Young-Seon
Ko
a,
Yun Haeng
Joe
b,
Mihwa
Seo
a,
Kipil
Lim
a,
Jungho
Hwang
b and
Kyoungja
Woo
*ac
aMolecular Recognition Research Center, Korea Institute of Science and Technology, P. O. Box 131, Cheongryang, Seoul 130-650, Korea. E-mail: kjwoo@kist.re.kr
bSchool of Mechanical Engineering, Yonsei University, Seoul 120-749, Korea
cKorea University of Science and Technology, Seoul 136-701, Korea
First published on 8th August 2014
There is a significant need for materials that promptly exhibit antimicrobial activity upon contact. The large-scale fabrication of monodisperse silver nanoparticle (AgNP)-decorated silica (AgNP@SiO2) hybrid particles, and their prompt and synergistic antibacterial activity against both the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus epidermidis on air filtration units are presented. Monodisperse aminopropyl-functionalized silica colloids (406 nm) were used as a support material and were hybridized with AgNPs using a seeding, sorting-out, and growing strategy with Ag seeds (1–2 nm) into ∼30 nm AgNPs, successfully yielding 51 g of AgNP@SiO2 hybrid particles. Medium filter samples (glass fiber material, 4 × 4 cm2) were coated with AgNP@SiO2 particles and tested for antibacterial efficacy. SEM characterization of the bacterial morphology suggested prompt and synergistic antibacterial activity against both classes of bacteria. Moreover, antibacterial efficacies >99.99% for both bacteria were obtained using a filter sample with a coating areal density of 1 × 108 particles per cm2. Solutions of AgNP@SiO2 at 1.3% were stable even after 8 months. The hybrid particle AgNP@SiO2 and the air filter system coated with the particles are expected to be useful for future green environment applications.
Silver and silver containing materials such as silver nanoparticles (AgNPs) and Ag+ ions are known to be potent antimicrobial agents.4–6,8–13 In particular, Ag+ ions in aqueous solution have shown great antimicrobial activity, and their antimicrobial mechanism has been studied widely.5,8 Ag+ ions interact with the proteins on the bacterial membrane and penetrate into the cell, where they interact with DNA and RNA. These interactions change the structure and function of target molecules and eventually lead to cell death. Antimicrobial efficacy normally appears after treatment for 2 h to several days, depending on the Ag+ concentration. On the other hand, the antimicrobial activity of AgNPs has been debated. A clear-cut distinction between the antimicrobial activity of AgNP and that of Ag+ has been difficult because AgNPs are oxidized by oxygen and release Ag+ ions. Therefore, AgNPs and Ag+ ions always coexist in aerobic aqueous solutions.14–16 This issue was recently investigated using poly(ethylene glycol)–thiol-coated AgNPs in an anaerobic aqueous solution to prohibit the release of Ag+ ions.16 It was reported that the antibacterial activity of AgNPs in the anaerobic aqueous solution mainly comes from the previously oxidized and dissolved Ag+ ions and that the particle-specific antibacterial activity is negligible.16 Prior to this, Sondi et al. reported that AgNPs make pits on the membranes of Gram-negative bacteria (Escherichia coli) and lead to cell death in aerobic aqueous solutions.9 Supporting reports have indicated an enhanced antibacterial effect under the co-existence of AgNPs and dissolved Ag+ ions, rather than Ag+ ions alone at constant concentrations.17–19 In particular, comparable antibacterial activities of AgNPs and the released Ag+ ions have been observed for AgNPs larger than ∼15 nm,18,19 whereas the antibacterial activity is dominated by the released Ag+ ions for AgNPs smaller than ∼10 nm.18 It is noteworthy that AgNPs9,17–19 which have shown enhanced antibacterial activity, in addition to the antibacterial activity of Ag+ ions, have had a partially naked surface due to their labile ligands or their hybrid structure. Unfortunately, fully naked AgNPs cannot exist as a colloidal solution due to their aggregation caused by high surface energy.
A silica hybrid colloid encapsulating a magnetite core and decorated with 30 nm-sized AgNPs (AgNP@SiO2/Fe3O4)20 and a magnetite-silica Janus nanorod decorated with 5 nm-sized AgNPs (AgNP@Fe3O4–SiO2)21 have been reported as magnetically collectable antimicrobial materials in water. Both reports used the silica material as a biocompatible, water-dispersible, and physico-chemically stable inorganic support for the naked AgNPs. The former colloid exhibited immediate and synergistic antibacterial activity compared to the latter Janus nanorod due to the large (30 nm vs. 5 nm) and many AgNPs in a three-dimensional structure on a silica support.20 However, hybridization of silica with magnetic material encumbers a synthesis process with extra complexity, particularly for a large-scale fabrication. Moreover, the magnetic property is not necessary for a coating material, which is applied on appliances such as air filters and air conditioners. Thus, monodisperse silica hybrid particles decorated with AgNPs of ∼30 nm may be a reliable candidate as a prompt antibacterial coating material.
Researchers have coated Ag+ ion-containing materials and AgNPs or their carbon composites on glasses and air filters using various methods and have reported antibacterial efficacy by the colony count method (CCM) or by the disk diffusion method (DDM), which are generally used worldwide.4,5,13,22–26 The CCM is a method to count the bacterial colony number after spreading a specimen-treated bacterial solution on an agar plate and incubating for approximately 24 h. The DDM is based on the appearance of a belt-like inhibition zone surrounding the specimen after placing it on the nutrient agar in bacterial solution and incubating for approximately 24 h. Both methods evaluate antibacterial activity after incubation of aqueous bacterial solution with the Ag+- or AgNP-coated specimen. Fundamentally, these evaluation methods do not differentiate the antibacterial effect of Ag+ ions from that of AgNPs which release Ag+ ions into aerobic water. To the best of our knowledge, there has been no substantiated coating material showing a prompt antibacterial activity in air. Therefore, a material that can promptly show antibacterial activity in air at its coated state is greatly needed.
In this report, the large-scale (51 g) fabrication of monodisperse silica hybrid particles decorated with ∼30 nm-sized AgNPs (AgNP@SiO2), and their prompt and synergistic antibacterial activity against both the Gram-negative bacteria Escherichia coli (E. coli) and the Gram-positive bacteria Staphylococcus epidermidis (S. epidermidis) on air filtration units are presented.
The average size of the aerosols was determined to be ∼430 nm by SMPS. The particle concentrations upstream and downstream from the filter samples were 1.78 × 105 particles (#) per cm3 and 1.75 × 104 # per cm3, respectively. The coating areal density (ρareal) in # per cm2 was calculated using eqn (1) for each coating time t:
![]() | (1) |
Coating time t (min) | Particle concentration (# per cm3) | Volume flow rate Q (cm3 min−1) | Effective area A (cm2) | Coating areal density ρareal (# per cm2) | |
---|---|---|---|---|---|
C up | C down | ||||
1 | 1.78 × 105 | 1.75 × 104 | 2000 | 16 | 2 × 107 |
3 | 6 × 107 | ||||
5 | 1 × 108 | ||||
10 | 2 × 108 |
The results of each experimental method were averaged from a dozen replications. Antibacterial efficiency (ηCCM) was calculated by the following eqn (2):
![]() | (2) |
The overall process for the hybridization reaction is illustrated in Scheme 1. Fig. 1b(i–iv) show TEM images of a magnified part of each hybrid particle through the fabrication process. Corresponding TEM images of the whole hybrid particles are included in Fig. S4.†Fig. 1b(i) shows the smooth surface of a part of APSiO2 and corresponds to Scheme 1a. Fig. 1b(ii) shows densely self-assembled Ag seeds on APSiO2 (Agseed@SiO2) and corresponds to Scheme 1b. The reaction for the self-assembly of Ag seeds on APSiO2 was delicate and different from that of Ag seeds on APSiO2/Fe3O4 (ref. 20) or from that of Au seeds on APSiO2.29 The labile property of a bond between an amine and an Ag seed is not considered to allow seeding by coordinative bonding, which works nicely in the case of Au seeds.29–31 Thus, an electrostatic interaction was used to self-assemble negatively charged THPC-protected Ag seeds on a positively charged APSiO2 at pH ∼ 4. In the case of Ag seeding on APSiO2/Fe3O4, the Fe3O4 core seems to play a role as a stabilizer for the seeded hybrid structure of Agseed@SiO2/Fe3O4 thanks to its reducing property32 with respect to Ag+ to Ag. However, large-scale fabrication with a monodisperse size distribution is limited in the case of AgNP@SiO2/Fe3O4 hybrid particles, because the silica-encapsulation reaction of the Fe3O4 core requires a highly diluted condition to avoid aggregation and the resultant product SiO2/Fe3O4 exhibits a range (0.5–0.7 μm) of size distribution.20 For a successful seeding on APSiO2, a large portion of well-controlled Ag seeds in the 1–2 nm range was required. These seeds were obtained by optimizing the molar ratio of THPC–Ag to 1:
1 and by adding Ag+ into a THPC solution rather than the vice versa. The APSiO2 solution (pH ∼ 4) was poured into the alkaline THPC-protected Ag seed solution and gently swirled intermittently for seeding. When the centrifuged and re-dispersed Agseed@SiO2 solution was poured into an Ag+ solution containing NH4OH and stirred, only the relatively larger seeds were displaced from the amine moieties and coalesced together as shown in Fig. 1b(iii) and Scheme 1c. The coalesced particles departed from the silica particle due to their lager inertia during stirring and at the end of the reaction they were removed by centrifugation. The stirring time of Agseed@SiO2 with the Ag+ solution containing NH4OH was critical and was adjusted empirically. This kind of sorting-out step guaranteed some distance between the relatively small remaining seeds. This distance was necessary for the seeds to grow into the nanoparticles shown in the Fig. 1b(iv) and Scheme 1d. During the early stage of the growth step, the remaining Ag seeds are likely to grow in all directions. During the later stage, the seeds grow upwards and sideways because there is not much space downward. Through the growth step, AP moieties are expected to be embedded in AgNPs, similar to the case of AgNPs grown from the Au seeds.29 Thus, the AgNPs on the APSiO2 sphere exhibit a hemisphere-like shape, which is key for the fixation of the AgNPs on APSiO2 spheres. Without the sorting-out step, the densely self-assembled Ag seeds are likely to grow to a smooth and complete shell on APSiO2.29,33 In the case of using Au seeds, instead of Ag seeds, growth of the AgNPs is limited up to ∼15 nm because the Au seeds pass through a different reaction pathway29 than the Ag seeds and the resultant hybrid particles show much lower antimicrobial effects than their corresponding hybrid particles with ∼30 nm AgNPs.20 The TEM image of AgNP@SiO2 particles exhibits gray and black areas, which correspond to silica and AgNPs, respectively.
Fig. 1c shows the stability of the hybrid structure of AgNP@SiO2 colloids with varying times and concentrations in aqueous solution. The initial morphology of a 1.3% AgNP@SiO2 solution was preserved after 8 months (i). In the case of a 5% solution, the initial morphology was preserved even after 1 year (data not shown). Therefore, the hybrid structure of AgNP@SiO2 colloids in solution seems to be stable for use as an air filter coating, considering that the concentration we applied in the atomizer for air filter coating was 1.3% and a higher concentration would lead to higher throughput in the coating process. However, the 10-fold diluted hybrid colloids (0.13%) lost their integral morphology after only 1 month (ii). This morphological change was likely caused by oxidation and dissolution of AgNPs in the diluted aerobic solution. Interestingly, when SiO2/Fe3O4 particles were added at 5% by particle number, the morphology was preserved (iii). The preservation of the hybrid structure of AgNP@SiO2 colloids in (iii) can be attributed to the sacrificial oxidation of Fe2+ to Fe3+ in the Fe3O4 core prior to oxidation of AgNPs because the standard reduction potentials of Ag+ to Ag and Fe3+ to Fe2+ are 0.80 V and 0.77 V, respectively.34
Fig. 1d shows the XPS result of an AgNP@SiO2 sample. The carbon 1 s line was used as a reference for the binding energy scale (284.6 eV). Every line position in (i) is similar to those of AgNP@ SiO2/Fe3O4,20 as expected, because the magnetite core is located deep at the center of the hybrid sphere with a silica shell thickness of ∼0.1 μm. Interestingly, however, the line positions of Ag 3d5/2 and 3d3/2 in the high resolution spectrum (ii) appear at 368.3 and 374.3 eV, which are 0.5 eV higher than those in AgNP@SiO2/Fe3O4. This result indicates that partial oxidation of the AgNPs proceeded further on SiO2 than on SiO2/Fe3O4. Fortunately, it has been reported that partially oxidized AgNPs still exhibit antibacterial activity,13,14 which will be shown later. The XRD pattern of AgNP@SiO2 (Fig. 1e) exhibits only the face centered cubic crystalline phase of the Ag metal (JCPDS File no. 04-0783) because silica is amorphous. The UV-Vis absorption spectrum of AgNP@SiO2 (Fig. S5†) shows a typical plasmonic peak33 of AgNPs at 410 nm as well as the associated peak at ∼600 nm.
As an example for application, AgNP@SiO2 aerosols were generated from AgNP@SiO2 colloidal solution via a Collison atomizer and coated on the air filter samples using the experimental setup in Fig. S2.† The coated filter samples with varying coating times and particle concentrations are summarized in Table 1. First, we wanted to examine whether the coated AgNP@SiO2 particles exhibit antibacterial activity on air filtration units. For this purpose, the selected bacterial aerosols carried by clean air at 2 L min−1 were deposited on the AgNP@SiO2-coated filter samples with a coating areal density of 2 × 108 # per cm2. SEM images of these samples with different magnifications are displayed in Fig. 2a and b for E. coli and S. epidermidis, respectively. The coated AgNP@SiO2 aerosols show a relatively homogeneous distribution on the filter. Relatively larger ellipsoids (∼0.6 μm) in a and spheres (∼0.8 μm) in b represent E. coli and S. epidermidis, respectively, which agrees with the published data.35,36 Strikingly, both types of bacteria are trapped by AgNP@SiO2 hybrid particles coated on the filter fiber and a single AgNP@SiO2 hybrid particle is enough to trap each bacterium. Furthermore, the trapped bacteria do not seem to be viable, possibly because the trap is so strong that the bacteria cannot escape without rupturing their cellular membrane or cell wall. Many AgNPs protruding from a silica support are considered to play a key role as many teeth to bite bacteria, leading to cell death by chemsorption of Mg2+ or Ca2+ ions from bacterial surface as will be discussed later. To the best of our knowledge, this is the first explicit evidence for a coated material showing prompt antibacterial activity on air filtration.
It would be useful to know whether the interaction between AgNP@SiO2 hybrid particles and bacteria in air is the same as in water. Thus, the temporal morphology of the bacteria was investigated while incubating their aqueous mixtures with AgNP@SiO2 hybrid colloids. SEM images from this experiment are displayed in Fig. 2c and d and S6.† In the case of E. coli, their morphology (ellipsoid, a) trapped on the filter looks different from that (ci) dropped on the silicon wafer. The morphology of E. coli on the filter has been preserved during Pd/Pt sputtering for SEM sample preparation because they were deposited on the filter fiber in three-dimensional space after passing through a diffusion dryer. On the other hand, the dropped and dried E. coli on the silicon wafer with a two-dimensional surface exhibits rich surface morphology, as reported,37 due to a chemical etching by plasma during Pd/Pt sputtering. Plasma is known to etch away the outer membrane according to the treatment time and cause shrinkage of E. coli (ATCC 11775).37 The E. coli bacteria seem to be disrupted promptly upon contact with AgNP@SiO2 colloids. No pristine bacteria were present, even at 10 min. In the case of S. epidermidis, most AgNP@SiO2 colloids seem to bite bacteria promptly at their first contact (10 min image). In contrast to the morphology (sphere) of bacteria trapped on the coated air filter, the morphology of S. epidermidis on the silicon wafer exhibited an additionally flattened disk shape, which seems to be caused by the Pd/Pt sputtering on the wafer for SEM sample preparation.37 Bitten traces are indicated as an arrow in Fig. 2d and S6b.† The bitten traces appear to be separated from the AgNPs on the silica sphere by the sputtering impact. Therefore, we deduced that the bacteria trapped on the AgNP@SiO2 hybrid particles coated on the air filter will lead to cell disruption, indicating cell death and that many AgNPs decorated on the silica sphere play that role promptly and synergistically; i.e., a single AgNP with partially naked surface can make a pit on the Gram-negative bacterial membrane,9 whereas a single AgNP@SiO2 hybrid particle can disrupt a bacterial membrane or cell wall upon contact. Cell viability tests that will be shown later with Fig. 3b and c can support our deduction. This type of prompt antibacterial effect from AgNP@SiO2 hybrid particles may be particularly promising for treatment of antibiotic-resistant microorganisms such as super-bacteria.
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Fig. 3 (a) Pressure drop and antibacterial efficacy of AgNP@SiO2-coated air filter samples for (b) E. coli and (c) S. epidermidis. |
The prompt and fatal antibacterial activity of AgNP@SiO2 hybrid particles is attributed to the naked AgNPs and their teeth-like synergistic effect in a three-dimensional structure. Much like the case of AgNP@SiO2/Fe3O4 hybrid particles,20 the naked AgNPs chemically adsorb Mg2+ or Ca2+ ions from the Gram-negative bacterial membrane that contains tightly packed lipopolysaccharides (LPS). Within the membrane, metal ions such as Mg2+ and Ca2+ play an essential role in preserving the structure by strengthening the LPS interactions.38 The same mechanism is also likely responsible for the efficacy against the Gram-positive bacteria. In the case of Gram-positive bacteria, the thick peptidoglycan layer of the cell wall is known to be resistant to the penetration of Ag+ ions.8 However, the peptidoglycan layer is also strengthened by metal ions, as Mg2+ and Ca2+ ions are known to form bridges across phosphate groups in adjacent teichoic acid chains in the Gram-positive bacterial cell wall.39 Thus, the naked AgNPs decorated on the silica sphere seem to pressurize the spots where the metal ions are bridged in the thick bacterial cell wall and chemically adsorb Mg2+ or Ca2+ ions, as observed on the right top side of Fig. 2b. Due to the thick bacterial cell wall, the bacteria stay attached to the AgNP@SiO2 hybrid particles until they are disrupted by an external force, as indicated by the arrows in Fig. 2d and S6b.† From these images and the viability test in Fig. 3b and c, the trapped bacteria are assumed to be dead. Therefore, in both Gram-negative and Gram-positive bacteria, the bacterial trapping by AgNP@SiO2 hybrid particles indicates cell death.
The air filter samples summarized in Table 1 all showed a coating efficiency of AgNP@SiO2 particles greater than 90%. No meaningful detachments of AgNP@SiO2 particles were observed from the coated air filter samples, even when a clean air flow of 10 L min−1 was forced onto them. In Fig. 3a, the pressure drops caused by the coating increased linearly with the increasing coating areal density at constant media velocity but are acceptable because the variation was generally within 5%. At a media velocity of 0.025 m s−1, the filtration efficiencies of bioaerosols were 91% and 95% for the air filter samples with the coating areal densities of 6 × 107 and 2 × 108 # per cm2, respectively. Although the CCM or the DDM do not exactly reflect antibacterial activity in air, we performed cell viability test using the CCM because there is no better alternative in the present case. In Fig. 3b and c, the antibacterial efficacies determined by the CCM were high (i.e., >99.99%) against both E. coli and S. epidermidis in solutions containing the filter samples with a coating areal density ≥1 × 108 # per cm2.
Footnote |
† Electronic supplementary information (ESI) available: A photograph of tailor-made 50 L reactor, experimental setup for fabrication of antibacterial air filters, TEM and SEM images, UV-Vis absorption spectrum. See DOI: 10.1039/c4tb01068j |
This journal is © The Royal Society of Chemistry 2014 |