Atomic Force Microscopy and Angular-Dependent X-ray Photoelectron

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AFM and Angular Dependent XPS Studies of Anchored Quaternary Ammonium Salt Biocides on Quartz Surfaces Rachel Mathew, Ralph P. Cooney, Jenny Malmstrom, and Colin S. Doyle Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00535 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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AFM and Angular Dependent XPS Studies of Anchored Quaternary Ammonium Salt Biocides on Quartz Surfaces Rachel T.Mathew1, Ralph P. Cooney*1, Jenny Malmstrom1, 2 and Colin S. Doyle2 1

MBIE The Biocide Toolbox Programme, School of Chemical Sciences, The University of

Auckland, Private Bag 92019, Auckland, New Zealand. 2

Department of Chemical and Materials Engineering, The University of Auckland, Private Bag

92019, Auckland, New Zealand. Keywords: Biocides, QAS, Chemisorption, Island Films, Quartz, AFM, XPS, Bacteria, Escherichia coli, Staphylococcus aureus ABSTRACT A siloxane surface-anchored quaternary ammonium salt (AQAS: Biosafe HM4100 in this study) has been chemisorbed onto a quartz substrate. The aim of this study is to elucidate, using Atomic Force Microscopy (AFM) and X-Ray Photoelectron Spectroscopy (XPS), the structure of the chemisorbed AQAS layers. The AQAS biocide includes a C18 alkyl chain previously invoked in lysis potency. The AQAS coverage appears in zones on the surface which include a first layer (2.6 ±0.1nm) and multi-layering which were explored using AFM. The X-ray Photoelectron Spectroscopy (XPS) data exhibited two N1s signals at about 402 and 399eV, with only the former exhibiting angular dependence. This signal at 402eV was assigned to the first anchored layer with perpendicular orientation determined by the AQAS anchoring to the surface. In preliminary AFM studies of bacteria on these AQAS surfaces, perturbations on the Staphylococcus aureus cells, and the degradation of Escherichia coli cells, suggest lysis potency.

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INTRODUCTION The global market trend towards greener biocides has been driven by consumer demand for improved hygiene with reduced risks to human health and background environmental impact1. This trend is reflected in the incorporation of greener biocides in commercial products across several niche sectors including healthcare, construction, packaging, water and air quality, etc. The biocide types that can be presented at mono-molecular or nanoscale coverage levels have significant advantages over conventional thicker coating technologies, especially if the former are strongly anchored to the substrates and therefore are able to provide persistent non-leachable hygienic protection over an extended period. The advantages of anchored biocides include ideally optimized hygiene at very low mono-molecular coverage dosage levels which are several orders of magnitude less than conventional coating technologies. In addition to the reduced risk of toxicity with very low dosage levels, this also substantially simplifies the product life-cycle, in particular in regards to safe substrate recycling processes. One of the more important, and established, anchored biocides is the commercial product, Biosafe HM4100, an Anchored Quaternary Ammonium Salt (AQAS) incorporating a siloxane anchoring group. The mechanism of its potency is invoked by its manufacturers to be lysis (cell wall penetration) by the C18 chain, which is attached to the positively charged quaternary nitrogen2-4. A contrasting aspect is that the common, potent non-anchoring Quaternary Ammonium Salt (QAS) biocides are usually considered to act by ion-exchange with alkaline earth cations in the cellular membrane surface5, 6. Such a cation-exchange mechanism is not feasible for surface-anchored QAS.


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The overall aim of this paper is to elucidate, using surface-analytical techniques, the fundamental morphology, structure, orientation and surface chemical behavior of AQAS biocide monolayers and multilayers on a flat quartz surface with known silanol-anchoring capacity. The surfaceanalytical techniques employed are Atomic Force Microscopy (AFM) and angular-dependence X-Ray Photoelectron Spectroscopy (XPS) with some additional molecular surface-region analysis from Attenuated Total Reflectance Infrared Spectroscopy (ATR FTIR). The secondary aim using AFM is to investigate the extent of cellular disruption for common Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria in surface contact with the surface layers of AQAS on Quartz. The latter recognizes the limitations of AFM to probe the actual bacteria-AQAS contact surface and so focuses on evidence of perturbation on the outer bacterial surfaces. There have been recent developments in the literature for the types of systems in this study, viz., Anchored QAS film/Substrate and Bacteria/Anchored QAS film/Substrate systems. Some but not all of these reports involve the combination of surface analysis methods, Atomic Force Microscopy (AFM) and X-Ray Photoelectron spectroscopy (XPS)7-22. The extensive review of the literature prior to 2015 by Xue et al.13 includes a comparison of ammonium and phosphonium salt biocides. The recent literature papers can be summarized in relation to the relevance of key aspects to the aim of the present study: AFM and XPS studies of similar surface films not including anchored QAS

7-9, 20

; QAS type species on or in substrates such as co-polymers, polymers, resins or

membranes10, 12, 14-17, 21 ; AQAS surface studies but not deploying both AFM and XPS analysis10, 11, 14-17, 22

, and binary systems such as chitosan/silica or silver (which is itself a potent established

biocide)19, 21.


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The important challenge defined by van de Lagemaat et al.10 seeking new improved direct methods in evaluating and understanding anti-bacterial activity is relevant to this study, and for the field in general. Iarikov et al.18describe a system involving a different form of quaternary salt prepared in situ for which a nano-scale layer thickness is observed. Also, the XPS study by Poverenov et al.16 discusses N1s peak assignments for a related system. Paterakis et al.7 and the important early study by Schwartz et al.20 have identified island structures in AFM studies of conducting polymers and organo-silanes respectively on various surfaces. The originality in this study can be defined in terms of the literature analysis above and specifically with reference to the challenge posed by Lagemaat et al.10 It is the first in-depth AFM and angular dependence XPS study of the fundamental surface chemistry of the important Bacteria/Anchored QAS/Quartz system, including coverage, structure, ordering and disordering of the widely deployed type of anchored QAS biocide (Biosafe HM4100). The biocide has as its active component: 3 (trihyroxysilyl) propyl dimethyloctadecyl ammonium chloride (Fig.1) which forms a covalently bonded AQAS layer (Fig. 2). This is the layer that is the primary focus of this paper. EXPERIMENTAL Materials and Reagents The Anchored Quaternary Ammonium Salt used (AQAS: Biosafe HM4100) was obtained from Biosafe Inc. (Pittsburg PA) in the initial stages of the experiment and subsequently from Gelest Inc. U.S.A. Quartz microscope slides G381 were purchased from ProSciTech Pty Ltd (Australia). Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538) cultures were prepared in the Medical Microbiology Laboratory at the University of Auckland (Grafton


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Campus). Tryptic Soya Broth (TSB) was purchased from Fort Richard (Bacto). Tris hydroxymethylmethylamine SA buffer (Tris) was purchased from Romil Ltd. AFM cantilevers were purchased from Budget Sensors and Bruker International. Poly- L- lysine was purchased from Sigma Aldrich. Nucleic Acid stains (Propidium Iodide/Syto 9) were purchased as BacLight™ Bacterial Viability Kit from Thermo Fisher Scientific. The AQAS product, Biosafe HM4100 is approved by the EPA (Reg. No. 83019-1) and FDA and is listed as modifier of plastic substrates. It is an established antimicrobial product which finds application both as a surface anchored system as well as in bulk systems such as polymer resins. The AQAS product is thermally stable up to 250◦C in inert conditions4 and up to 200°C under air/ambient conditions. The potencies of a stable layer of Biosafe 4100 against various organisms have been specified by an ISO 17025 accredited medical-testing laboratory and these potencies have been reported by the manufacturers23 (Gelest/Biosafe) and in published literature24. These include rapid potency (99.999% killing within 5 mins) against Staphylococcus aureus. Potency against Escherichia coli has been reported separately in the scientific literature24, 25. Chemisorption of AQAS onto quartz substrates The preparation of a stable chemisorbed AQAS layer follows the protocols given by the manufacturers (Gelest/Biosafe). The mechanism of film formation is expected to involve two steps: the silanol terminating groups of the AQAS (Biosafe HM4100) initially hydrogen bonds with the terminal silanol groups of the quartz surface, which after curing at 160°C leads to a stable persistent covalently bonded AQAS layer (Fig.2).


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Pre-Cleaning of the Quartz Slide Surface: The quartz slides were washed in a 1 % (w/w) nonionic surfactant Triton X 100 solution, and were rinsed several times in de-ionized water under sonication for 15 minutes, each at 45°C. This was allowed to dry in air, followed by standing in 35% hydrogen peroxide under ambient conditions to remove any pre-existent carbonaceous surface impurities on the slides. The slides were further rinsed extensively in de-ionized water and air-dried for deposition of AQAS onto the surface. The same pre-cleaning process was carried out for preparation of the control bacterial surface. The duration between pre cleaning step and AQAS deposition was always minimized (99%. The error margins are the standard error of the percentage of coverage. It is important to note that the monolayer exists within island films (extended layers of AQAS clusters separated by zones of clear quartz surface voids) and not as isolated dispersed units of AQAS. This is consistent with AQAS-AQAS mat-like hydrogen-bonding across the


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quartz surface in addition to the primary covalent anchoring between the quartz surface and the AQAS mat. While the samples at higher concentrations (>0.01%) generated a very stable mono-molecular layer (Fig.7) and were the primary subjects of our study, the samples with the lowest concentration, 0.001%, revealed an unstable surface coating which was largely and easily removed by washing with water, and this type of sample was therefore not explored in detail. This leads to the conclusion that the lowest practical threshold dipping concentration for a stable island film formation on the surface is about 0.01% (w/w). Yarovsky et al.40 previously studied conformational chain lengths of the C18 alkyl chain of adsorbates bonded on silica surfaces. A C18 long alkyl chain in an all-trans conformation is approximately 2.2 nm in length and a folded conformation gives a length of 1.6 nm. Assuming the perpendicular height above the quartz surface of the siloxane and quaternary nitrogen centre (estimated from the bond lengths and angles) to be approximately 1 nm41, 42, we anticipate a total height of approximately 2.6-3.2 nm for the AQAS monolayer. This height is comparable to the maximum height (2.6 ± 0.1 nm) of the AQAS monolayer measured by AFM. The difference is probably attributable to other secondary disordering or folding of the C18 alkyl chain as suggested by the XPS N1s angular dependence data showing evidence for significant AQAS disordering effects (see XPS data later). Prior studies by Wasserman et al.43 has reported the thickness of complete monolayer coverage of alkylsiloxanes including a C18 chain length to be 2.6nm as measured by ellipsometry and low angle X-ray reflectivity.


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X-Ray Photoelectron Spectroscopy (XPS): AQAS Layers XPS spectra (sampling depth ~ 10nm) is a preferred surface analytical method for molecular surface layers. XPS N1s angular dependent spectra of the AQAS surface on quartz reveal two distinct peaks (399eV and 402eV) in the N1s region (Fig.8, 9). The 402eV peak exhibits distinct angular dependence indicating an orientation ordered species (Fig. 10). An additional sub-band appears at 404eV at relatively lower surface coverage. The comparison of XPS data in the present study between AQAS/quartz surfaces formed from dipping solutions at pH 4.02 and 1.17 indicates that the ratio of the two nitrogen peak components is relatively insensitive to this pH change (Fig.11). The assignment of these peaks to AQAS-related species

44, 45

is consistent with XPS data

recorded for bulk solid Biosafe HM4100, which has its singular N1s peak at 401eV (Fig.8). The 402eV peak, given its clear angular dependence behavior, is therefore assigned to a highly ordered surface species and the obvious prospect is the AQAS species in the first covalently anchored surface layer, where the anchoring process orients the first layer AQAS (2.6 ± 0.1 nm in thickness from the earlier AFM images) perpendicular to the quartz surface. The assignment of the second peak at 399eV, which does not exhibit comparable angular dependence, is a little more complex. The peak at 399eV would appear, on the basis of the angular dependence insensitivity, to arise from a disordered surface species free of the anchoring influence of the quartz surface. Poverenov et al.16 have assigned a similar peak observed for a range of polymer and glass substrates to a neutral N species, or in other words, an amine45-47 rather than a QAS N+. A small change of the relative ratio of the two nitrogen peak components from 1.8 % to 2.1% is observed with adjustment of pH from 4.02 to 1.17, which argues against an assignment of 399eV to a neutral surface amine. A neutral amine would be expected to be protonated forming a


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quaternary ammonium species at very low pH and this would lead to the expectation that the 399eV peak would decrease significantly in relative intensity, but which is not observed. It has also been established by Newkome et al.48 that the de-alkylation of quaternary ammonium salts is not likely to happen under mild conditions such as those in our adsorption processes. We are left with the conclusion that the 399eV species is very likely to be associated with multi-layering of AQAS on the surface which is not anchored to the quartz and is present in a disordered form. The disordered nature of this species also appears to rule out the prospect of ordered liquidcrystal type structures in the higher AQAS layers. The options for this disordered species are limited in this type of basic system and the key question is: are these disordered AQAS in the first or higher layers and if in the first layer why are these disordered species not surfaceanchored and therefore oriented? In the absence of definitive further evidence we conclude that this 399eV species is mainly resident in higher (second, third, etc.) layers. The relative ratio of the peak intensity of 399eV to that of the 402eV decreases with decreasing surface coverage (see Fig. 9) and with an associated decrease in the proportion of the higher AQAS layers. However we do not rule out a contribution from C18 chains on the periphery of anchored first-layer AQAS island films, where the film-edge stacking forces would be asymmetric so that these peripheral AQAS units exhibit either outward or random flexing49 of the C18 chains. Such flexing of the C18 chains would involve the introduction of gauche conformational groupings (e.g. TGT, GTG and others) into peripheral zones of AQAS C18 chains, while mainly all trans (TTT) conformational groupings are expected to occur in ordered tight packed chains in the core of the AQAS island films40, 50. The conformational changes of the C18 alkyl chains may cause them either to collapse around the N+ centre leading to a shielding effect49, or to flex outward, thereby further exposing


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the N+. In either case, such changes would be expected to have a significant effect on the binding energies51. The appearance of the weak 404eV sub-band (see Fig.9) observed at lower coverage is tentatively attributed to peripheral AQAS units on the edges of the island films. It is assumed that packing densities within the inner cores of the AQAS island films would not vary much as the coverage changes, because the existence of island films (mat like structures) implies AQASAQAS bonding across the surface. One final piece of evidence provides an important insight into the nature of the higher-layer AQAS molecules: washing the multilayered surface with water does not lead to the easy removal of these higher layers. This suggests that AQAS-AQAS bonding of the type reported for the first anchored layer (above) also occurs for the higher non-anchored layers. In these higher layers the AQAS-AQAS bonding via siloxane groupings is likely to be mixed oligomeric and clustered in nature which would explain the low water leachability (above) and the disordered nature of this speciation as revealed in the angular dependence XPS data. The differences in peak positions (402eV to 399eV including the 404eV sub-band) can be attributed to charge and conformation changes within the AQAS layers. Changes in the conformation of the C18 alkyl chain of the AQAS49 resulting from adsorption may also lead to changes in shielding of the N+ charge, and hence cause a small shift of a couple of eV in the N1s signal51. The possibility also exists that the greater chemical shift for the 404eV sub-band at lower surface coverage (above) is due to C18 chain disordering within the island film peripheral zone. The proposed conclusions regarding the assignments of the XPS peaks observed for the AQAS on quartz system (Fig.9) are summarised in Table 2.


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The total atomic % N for all of our AQAS sample surfaces arising from quaternary nitrogen species is 0.96% with a standard error of ± 0.1.This value is well above the threshold atomic percentage of quaternary nitrogen (N1s) required for bacterial contact killing (0.45%) as proposed by Roest et al. 52 Bacteria on AQAS Surface (AFM) AQAS are well known for their broad biocidal activity, as revealed by the manufacturers specifications

based

on

ISO17025

laboratory

potencies23.

These

potencies

include

Staphylococcus aureus and several others. Potency against Escherichia coli has been reported separately by D’Antonio et al.25 Gram positive and Gram negative bacteria have been employed in this study to confirm that the observed cell disruption had some more general significance. Also, the absence of an outer cell membrane which exists in Gram negative bacteria, but not in Gram positive bacteria, provided a confirmation that these two somewhat different types of cellular target surfaces exhibited similar potency outcomes. The mechanism most frequently invoked is a form of lysis which involves as its first step, the positive quaternary nitrogen center on the AQAS attracting the negatively charged bacterium; and in the subsequent step, the C18 chain penetrates the outer bacterial membrane, causing leakage of the intercellular contents. However, when higher layers of AQAS can be removed by washing then diffusion-dependent potency mechanisms such as ion exchange with cations in the cell membranes may become possible. The AQAS surfaces formed from a 0.01% concentration of the biocide have been adopted as our archetypal case history for AQAS activity using the AFM. The bacterial topography on the biocidal surface was observed in comparison to bacteria on a control (clean quartz) surface to which it was immobilized using the agent poly-L-lysine. The electronegative forces of attraction from the positively charged poly-L-lysine and the negatively charged


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bacterial cell wall causes the bacteria to adhere firmly to the substrate and aids in imaging the bacteria28. It is important to note that AFM is effective at monitoring changes in the top surfaces of the bacterial cells but is unable to observe changes on the interfacial interface with the AQAS layer where the lysis process actually occurs. It should also be noted that the bacterial images reported here are from dry surfaces (see experimental) as attempts to record images of bacteria on wet AQAS surfaces could not be readily obtained. It is also important to note that the industrial applications of AQAS surfaces are generally under dry conditions53. E. coli on AQAS surfaces The AFM studies on E. coli 25922 on the control surface and on the AQAS surface revealed the removal of bacterial pili indicating the disruption of outer membrane and further damage resulting in total cell rupture (Fig.12). The piliar destruction could be indicative of the onset of disruption of outer membrane and further damages resulting in cell lysis. Studies by da Silva et al. have reported the removal of piliar structures on E. coli by an antimicrobial peptide, PGLa54, 55

. An E. coli surface under normal conditions have been previously reported to have wrinkled

morphologies by Umeda et al.56 due to the shrinkage of the outer membrane during the drying procedure. This is also evident in the current study as seen in Fig.12a. This shape has been observed several times, it has typical E. coli dimensions compared to literature images54, 56, 57, and appears only after E. coli was introduced onto the AQAS/Quartz surface. It can be seen from the subsequent images (Fig.12 b & c ) that these wrinkles start disappearing when the bacteria rests on the biocidal surface giving out a smooth morphology owing to the turgor pressure build up inside the cellular environment. Such smoothing of cell surfaces has been reported in previous


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studies and the turgid states of bacterial cells are indicative of a breakdown of the osmoregulatory capacity of the bacterial cells58. This would further proceed to a complete leakage of intracellular periplasmic contents and final death of the bacteria. The AQAS layer was observed to be intact after the bacterial debris was washed off from the surface indicating the stability and reusability of the monomolecular layer. There was no background debris on either of the two bacteria / poly-l-lysine surfaces. The only surfaces where debris appeared were bacteria on the AQAS/Quartz surfaces. While following the degradation of E. coli on the biocidal surfaces, a small proportion of the bacterial species on the surface were consistently observed to show a changed shape whereby the rod shaped morphologies seemed to expand in a balloon-like fashion at the longitudinal axis of the single bacterium. This led to a lemon-shaped structure which resembled a bloated membrane, and appeared ready to rupture (Fig.13). A study by Huang et al.59 has reported similar change in cell wall morphologies due to defects in the cell wall segment synthesis. Increased substitution of glycan–peptide–glycan segments in place of glycans near the mid-plane of the cell was observed to create a swelled cylinder, similar to the lemon shape of cylindrical Gram-negative bacteria after treatment with the chemical A22, an inhibitor of the cytoskeletal protein MreB. The current study also provides evidence of rupturing of the membrane at the periphery of the cells, from where the cellular leakage originates. It is also important to note at this point that a 100% complete coverage is not necessary for biocidal activity as evident from the discontinuous AQAS film layer observed in the foreground of Fig.13b. This discontinuous film structure of the biocidal layer would not hinder the proposed bacterial lysis provided the dimensions of bacteria in the 1-2 µm range such as E. coli are larger than the surface voids of smaller dimensions (< 0.3 µm) that permit bridging of film zones. This


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observation reinstates the fact that a minimum required dose for effective antibacterial potency can be achieved by an anchored surface presented biocide. Henceforth it will greatly reduce the ecological toxicity currently encountered by superfluous use of biocides in the environment. S. aureus on AQAS surfaces AFM studies on S. aureus cells also were carried out after the former were adsorbed on AQAS biocidal surfaces (Fig.14. b and c). An examination of the interface between adjacent S. aureus bacterial cells as shown in (Fig.14.a and b) shows clear difference between the control and biocidal surfaces. The control gives clear sharp intercellular interfaces with no known protrusions, while the bacteria deposited on the biocidal surface always show formation of vesicles at the periphery of cells (Fig.14. b and c) prior to a complete lysis followed by eventual periplasmic leakage approximately after 1 hour. The appearance of vesicles were observed 100% of the time on the S. aureus bacteria deposited on AQAS surfaces. Similar outer membrane vesicles appear on Gram-negative bacteria, E. coli, upon attack by silver ions, has been reported previously as a novel envelope stress response57. Increased vesicles were indicative of a weakened outer membrane, thus exposing the thin peptidoglycan layer to mutilation and subsequent damage of inside cytoplasmic membrane or intracellular metabolic activity. It is of interest to note here, the mechanism of anti-bacterial potency for non-anchored QAS and anchored AQAS systems follow different courses. The former usually involves the cation exchange5, 60 of the positively charged soluble QAS with alkaline earth cations within the cell membrane. This aqueous cation exchange mechanism is not possible for an anchored QAS, as shown by prior studies61,

62

that report the non-antibacterial nature of hydrogen bonded

polyallylamine system lacking free poly cationic species. Therefore a form of lysis involving cell penetration by the C18 chain is invoked in the present study. This might be inducing progressive


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damage to the cell membrane which is manifested in the form of vesicular structures observed on the edges of the cells. The importance of the chain length of the hydrophobic alkyl tail, in imparting antimicrobial efficacy2, 11, 15, 17 supports this inference. Confocal Microscopy Study of E. coli on AQAS surfaces The purpose of using confocal microscopy was to confirm the potency of the AQAS surface under wet conditions, and to observe the dynamics between the solution bacteria and the biocide surface. A schematic depiction of the experimental design is shown in Fig.15. The result gives a clear depiction of the spread of live (green) and dead (red) bacteria on the sample surface and further away from the sample (Fig.15) thus confirming the potency of the AQAS surface. The dead bacteria (red) are more dominant close to the surface while further away from the surface, the live bacteria (green) are dominant. The gradient of red to green regions evident in the image reflects the net solution dynamics of live bacteria moving towards and dead bacteria moving away from the biocidal surface. The mechanism of potency is presumed to be lysis (cell wall perforation by the C18 chain of the AQAS) but has not been able to be elucidated directly by the imaging techniques (AFM or Confocal Microscopy) deployed in this research. It nevertheless remains an objective for future work using an alternative imaging technique. A key outcome of this confocal microscopy work is the confirmation of the potency of the AQAS (Biosafe) surface indicated by the specified potencies of Biosafe HM4100 conveyed by the manufacturers23.


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SUMMARY & CONCLUSION The recent challenge by Lagermaat et al.4 that new approaches need to be developed to understand the modes of action of anti-bacterial systems has significantly inspired this work. This type of system, viz., bacteria /AQAS is of increasing importance in global industrial and healthcare hygiene applications in comparison with conventional biocide coatings and blends, because of its “green” advantages which involve, primarily, extremely low dosage levels associated with mono-layers or even multi-layers. A detailed and holistic picture of the fundamental surface chemistry of the system {bacteria/AQAS/quartz} has emerged from this interfacial study. The AQAS units in the dipping solution appear to interact to form hydrogenbonded clusters which are expected to be the precursors to the initial physically adsorbed AQAS attached by hydrogen bonding to the surface silanols of the quartz substrate. The curing process at 160°C then converts the silanol-silanol hydrogen-bonded interaction into a covalent, stable, persistent biocide first layer via a condensation reaction. This covalent layer exists on the quartz surface within micro-zones, assumed to be influenced by quartz surface hydrophilicity, which include island film structures. The disordered higher AQAS layers are not easily removed by aqueous washing and so are expected to involve AQAS-AQAS clustering (oligomers) via their silanol groups. This also explains aspects of the XPS data. Some disordering from asymmetric packing forces inducing conformational disorder in the C18 chain of the AQAS units may even occur within the first layer in the peripheral zones of the island films. The voids (gaps) within the island film structures are smaller than the dimensions of most bacteria and so should not limit the potency of the biocide layer. The adsorption of bacteria (most likely involving C18 penetration lysis) on to this AQAS surface immobilizes the organisms and makes AFM imaging possible. The AFM images of both bacteria studied in these bacteria/AQAS systems reveal clear evidence


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of cellular perturbation or degradation. The challenge remains now to deploy new or alternative direct methods (beyond AFM) that can explore the bacteria/AQAS interface and especially the detailed fundamental mechanism of lysis potency. While lysis appears the most probable mechanism of bacterial potency for a covalently anchored biocide incorporating a C18 chain, we do not rule out the possibility that diffusion of AQAS arising from dissolution of weakly-held higher layers may involve some exchange with surface alkali earth cations in the bacterial cells. FIGURES

Figure 1. Biosafe HM4100 AQAS chemical structure


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Figure 2. Hydrogen bonding, condensation and siloxy bridging of AQAS molecules on the –OH rich quartz surface

Figure 3. FTIR spectra of bulk solid AQAS and centrifuged material from AQAS material in solution


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Figure 4. AFM image of a clean quartz surface (tapping mode)


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Figure 5. AFM images of AQAS Surface monolayers: Surface-coverage (90±1.4)) at different Scales AQAS solution concentration 0.1% (w/w)

Figure 6. AFM images of AQAS surface layers from different solution concentrations - Coverage variation with AQAS solution concentration. The layer heights for the blue section lines in the AFM images (top) are given in the surface profiles (bottom).


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Figure 7. AFM images of surface deposited from 0.01% (w/w) solution concentration: a) surface before rinsing with water b) surface after rinsing with water

Biosafe HM4100 (bulk) N (1s)

Biosafe HM4100 coated on quartz (AQAS) N (1s)

Figure 8. XPS N (1s) spectra of Biosafe HM4100 AQAS in bulk form and as deposited on quartz surface


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a

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b

c

Figure 9. N (1s) XPS spectra of Biosafe HM4100 AQAS deposited on quartz surfaces from the following dipping concentrations (w/w): a) 0.1% b) 0.05% c) 0.01%; Respective surface coverage %: a) 90 ± 1.4; b) 68 ± 5; c) 57 ± 1.9

a

Figure 10. Angular Variance of N (1s) spectra of AQAS surfaces deposited from 0.1% dipping solution. Black curve represents a normal angle of emission (90° take off angle) and blue curve represents an angular emission (60° take off angle)


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Figure 11. N (1s) XPS N (1s) spectra of pH varied AQAS on quartz with:a) AQAS solution at pH 4.02 b) AQAS solution at pH 1.17

a

b

c

Figure 12. AFM Amplitude Images of E. coli on a) control-Poly-L-Lysine surface; b) degradation of E. coli on AQAS surface; and c) E.coli cells shrinking in dimension and cellular detritus of cells on AQAS surface.


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Figure 13. (a) Altered shape of E. coli bacteria prior to degradation. (b) Note: Scale of surface voids on the AQAS layer indicating that cell dimensions exceed the scale of the voids

Figure 14. S. aureus cells on : a) control Poly-L-Lysine surfaces with uniform perimeters ; b) AQAS surfaces revealing vesicle-like formations on the periphery of cells; and c) AQAS surfaces revealing cellular degradation.


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Figure 15. a) Schematic of the confocal microscopy experimental configuration and b) live (green)-dead (red) fluorescence from the bacterial population adjacent to the AQAS surface.


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TABLES. Concentration Of AQAS solution

Surface Coverage %

Avg. Surface Layer Height nm

(w/w) %

(AFM)

(AFM)

0.1

90 ± 1.4

2.6 ± 0.1

0.01

57 ± 1.9

2.8 ± 0.2

0.001

25 ± 2.6

2.0 ± 0.1

Table 1: Surface Coverage of AQAS layers with various concentrations of AQAS (Biosafe HM4100)

XPS Angular Dependence

AQAS Concentration

Surface Coverage (%)

(w/w) %

from AFM data

Disordered (399 eV)

0.10

90 ± 1.4

More higher-layers, less

0.05

68 ± 5

edge units

Ordered (402 eV)

Surface anchored monolayer More edge units, 0.01

57 ± 1.9 less higher-layers

Table 2: Proposed Assignments of XPS Peaks to AQAS layer types


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AUTHOR INFORMATION Corresponding Author *Ralph Paul Cooney FRSNZ, DSc, PhD, Bsc (Hons), FRACI, FNZIC Professor of Chemistry Senior Advisor to The Biocide Toolbox (also founding Director) The School of Chemical Sciences The University of Auckland Newmarket Campus, Auckland 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 Funding Sources New Zealand Ministry of Business, Innovation and Employment (MBIE), The Biocide Toolbox (Contract UOA1410)


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ACKNOWLEDGMENTS The authors are grateful to the New Zealand Ministry of Business, Innovation and Employment (MBIE) for funding for the multi-year programme “The Biocide Toolbox” (Contract UOA1410). Rachel Mathew is grateful to The Biocide Toolbox for a PhD scholarship to undertake this and related research. The authors acknowledge the valuable scientific support and advice received from Professor Michael Higgins (University of Wollongong, Australia), Professor Bryony James (Faculty of Engineering, University of Auckland, NZ), Dr Sudip Ray (MBIE, The Biocide Toolbox, University of Auckland), Jacqui Ross (BIRU, University of Auckland, NZ) and Associate Professor Simon Swift (Medical Microbiology, University of Auckland, NZ) and to other members of the Biocide Toolbox Team ABBREVIATIONS AQAS: Anchored Quaternary Ammonium Salts AFM: Atomic Force Microscopy XPS: X-Ray Photo Electron Spectroscopy FTIR: Fourier Transform Infrared Spectroscopy E. coli: Escherichia coli S. aureus: Staphylococcus aureus


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TABLE OF CONTENTS GRAPHIC


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Atomic Force Microscopy and Angular-Dependent X-ray Photoelectron

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