials developed for this study show an approximately 10-fold enhancement of biocidal activity against the clinically relevant microorganisms: S. aureus, MRSA and E. coli O157:H7 compared to our previous work [15]. Additionally, we also demonstrate that samples of u-g-C3N4 possess sufficient photochemical reactivity to deactivate B. anthracis endospores. The results of this study reinforce the utility of g-C3N4-based materials for combatting hospital acquired infections by controlling populations of potentially pathogenic microorganisms in the environment. 2. Experimental 2.1. General All chemicals and media components were purchased from either Sigma-Aldrich Corp. (St. Louis, MO) or from Fisher Scientific (Pittsburgh, PA). As part of this study, comparative samples of gC3N4 were prepared from dicyandiamide as previously reported [15]. Samples of urea were dried overnight at 80 °C prior to use. Pure strain samples of E. coli O157:H7 (ATCC #43894) and S. aureus (methicillin sensitive, ATCC #6538) were purchased from ATCC (Mannassas, VA). Samples of B. anthracis (Sterne 34F2) were obtained from Colorado Serum Co. (Denver, CO). A clinical isolate of methicillin resistant S. aureus (MRSA) was the kind gift of Dr. Chris Ball, Idaho Bureau of Labs (Boise, ID). 2.2. Preparation of u-g-C3N4 Samples of u-g-C3N4 were prepared by a modification of the reported procedures [30]. Briefly, a sample of 10 g of dried urea was placed in a covered 50 mL porcelain crucible and heated from 25 °C to 575 °C at a rate of 175 °C/h in a muffle furnace. The sample subsequently dwelled at 575 °C for four hours and then was cooled to ambient temperature over 18 h. The resulting pale yellow solid was collected and ground to produce approximately 0.5 g of u-gC3N4 as a free flowing powder. 2.3. Spectroscopic characterization u-g-C3N4 Infrared spectra were collected on a Thermo-Nicolet Avatar 360 FT-IR spectrophotometer equipped with a single reflection Smart Orbit diamond ATR aperture in the range of 4000–400 cm1. Diffuse reflectance UV–Vis spectroscopy measurements were collected on powder samples using a Cary 5000 spectrophotometer. Bandgap values are estimated from diffuse reflectance data using Kubelka-Munk theory [34,35]. X-ray photoelectron spectroscopy was conducted on a PHI 5000 Versaprobe II Scanning ESCA microprobe using a monochromatic Al Ka X-ray source (1486.6 eV). The base vacuum in the chamber was better than 1.5  1010 torr. The samples used in this study were probed by an X-ray source with a power of 100 W and a beam diameter of 150 lm. Survey scans were collected on several different areas to study the relative composition of the sample. High resolutions scans were performed on each elemental region to improve the signal-to-noise ratio. Sample charging effects were minimized using a low energy electron gun and Ar+ ions. The binding energy scale was referenced to the C1s peak (284.8 eV) to accommodate peak shifts as a consequence of sample charging effects. Samples for photoluminescence and time resolved spectroscopy experiments were prepared by mixing with BaSO4. Steady-state photoluminescence spectra were recorded on a Horiba-Jovin Yvon Fluorolog 3. The excitation was at 371 nm and the emission was measured from 390 to 630 nm with a slit size of 1 nm using Front Face detection. The photoluminescence

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lifetime measurements were performed on an Edinburgh Instruments OD470 single-photon counting spectrometer equipped with a high speed red detector and a 371 nm picosecond pulse diode laser. Lifetime was calculated by iterative reconvolution of the measured decay and the instrument response function (IR). Fluorescence experiments were conducted on a Synergy H1 96 well plate reader using a xenon flash lamp oriented in the top position. The probe distance (Z axis) was 5 mm and a 100 ms delay was employed. 2.4. Physical characterization of u-g-C3N4 Transmission electron images of u-g-C3N4 were recorded on a Hitachi S-3400N analytical transmission electron microscope operating at an accelerating potential of 120.0 kV. The hydrodynamic radius of the materials developed for this study was measured using dynamic light scattering (DLS) measurements on a Zetasizer Nano ZS Zetapotential/Particle size analyzer (Malvern Instruments) equipped with a monochromatic and coherent light beam (633 nm He-Ne laser, 4 mW). Powdered samples of u-g-C3N4 were dispersed in deionized water to get a pre-determined concentration of 0.001 wt% aqueous solution. The samples used for analysis were subjected to ultrasonication for 10 min and then immediately transferred to a zeta cell for measurements. The particle size scans were averaged to obtain size distribution data. Powder X-ray diffraction data was collected on a Rigaku Miniflex 600 powder X-ray diffractometer using Cu Ka radiation (graphite monochromator, k = 1.5418 Å). Data sets were collected between 2.0° and 65.0° on 2h with a step size of 0.01°. Elemental analyses were performed by Galbraith Corporation (Knoxville, TN). 2.5. Irradiation procedures Irradiation experiments were carried out by illuminating the sample mixture with an ozone free xenon light source operating at 270 W (Power source: Newport # 69911). The lamp housing (Newport # 67001) contained an F/2.2 fused silica condenser and rear reflector (1.6 correction factor). The photon flux was stripped of infra-red radiation using a temperature-controlled, recirculating water filter. Similarly, UV radiation was removed using a k = 400 nm cut-off filter (Oriel # FSQ-GG400). Total applied radiation doses were calculated using the equations and procedures described previously [15]. 2.6. Determination of ROS production ROS production was monitored via production of the fluorescent molecule 2-hydroxy-terepthallic acid (2-OH-TA) from terepthallic acid (TA). In a typical experiment, a 10 mg sample of u-g-C3N4 was suspended in 10 mL of a solution that was 0.5 mM in TA and 2.0 mM in NaOH [36]. The mixture was stirred at 260 rpm in the dark for 30 min and then irradiated. Aliquots of the reaction mixture were collected prior to the start of irradiation (t = 0) and at five minute intervals throughout the experiment. Residual photocatalyst was removed from the aliquots by passage through a Teflon syringe filter (0.2 mm). The fluorescence intensity of the resulting solution was measured using an excitation wavelength of 315 nm and an emission wavelength of 425 nm. Studies employing charge carrier scavengers were conducted as described above, but the reaction mixture also contained 10 mM Na2CrO44H2O (e- scavenger) or 10 mM Na2C2O4 (h+ scavenger) [37,38]. 2.7. Antimicrobial experiments Several isolated colonies of E. coli O157:H7 or S. aureus (methicillin sensitive or methicillin resistant) from overnight cultures on

LB agar were suspended in 5 mL 0.9% sterile saline. The volume of the suspension was adjusted to achieve a final absorbance (k = 600 nm) to 0.5. The resulting solution was serially diluted to 106 using ice-cold sterile saline [39]. A 1.0 mL portion of the 106 dilution was combined with 20 mL of sterile 0.9% saline containing 10 mg u-g-C3N4 and the resulting mixture vacuum filtered onto a 0.45 mm nitrocellulose filter disk (47 mm disk diameter). These experimental conditions resulted in measured bacterial loading densities of 14–15 CFU/cm2. In all cases, the microbial loading density was selected to be 4–5 times higher than the average density of MRSA reported to be present on elevated surfaces in a hospital environment (3.5 CFU/cm2) [40]. Inoculated nitrocellulose filter disks were placed on pieces of sterile saline-dampened Whatman #1 filters in a glass petri dish placed on an ice bath, and then subjected to visible radiation (0–0.19 J total dose of applied radiation). Controls samples consisted of similarly prepared filters that received no irradiation (dark control). To measure nonspecific cytotoxicity (e.g. heating effects), replicate filters were prepared that contained bacteria, but lacked u-g-C3N4. After irradiation, the nitrocellulose filter disk was transferred to a LB agar plate and incubated for 24 h at 37 °C, and the colony forming units (CFU) on the disk counted. The % viability of each sample was calculated using the formula:

% Total CFU ¼

Test CFU  100 Total CFU ðt ¼ 0Þ

ð1Þ

Experiments were performed twice, on separate days, with each time point tested in triplicate. The results of the experiments are expressed as the mean % viability ± SEM (standard error on the mean). 2.8. Sporicidal experiments 10 mL of Difco Sporulation Media (DSM) was inoculated using an overnight streak plate of B. anthracis and grown overnight at 37 °C. The culture was diluted 1:100 in 100 mL of DSM and incubated at 33 °C for 3 days shaking at 250 rpm. The resulting solution was centrifuged at 10,000g for 10 min and washed once with 20 mL of sterile dH2O. The pellet was re-suspended in 20 mL sterile 50% ethanol (v/v), and incubated at 22 °C for 12 h with shaking at 100 rpm. The spores were then collected by centrifugation (10,000g/10 min), washed twice with 20 mL sterile dH2O, and the resulting suspension stored at 80 °C. To determine the viability of the endospores, 100 mL of the suspension was serially diluted into sterile PBS and plated on LB agar. The plates were incubated overnight at 37 °C and the colonies counted to determine CFU/mL. The purity of the endospore suspension was assessed by heat fixing 20 mL of the suspension onto a glass slide and staining for endospores. The stain was visualized using an oil immersion microscope and the number of endospores and vegetative cells counted manually in four areas of the slide (one in each quadrant of the stain as described in the paper). The endospore purity was >90% in each area. 3. Results and discussion 3.1. Physical characterization of u-g-C3N4 photocatalyst The activity of a heterogeneous photocatalyst will vary as a function of numerous chemical and physical characteristics, including composition, particle size and morphology, surface area, electronic structure, charge carrier lifetime and the presence of defect sites. Accordingly, in exploring the potential utility of u-gC3N4 for light-driven antimicrobial and biocidal applications, we have characterized this material with respect to these factors.

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Where appropriate, samples of g-C3N4 produced from dicyandiamide (hereafter labeled d-g-C3N4) are included in the discussion for the purpose of comparison. Samples of u-g-C3N4 are readily produced via direct thermal polymerization of dry urea. Since carbon nitride is generally understood to be non-stoichiometric, the composition of the material developed for this study was explored in detail. Combustion elemental analysis reveals that u-g-C3N4, possesses a composition of 34.17% carbon, 1.73% hydrogen, 60.64% nitrogen, and 2.40% oxygen. This composition corresponds to a C:N molar ratio of 1:1.52 and deviates from the 1:1.33 ratio predicted for the ideal ‘‘C3N4” molecular formula, suggesting that the materials developed for this study are slightly carbon deficient. Variation in material composition as a function of depth was explored by comparing the surface elemental composition derived from X-ray photoelectron spectroscopy analysis to the bulk elemental analysis described above. The surface C:N ratio is 1:1.45 and is similar to the results of the bulk elemental analysis, suggesting that the compositional imperfections are distributed throughout the sample. The results of the elemental analysis are supported by X-ray photoelectron spectroscopy experiments. The high resolution C 1s peak of u-g-C3N4 is asymmetric and, upon deconvolution, observed to be caused by the superposition of two major components with binding energies of 287.5 eV and 288.4 eV (Fig. 1). These peaks are assigned to the presence of (C)3AN and NAC@N groups, respectively [41,42]. Similarly, the N 1s portion of the ug-C3N4 photoelectron spectrum was deconvoluted into three components with binding energies of 398.1 eV (C@NAC). 398.9 eV (NA (C)3) and 400.3 eV (NAH) [42–45] In agreement with the results of the elemental analysis, oxygen was detected in u-g-C3N4 during the XPS experiment and the resulting O 1s peak was deconvoluted into four components. Two peaks with binding energies of 530.2 eV and 533.3 eV were assigned to the presence of C@O and CAO groups in the material, respectively. Two additional peaks at 531.9 eV and 534.9 eV originate from surface hydroxyl groups on the sample holder and chemisorbed water molecules on the surface of u-g-C3N4. The solid-state structure of u-g-C3N4 was investigated by a combination of powder X-ray diffraction and absorption (FT-IR) spectroscopy experiments (Fig. 2). The successful formation of the desired graphitic phase is indicated by the characteristic (0 0 2) reflection in the powder X-ray diffraction pattern. This reflection, which is caused by the interplanar spacing of the material, is observed at 2h = 27.5° (d = 3.24 Å). Similarly, the (1 0 0) reflection is observed at 2h = 12.9° and closely matches the dimensions expected for the individual melon subunits (d = 6.94 Å) understood to be present in the polymeric structure of the

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material. Two higher angle reflections are also observed at 2h = 44.4° and 56.7°. These peaks have been previously reported for samples of g-C3N4 synthesized in open crucibles and suggests that the tri-s-triazine subunits of the u-g-C3N4 polymer are incompletely condensed [20]. No other impurity phases or evidence of unreacted starting material was detected in the powder diffraction pattern. The materials prepared for this study possess an average crystallite domain size of approximately 8.3 nm, which agrees well with previously reported studies on carbon nitride materials [46]. The strong similarities between the FT-IR spectra of samples of u-g-C3N4 and d-g-C3N4, indicate that the general spatial orientation of the two materials is largely coincident. Indeed, the spectrum of both compounds is dominated by the characteristic vibrational and breathing modes associated with the heptazine ring systems and the terminal amine functional groups anticipated to be present in the extended, polymeric structure of g-C3N4. (Fig. 2, right) The sharp absorbance observed at 807 cm1 originates from either striazine or from heptazine ring units, while the numerous peaks located between 750 cm1 and 1650 cm1 are generally in excellent agreement with samples of g-C3N4 produced via thermal polymerization reactions, including materials produced from oxygenated precursors [30,47,48]. However, the relatively well defined peaks at 1201 cm1, 1237 cm1 and 1319 cm1 have been reported to be associated with the presence of CANHAC groups as a consequence of incomplete condensation and polymerization of the individual heptazine unit [49]. The morphology of u-g-C3N4 was probed by transmission electron microscopy experiments and found to be comprised of highly folded aggregates of discrete carbon nitride sheets (Fig. 3, left). Further investigation of the samples at higher magnification (Fig. 3, right) reveals that the carbon nitride layers contain numerous, irregularly shaped in-plane void spaces. The presence of in-plane pores has been previously reported to be characteristic of samples of g-C3N4 produced from oxygenated precursors, including urea or thiourea, or samples of g-C3N4 that were synthesized in the presence of water [29,50]. No evidence of lattice fringes were observed at the boundaries of samples of u-g-C3N4, indicating that the samples of this material were amorphous [51]. The effective particle size and surface area of u-g-C3N4 was investigated by hydrodynamic radius and BET measurements. The surface area of the photocatalyst, as determined by BET surface area analysis, is 72.2 m2/g. Hydrodynamic radius measurements reveal that approximately 61% of the sample is composed of particles with dimensions of 615 ± 201 nm, while 39% of the material exists in the form of larger aggregates with calculated dimensions of 5.51 ± 0.80 lm (Fig. 3, inset). A small fraction of the sample (ca. 0.4%) has an average particle size of 129 ± 31 nm.

Fig. 1. High resolution XPS spectra of u-g-C3N4 detailing the C 1s core-level spectra (left), N 1s core-level spectra (middle) and O 1s core-level spectra (right). Peaks identified in the course of spectral deconvolution are presented as dashed lines. In all cases, the identified peaks in each region-of-interest are consistent with the chemical environments anticipated to be present in u-g-C3N4.

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Fig. 2. Powder X-ray diffraction pattern of u-g-C3N4 (left) and FT-IR spectrum (right) of u-g-C3N4 (black trace) and d-g-C3N4 (grey trace).

Fig. 3. Transmission electron micrographs detailing the morphology of u-g-C3N4 at low (left) and high (right) magnification levels. The inset image depicts the particle size distribution of u-g-C3N4, as determined by hydrodynamic radius measurements.

3.2. Electronic characterization of u-g-C3N4 photocatalyst The electronic structure of u-g-C3N4 and d-g-C3N4 was investigated by diffuse reflectance UV–visible absorption spectroscopy (Fig. 4, left). Analysis of the absorbance data indicates that u-gC3N4 possesses an indirect band gap of 2.86 ± 0.14 eV. This value is comparable to other samples of g-C3N4 prepared under similar conditions [41]. The valence band edge (VBE) in u-g-C3N4 was directly measured by XPS and lies at a potential of +1.29 V vs NHE (Fig. 4, right). Using this information, the potential of the conduction band edge (CBE) was calculated to lie at 1.57 V vs NHE. The VBE and CBE for d-g-C3N4 have been previously reported to

have potentials of +1.63 V and 1.17 V, respectively (Fig. 5, left) [15]. We anticipate that the physical and electronic properties of u-gC3N4 will enhance the utility of this material for antimicrobial applications by facilitating the photochemical production of ROS. Our previous research in this area clearly demonstrates that nanostructured samples of g-C3N4 are able to efficiently produce hydroxyl radicals (OH) in solution when subjected to appropriately energetic radiation [15]. We confirmed the photochemical production of ROS by u-g-C3N4 using the molecular probe TA. As described above, this molecule selectively scavenges hydroxyl radicals (OH) in solution to produce the fluorescent molecule 2-OH-TA [36].

Fig. 4. Tauc plot (left) and XPS valence band energy spectrum (right) of u-g-C3N4. The left inset image details the diffuse reflectance absorbance UV–vis absorbance spectrum of u-g-C3N4 (black trace) and d-g-C3N4 (grey trace).

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Fig. 5. Detail of the calculated band structures of u-g-C3N4 and d-g-C3N4 (left) and the observed change fluorescence (kex = 315 nm; kem = 425 nm) of irradiated mixtures of TA and u-g-C3N4, either alone ( ) or in the presence of the selective charge carrier scavenging agents CrO2 ) or C2O2 ). 4 ( 4 (

Irradiated reaction mixtures of u-g-C3N4 and TA produce 2-OH-TA and a concomitant increase in fluorescence emission intensity, indicating successful formation of hydroxyl radicals by the photocatalyst. (Fig. 5, right). The mechanism of formation of the detected hydroxyl radicals by u-g-C3N4 is a point of consideration. The spectroscopic results described above indicate that the potential of a photoelectron at 0 the u-g-C3N4 CBE can reduce O2 to O O2/O 2 (E 2 = 0.33 V). (Fig. 5, left) Importantly, as described in Eq. (2), the formation of O 2 can result in the production of a variety of other ROS, including the hydroperoxyl radical (HO2), hydrogen peroxide (H2O2) and the spectroscopically detected hydroxyl radical (OH). All ROS are highly reactive and potentially cytotoxic [52]. In contrast, a photogenerated h+ charge carrier located at the VBE of u-g-C3N4 is insufficiently reactive to oxidize water to produce hydroxyl radicals (single photon, single electron process) or hydrogen peroxide (two photon, two electron process). (E0 H2O/OH = +2.33 V; E0 H2O/H2O2 = +1.78 V) [53]. The electrochemical properties of u-g-C3N4 described here indicate that the observed photochemical formation of OH likely proceeds by way of reduction of elemental oxygen.   O2 ! O 2 $ HO2 ! H2 O2 $ HO

ð2Þ

We tested the above analysis by exploring the photochemical production of hydroxyl radicals by u-g-C3N4 in the presence of the selective charge carrier scavenging agents Na2CrO44H2O and Na2C2O4. Administration of the photoelectron scavenging agent Na2CrO44H2O to a u-g-C3N4/TA reaction mixture results in a ca. 98.3 ± 2.9% decline in the rate of formation of 2-OH-TA. (Fig. 5, right) In contrast, reaction mixtures containing the h+ specific scavenging agent Na2C2O4, experience a ca. 35.6 ± 1.1% decline in the rate of formation of 2-OH-TA. This data suggests that both charge carrier species are able to play a role in the photochemical production of ROS. The nearly complete lack of production of 2-OH-TA in the presence of the e scavenging agent indicates that reductive pathways are the major source of ROS formation by u-g-C3N4. This conclusion is supported by the successful formation of 2-OH-TA in the presence of the h+ specific scavenger, Na2C2O4, revealing that the h+ charge carrier has a reduced impact on ROS formation by u-g-C3N4. Several recent reports reinforce the significance of reductive processes for ROS formation and biocidal applications by gC3N4-based materials [23,24,54]. In the case of this study, we note that the u-g-C3N4 h+ is sufficiently oxidizing to convert water to elemental oxygen. (E0 H2O/O2 = +1.23 V) [53]. Such oxidation processes could promote ROS production, as indicated by the scavenger study, by increasing the concentration of oxygen in the region of the photocatalyst. The photophysical behavior of u-g-C3N4 was investigated by photoluminescence (PL) spectroscopy. The steady-state PL spec-

trum of d-g-C3N4 and u-g-C3N4 contain a broad peak due to the radiative recombination of charge carriers (Fig. 6, left) [55]. Relative to what is observed for d-g-C3N4, the total intensity of the emission peak is significantly reduced for samples of u-g-C3N4, indicating a reduced rate of radiative recombination events and a shorter lifetime of the photogenerated charge carriers in this material [49]. The wavelength of the emission maximum of the luminescent peak of d-g-C3N4 is observed at 439 nm and agrees well with the energy associated for the band-to-band transition in this material. In contrast, the PL emission peak maxima for u-g-C3N4 undergoes a bathochromic shift to 454 nm. The reduced energy of the maxima of the PL peak for samples of u-g-C3N4 is indicative of the presence of sub-gap defects in the material, possibly due to defects stemming from the previously described incomplete condensation and polymerization [49,56]. The observed reduction in recombination rate observed in the steady-state PL experiments can be caused by either rapid charge carrier migration with shorter total lifetimes or slower charge carrier recombination processes with consequent extended lifetimes [57]. In order to more completely explore the behavior and dynamics of the charge carriers in u-g-C3N4, we conducted time-resolved transient photoluminescent experiments (Fig. 6, right). The emission decay spectra for both d-g-C3N4 and u-g-C3N4 were modeled using triexponential kinetics for which the three individual components were derived. As detailed in the inset table presented in Fig. 6, all of the individual components for u-g-C3N4 are shorter than what is observed for d-g-C3N4. The intensity average lifetime, hsi, of u-g-C3N4 is also shorter than what is observed for d-g-C3N4. The combined shortening of transient lifetimes and reduced steady-state luminescence observed for samples of u-g-C3N4 can be attributed to the emergence of new, non-radiative decay pathways in the material. Non-radiative pathways compete with direct charge carrier recombination events and follow an inverse trend for fluorescence quantum yield [56]. The dramatic increase in the calculated weighting factor of s1 for u-g-C3N4 (22.01%), compared to d-g-C3N4 (8.41%), suggests that this alternate pathway proceeds via rapid charge carrier injection into various localized states, including those produced as a consequence of lattice defect sites [41,56,58]. Significantly, such localized defect states have previously been observed to serve as points of enhanced photochemical reactivity in samples of g-C3N4, even as they simultaneously facilitated a reduction in the total observed photoluminescent lifetime of the material [59]. 3.3. Antimicrobial experiments Samples of g-C3N4, independent of their molecular precursor, are established intermediate band gap semiconductors and will

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Fig. 6. Steady state (left) and transient (right) photoluminescent spectra of u-g-C3N4 (black trace) and d-g-C3N4 (grey trace). The employed excitation radiation was k = 371 nm for all experiments.

Fig. 7. Relative % viability for S. aureus 6538 or MRSA ( ) observed as a function of applied radiation dose (400 nm
k
426 nm). Traces for dark control studies with ( ) or without ( ) u-g-C3N4, or irradiated without u-g-C3N4 ( ) are also presented. Error bars represent the standard error of the mean (SEM) for at least three independent trials.

interact with appropriately energetic radiation to produce reactive charge carrier species [16,60]. The combined electronic, chemical and physical features of u-g-C3N4 described above, including high surface area, porous architecture, increased CBE reduction potential, facile ROS production and reduced incidence of radiative charge carrier recombination events, suggest that this material is well suited for antimicrobial applications. Accordingly, films of ug-C3N4 were fabricated and challenged with a variety of clinically relevant microorganisms in the presence of visible radiation. Administration of visible radiation to co-deposited samples of ug-C3N4 and the Gram positive coccus S. aureus resulted in dramatic decreases in total bacterial survivorship (Fig. 7). Both methicillin resistant (MRSA) and methicillin sensitive strains of S. aureus were tested in the course of this project. In the presence of visible radiation, coatings of u-g-C3N4 exhibited an IC50 value of 33.5 ± 0.2 mJ against MRSA and 42.7 ± 0.5 mJ against S. aureus (ATCC 6538). The observed IC50 value for MRSA represents a 90.6% reduction in total required input radiation dose relative to what has been previously reported [15]. Similar improvements in antimicrobial activity were realized in studies using E. coli O157:H7 (Fig. 8, left). As with the samples of S. aureus, co-deposited mixtures of u-g-C3N4 and E. coli O157:H7 exhibited a dose related response to visible radiation (IC50 = 14.1 ± 0.2 mJ). Complete eradication of E. coli O157:H7 CFUs was achieved upon administration of 96 mJ of visible radiation. The IC50 value of u-g-C3N4 against E. coli O157:H7 represents a 95.5% improvement in antimicrobial activity relative to what has been

reported for other nanostructured carbon nitride-based materials [15]. In all experiments involving S. aureus (methicillin resistant or sensitive) or E. coli O157:H7, coatings of u-g-C3N4 that were exposed to visible radiation were able to reduce the population density of the target microorganisms below 2.5 CFU/cm2, which is an accepted threshold for the successful decontamination of high touch frequency sites [61]. Non-irradiated filters with co-deposited u-g-C3N4 and bacterial cells (dark controls) failed to demonstrate any impact on bacterial survival, indicating that the material itself is not overtly toxic. Additionally, control experiments employing irradiated/nonirradiated filters without the catalyst generally exhibited no significant effects on bacterial survivorship. While some light toxicity was observed in the case of MRSA, these effects were not enough to account for the total loss in viability observed in experiments that included both u-g-C3N4 and visible radiation. Consequently, it is possible to conclude that the antimicrobial activity of u-gC3N4 films observed in these studies is not simply a product of radiation-induced cellular damage. It is significant to note that the observed rate of antimicrobial activity against both grampositive and gram-negative organisms by u-g-C3N4 compares favorably to results reported for TiO2 thin films, despite the fact that this present study employed only visible radiation as an excitation source [62,63]. The significant improvements in antimicrobial activity of the ug-C3N4-based films against samples of S. aureus and E. coli O157:H7 prompted us to explore the ability of this material to deactivate

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Fig. 8. Relative % viability for of E. coli O157:H7 or B. anthracis endospores ( ) observed as a function of applied radiation dose (400 nm
k
426 nm). Traces for dark control studies conducted with ( ) or without ( ) u-g-C3N4, or irradiated without u-g-C3N4 ( ) are also presented. Error bars represent the standard error of the mean (SEM) for at least three independent trials.

bacterial endospores. As anticipated, endospores of B. anthracis are much more resistant to damage than either the gram-positive or gram-negative microorganisms. (Fig. 8, right) However, there was a ca. 25% reduction observed in endospore viability upon administration of dose of visible radiation that was comparable to what was employed in the bacterial studies (0.2 J). The decline in survivorship of B. anthracis endospores exposed to a combination of visible radiation and u-g-C3N4 is significantly different (p < 0.05) from both the light and dark control runs. These results represent the first successful demonstration of sporicidal activity by carbon nitride-based materials. 4. Conclusion Films prepared from u-g-C3N4 demonstrate improved, visible light driven biocidal activity against both clinically significant gram-negative and gram-positive bacteria and demonstrable activity against B. anthracis endospores. The enhanced reactivity of u-gC3N4 is likely due to a combination of factors including the high surface area of the catalyst, the increased reducing power of the conduction band edge of the material, and a reduced incidence of radiative recombination events of photogenerated charge carriers. Importantly, no antibacterial activity was observed for u-g-C3N4 films that were not exposed to visible radiation. These results indicate that samples of u-g-C3N4 are not inherently toxic, despite the observed overall improvements in the reactivity of the material. This study clearly demonstrates that g-C3N4-based materials are viable candidates for antimicrobial and environmental remediation applications, including for the photochemical decontamination of high-touch frequency surfaces. As such, these materials can be envisioned to help develop new technologies to suppress the continued transmission of hospital-acquired infections. Acknowledgements The work described in this publication was made possible by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants #P20GM103408 and P20GM109095. We also acknowledge support from the Biomolecular Research Center at Boise State with funding from the National Science Foundation (Grants #0619793 and #0923535); the MJ Murdock Charitable Trust; and the Idaho State Board of Education. The authors gratefully acknowledge Prof. Angel Marti (Rice University, Houston, TX) for assistance with photoluminescence spectroscopy experiments and Dr. Madhu Kongara (Boise State University, Boise, ID) for assistance with DRFTS and hydrodynamic

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