Direct Force Measurement Between Bio-Colloidal Giardia lamblia

Force-separation measurements between Giardia lamblia cysts and an inorganic oxide (silicate glass) have been obtained by using an atomic force micros...
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Direct Force Measurement Between Bio-Colloidal Giardia lamblia Cysts and Colloidal Silicate Glass Particles Anne-Mari J. Virtanen,† Robert F. Considine,‡ David R. Dixon,§ Celesta Fong,∥ and Calum J. Drummond*,∥ †

Orion Corporation, Orion Pharma, Turku, 20101 Finland Melbourne Water Corporation, Docklands, VIC 3008, Australia § Department of Chemical and Biomolecular Engineering, University of Melbourne, Parkville, VIC 3010, Australia ∥ CSIRO Materials Science and Engineering, Private Bag 10, Clayton South, VIC 3169, Australia ‡

ABSTRACT: Force-separation measurements between Giardia lamblia cysts and an inorganic oxide (silicate glass) have been obtained by using an atomic force microscope (AFM). The cysts are compressible on the scale of the loads applied during force measurement, with the surface compressibility expressed in terms of an interfacial spring constant (Kint). The force of interaction prior to this Hookean region, on approach, is long-range and repulsive. The long-range force has been compared to models of the electrical double layer as well as an electrosteric layer. The comparison has led to the conclusion that the cyst surface can be described as a polyelectrolyte brush at intermediate separations (5−115 nm from linear compliance) with an electrical double layer often observed at larger separations. The dependence of the interaction force on surface retraction suggests that tethering between the cyst and siliceous surface can occur. The variation of the interaction with pH and upon variation with ionic strength has also been assessed. The information gained from the measurement of the interaction between G. lamblia and this model sandlike surface informs water treatment processes. Similar studies have been performed by us for the Cryptosporidium parvum (C. parvum) oocyst system to which this work is compared.

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INTRODUCTION Interest in the contamination of drinking water and the pollution of recreational water by protozoan such as Giardia is a major concern to water suppliers and regulatory agencies.1−3 Giardiasis has been implicated in waterborne outbreaks of gastroenteric disease worldwide,1,4,5,3,6,7,8 with Giardia lamblia (G. lamblia) responsible for ca. 35% of waterborne parasitic outbreaks that occurred between 2004 and 2010.3 Giardiasis is transmitted through the fecal-oral route via the infectious cyst stage which is environmentally robust, and can survive outside its mammalian host in surface waters for many months. The ubiquity of Giardia in water sources and the resulting seriousness of these outbreaks necessitate effective treatment and surveillance to safeguard clean drinking water. The former relies on a multistep approach that includes a regimen of sedimentation, flocculation, coagulation, filtration, and disinfection.9,10 The application of multiple barriers/treatment processes is advocated by the World Health Organisation’s Guidelines for Drinking Water Quality.8 The resistance of G. lamblia cysts to environmental stresses and various disinfection methodologies (e.g., UV exposure and ozonation) means that granular porous media such as sand-bed filters have been employed as physical barriers to help prevent contamination. Hence the physical removal of protozoa in the water treatment © 2012 American Chemical Society

process involves direct contact with the granular medium (filter). It follows then that the cyst surface properties play a critical role in controlling these interactions.10−14 The principles of colloid and surface science can provide insight into these interactions at the nanoscale, since these are a function of the surface chemical charge, functionality, and hydrophobicity of the cyst surface.15,16 Hence, there has been a growing research effort directed toward elucidating the factors that influence cyst−sand interactions.10−16 In particular, a previous study by this paper’s authors revealed that the force of interaction between cysts of G. lamblia and a model sandlike surface (AFM tip) is larger than considerations of electrical double-layer theory alone.17 This was rationalized in terms of the biomolecular composition of the cyst surface. This is made up of an inner, double membranous layer surrounded by a protective filamentous outer coating (ca. 0.25−0.30 μm thick) that is composed of carbohydrates and cysteine rich proteins.18,19,4,6,2021 The filaments are closely associated with certain proteins (cyst wall proteins, CWPs) that are leucine rich. This layer is Received: August 16, 2012 Revised: November 9, 2012 Published: November 28, 2012 17026

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measurements, from which the times were averaged. Mobilities (μ) were converted to the electrophoretic (ζ) potential by the Helmholtz−Smolouchowski equation.27 Approximately 1000 μL of the cyst dispersion (2.5 × 105 particles/ ml of PBS) was transferred to a 25 mL flask and diluted with the appropriate solution. Measurements were obtained as a function of pH in 0.2 and 1.0 mM KNO3 solutions. The pH adjustment was conducted by dropwise addition of either potassium hydroxide solution (pH 12) or nitric acid solution (pH 2). Atomic Force Microscopy. A Nanoscope III (Digital Instruments, CA), equipped with an EV (10 × 10 × 2.5 μm) piezoscanner, was used to measure topographical features and surface forces. The electric double layer (EDL) imaging mode28,29 was used to obtain images of G. lamblia cysts.14 All imaging was performed in 1 mM KNO3 at pH ca.8 to optimize the strong electrostatic repulsion between the AFM tip and the underlying surface. Commercially available silicon nitride oxide sharpened tips (Nanoprobes, Digital instruments, CA) with quoted spring constants of 0.58 N m‑1 were used for imaging in EDL mode. The glass bead−G. lamblia cyst force measurements were acquired in force-volume mode. The spring constant for force measurements was determined by measuring a loaded and unloaded frequency, and was found to be 0.27 ± 0.04 N/m.30 The AFM measurements were made in aqueous solution using a fluid cell (cleaned by UV exposure, 30 min). The piezoelectric ceramic was calibrated by laser interferometry.31 The probe-tip assembly was cleaned using an air/ water plasma (Harrick, PDC-329: at medium power setting, 0.5 mm Hg) for 30 s. A two-dimensional matrix of force relative to surface separation was acquired, with each force curve analyzed for the interfacial spring constant (Kint), decay length, and repulsive force magnitudes. We have measured force curves (each of 1024 data points including both the surface approach and surface retraction curves) to a resolution of 256 data points (16 × 16 matrix of force curves) across the surface. Only coaxially aligned force curves12 are reported in this work. All AFM force measurements reported herein correspond to measurements of G. lamblia cyst with a glass sphere mounted on an AFM cantilever (weak spring). Repeat measurements with a different cyst and sphere exhibited a similar trend in behavior. Data analysis followed the method developed by Considine and coworkers for compressible surfaces.10 The electrostatic part of the force of interaction was analyzed in terms of the interaction between dissimilarly charged surfaces.32,33 Ducker et al.25 proposed that cantilever deflection could be calibrated by comparing the measured gradient of linear compliance to the measured gradient of a hard surface. The surface compressibility can then be determined in terms of an interfacial spring constant (Kint)

responsible for the survival of the cysts in water for long periods of time and for resistance to disinfectants. It was proposed that ionizable moieties on the cyst surface behave like a polyelectrolyte brush, for which the protrusion into the surrounding medium and surface charge density may be influenced through manipulation of the aqueous environment. Similar interactions are present in the behavior of many microorganisms, and G. lamblia is not unique in possessing surface macromolecules that strongly influence their interaction with other surfaces.10−14,22,23 Here, we report a study of the interaction of individual G. lamblia cysts and a sandlike surface (glass bead), building on the foundations of our earlier AFM tip work by using a geometrically well-defined spherical, colloidal particle.17 Specifically, atomic force microscopy (AFM) has been used to measure the force of interaction between a G. lamblia cyst and silica-like sphere. The surface topography and compressibility are assessed, and the role of surface deformation in the force of interaction is quantified in terms of an interfacial spring constant (Kint). The effect of solution pH and ionic strength is examined, with the steric contribution to the repulsive force modeled using a polyelectrolyte brush to represent tethered surface proteins. We further present a single protein model to rationalize the behavior of the cyst wall surface proteins and their response to variable solution conditions. Finally, we compare the nature of these interactions with those observed for the related Cryptosporidium parvum (C. parvum) oocyst− silica colloid system that we have studied previously.

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MATERIALS AND METHODS

Chemicals. Electrolyte solutions were prepared using Milli-Q water. Analytical grade potassium nitrate, nitric acid, and potassium hydroxide were obtained from BDH Chemicals Ltd. (England) and glutaric dialdehyde solution from Aldrich Chemical Co., Inc. All chemicals were used without further purification. Cysts of Giardia lamblia. Giardia lamblia cysts supplied as a dispersion of 1 × 106 cysts in 4 mL of PBS (phosphate buffered saline) with penicillin, streptomycin, and gentamicin, were obtained from Waterborne Inc. Clinical and Environmental Parasitology Products. The cysts had been purified from the faeces of experimentally infected gerbils by sucrose and Percoll density gradient centrifugation and were stored at 4−6 °C. The cysts were anchored onto a smooth silicon wafer substrate that had previously been treated by plasma deposition of 3-aminopropylene (power = 20 W, frequency = 200 kHz). The plasma treated wafer was immediately incubated in a 0.5% w/w glutaric dialdehyde solution for approximately 1 h, rinsed with Milli-Q water, and then incubated overnight under vacuum (25 mm Hg) with the cyst dispersion in PBS. The dried cysts were washed with Milli-Q water to remove any crystallized PBS.12 Inorganic Oxide (Silica) Particles. The inorganic oxide particles were obtained from Polysciences, Inc. The glass beads were manufactured from soda lime glass and have a reported density of 2.48 g cm−3 (cf. 2.32 g cm−3 for cristobalite SiO2) with radius 3−10 μm. Glass beads of radius ca. 2 μm were selected for attachment to the AFM cantilever for force measurements.24 The isoelectric point (IEP) and ζ-potential as a function of pH are consistent with earlier reports of SiO2 surfaces. These are composed predominantly of silanol functionalities and provide a suitable model of a sand surface. The silicate glass beads were mounted on AFM cantilevers according to the method of Ducker et al.25,26 Microelectrophoresis. Microelectrophoretic mobility data was obtained using a RANK Bros Mark II apparatus. The velocity of particles in an applied electric field E (100 V cm−1) was measured. At each pH, the particle velocity v was measured at both stationary planes and with the electric field reversed, involving in excess of 40

K int =

Ks

(

Ch C int

)

−1

(1)

where Kint represents the interfacial spring constant (N m‑1), Ch is the gradient of linear compliance for a hard surface, Cint is the gradient of linear compliance against the interface undergoing deformation, and Ks is the measured spring constant of the cantilever. The reference “zero”, the gradient of linear compliance of hard wall contact (glass sphere on oxidized silicon wafer), is 0.020 ± 0.001 V nm−1. This is defined as the onset of linear compliance and provides a consistent definition that permits comparison of force measurements made between surfaces of different compressibility. Comparison of glutaric dialdehyde−glass bead and cyst−glass bead force curves confirmed that the immobilization protocol did not contaminate the scanned region of the cyst. In our calculations we have used Ch values measured (glass bead on flat silicon wafer) before G. lamblia force curve acquisition, without changing the focus location of the laser beam on the back of the AFM cantilever. 17027

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RESULTS AND DISCUSSION Microelectrophoresis ζ-Potentials. Representative microelectrophoretic mobility for the G. lamblia cysts as a function of pH at 0.2 and 1.0 mM KNO3 solutions are presented in Figure 1. The electrophoretic mobility of the glass

Figure 2. AFM image of conjoined cysts with root-mean-square (RMS) roughness of a 600 × 600 nm2, xy plane fitted scan ca. 7(±4) nm, and peak to valley height ca. 21(±4) nm.

The topography of the glass beads has previously been measured by these authors.39 These are smooth compared to their radius of curvature and the cysts, with RMS ca. 1−2 nm for a 2 μm, xy plane fitted scan. The roughness is distributed as gentle undulations of a few hundred nanometers in the lateral dimension and ca.10−20 nm in the vertical dimension. Cyst Force Curves. The force separation curves between the G. lamblia cysts and siliceous probe are shown in Figure 3 for both approach and retraction of the AFM cantilever (with siliceous probe). These were obtained at varying ionic strength, namely 0.2 mM and 1.0 mM KNO3 aqueous solutions. The measured approach force curves show a repulsive force except at low pH and ionic strength, where the force of interaction suggests attractive forces are present. At long range, the surfaces are well separated and there is zero interaction force. As the two surfaces approach, an exponential repulsive surface force is observed, corresponding to a positive deflection of the cantilever loaded probe at intermediate separations. Finally at close range, the cantilever deflects linearly with piezo movement, when the tip is in contact with the sample (Hookean region). This last region is defined as constant compliance, and the gradient of linear compliance may be used to assess the deformation undergone by the surface during force measurement. In addition, the onset of this region is used as a definition of zero separation. The gradient in the Hookean region for a cyst/silicate glass probe force curve is less than for the corresponding glutaric dialdehyde/silicate glass probe force curve. The surface retraction force curves, on the other hand, often showed a series of pull-off spikes at separations 0−500 nm from constant compliance, with the likelihood of adhesion increasing with decreasing pH and ionic strength. Similar trends have been observed with earlier published tip−cyst systems.17 Role of Biomolecules in Cyst-Surface Interactions: Origins of the Repulsive Force. At intermediate separations, we have demonstrated that the force of interaction may be approximated by Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory.17 DLVO40,41 assumes that the electrical double-layer and van der Waals interactions are additive and govern the overall interaction between two surfaces. In the current work, the electrical double layer interactions have been calculated as a function of separation by employing a nonlinearized Poisson−

Figure 1. ζ-Potentials of Giardia lamblia cysts as a function of pH in 0.2 mM KNO3 (○) and 1.0 mM KNO3 (●) solutions determined from microelectrophoretic mobility measurements. The ζ-potentials of glass beads measured by Considine et al.14 at 0.2 mM (Δ) and 1.0 mM (▲) have also been shown. The error bars correspond to the standard deviation for the total of forty velocities measured at each ionic strength.

beads, measured previously by Considine et al.,14 have also been presented for comparison. The glass beads exhibit an IEP of pH < 3, and a plateau ζ-potential ca. −80 to −90 mV at pH > 6. The IEP of silica-like surface is unchanged with ionic strength. The cysts exhibit an isoelectric point (IEP) at pH = ∼4 and a ζ-potential of ca. −30 to −35 mV at pH = ∼8. This is comparable to values cited in the literature where ζ-potentials of ca. −40 mV to −17 mV (at pH = ∼7) have been reported.3416 These values are also consistent with the values of most particles in natural water sources which lie in the range ζ = −15 to −30 mV.34 For most inorganic oxides, increasing the ionic strength of the solution reduces the absolute value of the ζ-potential. However, it is apparent that increasing the ionic strength does not significantly alter the ζ-potential or IEP of the cysts, although there is an increase in the absolute value of the ζ-potential with increasing pH. The molecular origin of the surface charge of the cysts is attributable to ionizable surface carbohydrate functionalities, and cyst wall proteins (CWPs) rich in leucine, serine, threonine, and cysteine amino acids.18,35−3820 These functional groups are also believed to be responsible for a steric component to the repulsive force of interaction.17 Atomic Force Microscopy. Surface Topography. The topographical features of G. lamblia cysts were measured in aqueous electrolyte in EDL mode. Figure 2 shows an AFM image of conjoined cysts with root-mean-square (RMS) roughness of a 600 × 600 nm2, xy plane fitted scan ca. 7 ± 4 nm, and peak to valley height ca. 21 ± 4 nm. Note, however, that for cyst-glass bead force interaction measurements only individual cysts have been used. 17028

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proteinaceous “hairy-like” structure of the cyst wall wherein ionizable moieties on the cyst surface behave like a polyelectrolyte brush that is responsive to the immediate aqueous environment, elongating with increasing charge density or decreasing salt concentration.10,42 Similar behavior has been observed in the related Cryptosporidium parvum system10−14,39 as well as some other microorganisms.11,22,23 In the current work we endeavor to directly account for the steric contribution arising from macromolecules tethered to the cyst wall surface. Specifically, we have calculated the force of interaction between surfaces bearing a polyelectrolyte brush using the Pincus model.43,44 Pincus quantified the interaction forces in terms of counterion osmotic pressure as a function of separation distance H Π≈

2fNBkT d 2H

(3)

where f is the fraction of ionicity (or fraction of monomers carrying an ionic charge), NB is the number of monomers within the hydrophilic block, d is the grafted interchain distance, k is the Boltzmann constant, and T (298 K) is the temperature. Abraham et al.42 showed that, upon integration of this equation and using the Derjaguin approximation, the energy of interaction between two electrolyte brushes of uniform monomer concentration profile throughout the brush width (LB) can be given by 4πkTNBf ⎡ 2L B ⎤ F ln⎢ = ⎣ H ⎥⎦ R d2

(4)

It should be noted that the above equation corresponds to the interaction of two surfaces, both bearing an absorbed polymer. However, as the probe surface is well described by electrical double layer theory,14,45 the brush is assumed to be entirely located on the cyst surface. Hence, the parameter 2LB has been taken to be the brush width on the cyst surface. For the purpose of Pincus theory calculations, d (= 0.95 nm) has been arbitrarily fixed to permit a basis for the comparison of various factors that contribute to the steric force interaction. The value of d = 0.95 nm corresponds to the longest linear dimension of leucine calculated using average bond lengths, and has been assigned on the basis of the known composition of cyst wall proteins (CWPs) which is leucine rich.18−20,35−38 The remaining terms in the equation correspond to the fraction of ionicity, f, and the number of monomers within the hydrophilic block, NB. Both these dimensionless terms describe the chemical nature of the extended brush. As these are related we have combined these as the single variable NB f, where large values correspond to a brush that is highly charged and predominantly hydrophilic. The measured normalized force-separation curves for the interaction between a G. lamblia cyst and glass bead, together with the DLVO and Pincus model fits, at pH = ∼8 and pH = ∼4, are shown in Figures 4 and 5, respectively, in 0.2 mM KNO3 aqueous electrolyte. For the DLVO model, a Hamaker constant of 1 × 10−20 J was used and displacement of the origin of the plane of fit in the DLVO calculation (from the onset of linear compliance) by ca. 154 nm was needed at pH = ∼8. Under these conditions, the force of interaction at separations from ca. 10−115 nm may be described as the collapse of a polyelectrolyte brush (Pincus model), and at greater separation (ca. 170 < d < 250 nm) the electrical interactions are important. The implication is that the proteinaceous “hairs” on the cyst

Figure 3. Cyst−colloidal sphere force separation curves on surface approach (←) and on surface retraction (→) measured at pH = ∼8 and pH = ∼4 in (a) 0.2 mM KNO3 and (b) 1.0 mM KNO3.

Boltzmann equation. Here the van der Waals interactions are described by −AH F = (2) R 6H2 where AH is nonretarded Hamaker constant (∼1 × 10−20 J for cyst−silica interaction) and H is the separation between surfaces. At the long-range separations under consideration, the van der Waals component to the overall interaction is negligible. Our previous cyst−tip work has shown that DLVO approximations alone do not adequately describe the force of interaction.17 Specifically, the magnitude of the calculated electrical double layer force was less than the measured force, requiring a displacement of the origin of the plane of fit in the DLVO calculation (from the point of contact) by ca. 39 nm (“pseudo-DLVO fit”). The discrepancy between the measured and calculated electrical double layer forces is considered to arise from a steric contribution to the interaction force. The combination of these two has been referred to as an “electrosteric” interaction. This has been attributed to the 17029

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Table 1. Fitting Parameters for the DLVO and Pincus Models for the Interaction of G. lamblia and Glass Bead as a Function of pHa I (mM)

pH

0.2

8.20 6.60 5.68 4.93 4.72 4.04 8.42 6.26 5.49 5.39 4.78 4.08

1.0

Ho DLVO (nm) 151 135 153 160 166 170 103 117 147 124 130 126

± ± ± ± ± ± ± ± ± ± ± ±

10 10 9 10 4 10 19 23 17 20 9 27

2LB1 (nm) 75 70 75 10 25 8 48 48 53 50 51 19

± ± ± ± ± ± ± ± ± ± ± ±

3 1 3 2 3 1 4 4 2 4 2 2

N B f1 70 65 60 65 51 61 78 80 68 81 76 46

± ± ± ± ± ± ± ± ± ± ± ±

5 4 6 7 4 4 3 4 8 4 5 2

a Ho denotes the DLVO shift in origin, 2LB denotes the Pincus brush width, and NB f denotes a dimensionless term describing the fraction of ionicity and relative hydrophilicity.

Figure 4. Normalized force of interaction between a cyst and glass bead in 0.2 mM KNO3 pH 8.2. The thin solid line corresponds to the DLVO fit with approximated Hamaker constant 1 × 10−20 J, with plane of charge origin shifted 154 nm to obtain an order of magnitude fit. The thick solid line corresponds to the result predicted from a Pincus model of a polyelectrolyte brush.

different gradients (Figures 6 and 7). This phenomenon occurred most commonly near the IEP of the cyst surface and

Figure 5. Normalized force of interaction between a cyst and glass bead in 0.2 mM KNO3 pH 4. The thin solid line corresponds to the DLVO fit with approximated Hamaker constant 1 × 10−20 J, with plane of charge origin shifted 170 nm to obtain an order of magnitude fit. The thick solid line corresponds to the result predicted from a Pincus model of a polyelectrolyte brush.

Figure 6. Normalized force of interaction between a cyst and glass bead at pH = ∼4 at 0.2 mM KNO3 showing a “double” poleyelectrolyte brush model overlaid with DLVO fit. Here the DLVO plane of charge origin has been shifted 117 nm to obtain an order of magnitude fit, and the origin of “Pincus 1” is shifted 19 nm.

surface act as a significant repulsive barrier at intermediate separations. In contrast, at pH = ∼4 neither model provides an adequate fit since an attractive interaction is observed between the glass bead (IEP pH < 3) and the cyst surface (IEP pH = ∼4). The Pincus model parameters are listed in Table 1 at 0.2 mM and 1.0 mM as a function of pH. The brush width (2LB) and NB f decrease with ionic strength and pH, exhibiting a sharp decrease near the IEP of the cyst surface at ca. pH 4. This is consistent with the hypothesis that the polyelectrolyte brush acts like a stretchable coil, collapsing in response to the decharging of ionizable moieties near the IEP of the cyst surface. Interestingly, we have also observed occasions for which the force−separation curves show two regions with distinctly

upon increasing ionic strength of the medium. In these instances we have described the force separation curves using two polyelectrolyte brushes, for which the origin of the second brush (Pincus 2) has been shifted. In addition, a “pseudo” DLVO fit has been performed, although, at low ionic strength (0.2 mM KNO3), the DLVO model is not a good fit. The inability to obtain a good fit with the DLVO model is probably the combined result of significant surface roughness of the cyst as well as the extension of the cyst surface proteins into the electrical double layer. In general, the displacement of the origin of the plane of fit in the DLVO calculation (Ho) decreased with ionic strength (Table 1). This double polyelectrolyte brush model may be rationalized as two different protein layers on the cyst surface that have 17030

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Figure 8. Interfacial spring constant as a function of pH in aqueous 0.2 mM (blue triangle), 0.5 mM (pink X), 1.0 mM (black solid circle), and 5 mM (red tilted square) KNO3 solutions. Figure 7. Normalized force of interaction between a cyst and glass bead at pH = ∼4 at 1.0 mM KNO3 showing a “double” poleyelectrolyte brush model overlaid with DLVO fit. Here the DLVO plane of charge origin has been shifted 94 nm to obtain an order of magnitude fit and the origin of “Pincus 1” is shifted 19 nm.

different charge densities. This is envisaged as comprising an inner layer that is less highly charged and more hydrophobic, with an outer layer that consists of more charged, hydrophilic proteins. Repulsive Surface Force. Six force curves were selected from the center of the cyst surface within the region in which the AFM probe and cyst are coaxially aligned, and these have been analyzed as a function of pH at several ionic strengths. Given the complexity of the cyst topography, force profiles obtained from noncoaxially aligned areas of the sample relative to the AFM probe cannot be compared properly with theory due to curvature artifacts, associated with an additional torsional component. It is not possible to resolve the surface force contribution from the torsional component arising from edge artifacts.12 Each force curve was analyzed and characterized by a number of parameters, including interfacial spring constant (Figure 8), decay lengths (Figure 9), and repulsive force magnitude (Figure 10). Interfacial Spring Constant (Kint). The interfacial spring constant (Kint) may be used to quantify the compressibility of the cyst surface. Kint values have been presented in Figure 8 as a function of pH at various ionic strengths for data in the range 15−50 nm from zero separation. Overall, these are higher than values obtained using a hydrolyzed silicon nitride tip−cyst that we determined previously.17 However, it is noted that the effective geometry of a silicon nitride tip is not as well-defined as the system reported herein for which a spherical bead of defined geometry has been attached to the AFM tip. The observed trends for tip and bead nevertheless are largely similar. Specifically, Kint values in 0.2 mM KNO3 increase from 0.06 to 0.09 N m−1 with decreasing pH, compared to 0.09−0.12 N m−1 at higher electrolyte concentration. In the present study, there is a general trend for higher interfacial spring constants at lower electrolyte concentrations and more acidic pH, and this is similar to the observations of AFM tip−Giardia lamblia system previously reported.17 This is attributed to the roughness of the cyst surface that is known to possess asperities that may be

Figure 9. Exponential repulsive force decay length measured as a function of pH in aqueous 0.2 mM (blue triangle), 0.5 mM (pink X), 1.0 mM (green circle), and 5 mM (red tilted square) KNO3 solutions.

compressed to a varying extent depending on their geometry and individual compressibility. The discrete compression events were observed as discontinuities in linear compliance. This may also be attributed to the complexity of the proteinaceous layer/ s as discussed above. The collapse of the proteinaceous layer/s is not necessarily a coherent process. Similar observations have been made for the C. parvum−probe system14 and for the interaction between surfactant bilayers adsorbed on silica.28 Interestingly, in our earlier study using cyst−silica tip, only hard wall compliance was observed. Presumably the smaller radius of curvature of the tip resulted in sufficient pressure to penetrate the protein layer(s) on the cyst surface. Repulsive Force Decay Length. The repulsive force on surface approach has been characterized by a decay length, which is a log linear (natural log) slope of the force at separations between 20 and 50 nm from the onset of the linear compliance. Repulsive force decay lengths have been presented as a function of pH at various electrolyte concentrations in Figure 9. In general there is a systematic decrease in the decay lengths as a function of ionic strength. However, the decay 17031

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1 ∝ Cs−0.29 κexp

(5)

The measured exponent is significantly smaller than anticipated from DLVO theory which according to the nonlinearized Poisson−Boltzmann equation predicts dependence between the Debye length (1/κDebye) and the electrolyte concentration 43,44 according to 1/κDebye ∝ C−0.5 predicted s . However, Pincus the force between absorbed polyelectrolyte layers to be less sensitive to Debye screening, wherein the calculated dependence of the brush width on the electrolyte concentration is LB ∝ C−0.33 . The measured exponent in this study is therefore s closer to that predicted by Pincus. This provides additional evidence for the presence of a steric contribution to the repulsive force and is consistent with the idea that cyst surface proteins act like a polyelectrolyte brush. These extend into the surrounding medium because of charge repulsion between ionizable groups on the proteins, and as the ionic strength is increased, the electrostatic interactions are screened, thereby reducing the repulsive force. Similarly as the pH is lowered, the number of ionized groups decreases (for pH ∼ IEP) resulting in fewer inter/intramolecular interactions. Repulsive Force Magnitude. The magnitude of the repulsive force has been defined as the intercept of the log linear (natural log) slope. The intercept has been extrapolated from separations between 20 and 50 nm from the onset of the linear compliance. Repulsive force magnitudes have been presented as a function of pH in Figure 10. The average magnitude values vary from 8 to 14 mN m−1. At high ionic strength (5 mM KNO3 at pH = ∼8) the magnitude is 12.0 ± 0.7 mN m−1 compared to 10.3 ± 0.9 mN m−1 at pH∼4. At low ionic strength (0.2 mM KNO3, pH = ∼8) the magnitude is 11.4 ± 0.9 mN m−1 versus 9.6 ± 0.5 mN m−1at pH = ∼4. While there appears to be a slight dependence of the magnitude with pH and ionic strength, the trends are weak. Therefore, like Kint, it may be that surface roughness smears the effect. Nevertheless, the observed repulsive force magnitude is larger than predicted by DLVO considerations.14 Nature of the Adhesion on Surface Retraction. Despite a large repulsive surface force on approach, adhesion between the cyst surface and probe is regularly observed, particularly at low ionic strength (Figure 3). These are manifest as a series of “pull-off” spikes (25−500 nm from hard wall contact) that are attributed to possible adhesion/tethering of cyst wall biomolecules to the colloid probe. Adhesion between surfaces and in biological materials has been correlated with acid−base interactions, with the presence of proteins on cell membrane having been found to mediate adhesion and promote steric repulsive forces on surface approach.28,46−48 Similar features have been observed for cyst−tip and C. parvum−tip(probe) measurements reported previously.10,12,14,39 In order to compare the force of adhesion for cysts and ionic strength, the adhesion has been analyzed in terms of the maximum extension for attachment between two surfaces, Hjumpout, and the corresponding force of adhesion given by

Figure 10. Exponential repulsive force extrapolated magnitude as a function of pH in aqueous 0.2 mM (blue triangle), 0.5 mM (pink X), 1.0 mM (green circle), and 5 mM (red tilted square) KNO3 solutions.

length is only slightly influenced by pH, and this effect decreases with increasing electrolyte concentration. This is consistent with the view that the cyst surface is composed of ionizable functionalities and acid groups. These observations are in line with previous observations of the G. lamblia−tip interactions.17 For comparison with DLVO and Pincus theories, the power law dependence of the experimentally measured decay length (1/kexp) with increasing electrolyte concentration (Cs) can be obtained from the slope of a log−log plot of the decay length versus electrolyte concentration (Figure 11). With four electrolyte concentrations at high pH the quality of power law fit was high (r2 = 0.9944) and gave the following power law dependence:

−K sHjumpout ⎛F⎞ ⎜ ⎟ = ⎝ R ⎠adhesion R

(6)

where Ks corresponds to the measured cantilever spring constant and R the harmonic mean of the radii of two surfaces. The surface forces on separation have been analyzed using the gradient of linear compliance obtained against the reference

Figure 11. Power law dependence of the experimentally measured decay length (1/kexp) with increasing electrolyte concentration (Cs). The line of best fit is y = 32.3x−0.295 with R2 = 0.994. 17032

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The use of physical barriers for the entrapment/filtration of these pathogens is an important step in water sanitation. The effectiveness of filtration relies on the adhesion of the (oo)cysts to the filter matrix, and this is influenced by surface charge that is measured as ζ-potential. Both G. lamblia and C. parvum are electronegative at about neutral pH, with IEP ca. pH 3−4, similar to natural particle assemblages in surface waters.34 Interestingly, while the ζ-potentials of C. parvum become less negative with increasing ionic strength,14 the ζ-potentials G. lamblia are more or less invariant. However, for both, the absolute values of ζ-potentials are less negative with decreasing solution pH, suggesting that in some respects these behave as “bio”-colloids. These findings are consistent with observations by Hsu et al.16,50 that suggest that the filtration efficiency of C. parvum oocysts was consistently higher than G. lamblia cysts over a broad range of ionic strength and pH. These authors also note that the hydrophobicity of the filtration media was a more important determinant of removal rate (vs surface charge) for G. lamblia cysts than for C. parvum oocysts. These findings highlight the importance of evaluating waterborne pathogens on a case by case basis. The cysts are compressible on the scale of the loads applied during force measurement, with the surface compressibility expressed in terms of the interfacial spring constant (Kint). In general the interfacial spring constants of G. lamblia cysts are slightly lower than that for C. parvum oocysts at similar pH. Further, there appears to be an increase in Kint for G. lamblia cysts at low ionic strength which is attributed to the collapse of surface proteins. A similar phenomenon was not observed in the oocysts−glass bead system as the interactions were measured above the isoelectric point.14 Given the size, shape, and charge of these protozoa it would be anticipated that (oo)cyst−surface interactions would closely follow the DLVO theory of colloidal stability; however, DLVO can only partially account for this. A fundamental assumption of DLVO theory is that the geometry and topography of the interacting surfaces may be approximated in a straightforward manner. However, as we have observed for G. lamblia in this work, surface roughness and the compressibility of the surface will cause deviations from DLVO predictions. Specifically, C. parvum oocysts are rounded microspheres whereas G. lamblia cysts are typically ovoid, although deformations are common. Asperities have been observed on the surface of oocysts,10−12,14 although cysts have greater RMS roughness.17 In both cases, the force of interaction is long-range and repulsive with a Hookean region at closer separations. The long-range force has been compared to models of the electrical double layer as well as steric interaction. The surface can be described as a polyelectrolyte brush at intermediate separation viz. ∼10−30 nm14 and ∼10−70 nm (from linear compliance in 1 mM KNO3) for C. parvum oocysts and G. lamblia cysts, respectively. We have calculated the force of interaction between surfaces bearing a polyelectrolyte brush using the Pincus model for which NB f describes the chemical nature of the extended brush. NB f for the G. lamblia cyst surface is much higher than for the C. parvum oocyst. This is due in part to the different way in which the interchain distance (d) values were determined. In this study the linear dimension of amino acids has been calculated using the average bond lengths, whereas in previous work with C. parvum oocysts, this was derived using the van der Waals radius of the constituent atoms. Despite a large repulsive surface force on approach, adhesion between the (oo)cyst surface and glass bead is regularly

hard surface (silicon wafer). The value of zero separation obtained on surface approach (i.e., onset of linear compliance) has been used to reference the force−separation data on separation. The values of Hjumpout and (F/R)adhesion are presented in Figure 12 for three cysts at four ionic strengths

Figure 12. Normalized force of adhesion derived from corresponding jump-out distances as a function of pH at varying ionic strength: 0.2 mM (blue triangle), 0.5 mM (pink X), 1.0 mM (green circle), and 5 mM (red tilted square) KNO3 solutions.

and show that there is substantial variation between cysts. The difference between Hjumpout at various electrolyte concentrations is negligible, as the standard deviation is so high. Note that the standard deviation also increases with decreasing ionic strength and pH. However, it is qualitatively observed that adhesion is greater at low ionic concentration and low pH. This is rationalized in terms of the greater positive charge associated with cyst wall proteins under these conditions (see below), which therefore encourage adhesion to the negatively charged AFM probe. In practice, surface roughness can significantly change the contact area of the AFM probe and hence adhesion.49 Giardia lamblia Cyst−Glass Bead Force Interactions Compared to the Cryptosporidium parvum Oocysts−Silica Interactions. Considine et al. have studied the force of interaction between Cryptosporidium parvum (C. parvum) oocysts and silica surfaces.10,12−14,39 As with G. lamblia, C. parvum is a common pathogen in drinking water, and the two share many similarities with subtle differences with respect to their physicochemical characteristics and behavior. We now examine the forces of interaction of both G. lamblia and C. parvum side by side to compare and contrast their properties at the nanoscale. This will potentially inform differences in water treatment protocols for their removal. Many of these differences may in part, be accounted for by the composition of the (oo)cyst wall. The cyst wall of G. lamblia is composed of ionizable surface carbohydrate functionalities, mainly in the form of galactosamine homopolymers with strong interchain interactions, and cyst wall proteins (CWPs) rich in leucine, a nonpolar and nonionic amino acid (44%).36 In contrast, the wall protein of C. parvum contains more ionic amino acids (22%)10 that are glucose rich. 17033

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observed, particularly at low ionic strength, and is attributed to possible adhesion/tethering of cyst wall biomolecules to the glass bead. The force of adhesion appears to be greater for G. lamblia cysts, especially at low pH.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +61 3 9545 2050.

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Notes

The authors declare no competing financial interest.

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SUMMARY AND CONCLUSION The G. lamblia cyst wall plays a crucial role in forming a highly resistant barrier to physicochemical stresses. In the current study AFM has been used to directly measure the force of interaction between G. lamblia cysts and an amorphous siliceous surface (silica-like probe). For the G. lamblia/silicalike sphere system, the two main repulsive interactions are electrical and steric in origin. The electrical double layer arises from the presence of charged groups (e.g., ionized organic acids, amines) while the steric interaction originates from surface associated biomolecules (e.g., proteins and/or carbohydrates).49 The electrical double layer has been considered within DLVO theory. However, the DLVO approach alone has failed to attain quantitative agreement over the entire range of investigated conditions, and this is attributed to factors that include surface roughness, the compressibility of the surface, as well as a steric component arising from the interaction of tethered, long chain biomolecules. In the case of layers of end tethered high graft density macromolecules, these have been shown to adopt a “brush-like” conformation.51,52 As the brush is compressed, an additional non-DLVO repulsive force is observed that has been analyzed using the Pincus model43,44 in the current work. The comparison has led to the conclusion that the surface can be described as a polyelectrolyte brush at intermediate separations (5−115 nm from linear compliance) with an electrical double layer often observed at greater separations. This situation describes many biological systems11,22,23 including that of another waterborne pathogen, C. parvum.14 Despite a large repulsive surface force, adhesion between the cyst surface and glass bead is also observed, particularly at low ionic strength. “Pull-off” spikes in the retraction curves indicate adhesion events that are associated with tethering of cyst wall biomolecules to the AFM probe.1246−48,53 Similar features have been observed for cyst−tip and C. parvum−tip(probe) measurements reported previously.10,12,14,39 We have proposed a simple protein chain model of the cyst surface that qualitatively rationalizes many of our observations. This model supposes that the cyst wall protein chain of G. lamblia is negatively charged under the conditions of the water treatment plant (pIEP (4) < pH > pKa (−NH2)). Under these conditions the protein chain is elongated and electrosteric interactions are in play. However, when the pH is below the net isoelectric point of the cyst surface, carboxylic groups on acidic amino acid residues are protonated, and there is a slight electropositive charge overall, leading to the collapse of surface protein chain. A better fundamental understanding of the interaction between G. lamblia cysts and sandlike siliceous materials is required to inform water treatment processes that use sand bed filtration for their physical removal. In the current work we have demonstrated how the principles of colloid and surface science can provide a handle on how to manipulate the aqueous environment to provide general predictions for cyst removal. However, much remains to be done to investigate the impact of other components in the system (e.g., dissolved natural organic matter and coagulants).

ACKNOWLEDGMENTS A.-M.J.V. would like to thank CSIRO for providing the opportunity and funding as a visiting student. A.-M.J.V. would also like to acknowledge the IAESTE (International Association for the Exchange of Students for Technical Experience) program for their support. She would like to thank Drs. Considine and Drummond for their assistance and knowledge.

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REFERENCES

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