Laterally-Resolved Force Microscopy of Biological

CSIRO Molecular Science, Bag 10, Clayton South, Victoria 3169, Australia, and Cooperative. Research Centre for Water Quality and Treatment, Bag 3, Sal...
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Langmuir 2000, 16, 1323-1330

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Laterally-Resolved Force Microscopy of Biological MicrospheressOocysts of Cryptosporidium Parvum Robert F. Considine, David R. Dixon, and Calum J. Drummond* CSIRO Molecular Science, Bag 10, Clayton South, Victoria 3169, Australia, and Cooperative Research Centre for Water Quality and Treatment, Bag 3, Salisbury, South Australia 5108, Australia Received February 23, 1999. In Final Form: August 24, 1999 An atomic force microscope (AFM) has been used to measure the force of interaction between individual biological microspheres (oocysts of Cryptosporidium parvum) and an amorphous silica surface (hydrolyzed AFM tip). One of the main barriers to oocyst contamination of drinking waters is provided by sand-bed filtration. The AFM tip has been functionalized to silica in order to investigate the interaction between oocysts and model sand (siliceous) particles at the most fundamental level. The AFM force curves have been compared and contrasted with the ζ-potentials of silica particles and oocysts obtained from electrophoretic mobility measurements. It has been concluded that there is a steric interaction between the silica surface and the oocyst material, in addition to electrical double-layer and van der Waals interaction. The proteinaceous materials on the surface of the oocysts are considered to be responsible for the steric interaction. The magnitude of the steric interaction is little changed by varying the pH and electrolyte concentration (i.e. changing the charge characteristics of surfaces has little effect on the magnitude of the interaction). Force curves suggest that once the silica and oocyst surfaces contact one another, proteinlinked tethering can occur. Therefore, despite the large steric barrier, adhesion between oocysts and silica may occur. A measurement of the hardness of the Cryptosporidium oocyst surface has shown that the surface has silica-like hardness, which may explain why the oocysts are so resistant to disinfection. Finally, the effect on the surface force measurement of alignment between two curved surfaces has been reported. The findings have implications for all studies that force map surfaces that possess curvature.

Introduction Cryptosporidium is a coccidian parasite known to infect humans. First described early this century,1 the organism remained largely ignored for around 60 years. However, the status of cryptosporidiosis has changed over the past 20 years from that of a rare, largely asymptomatic infection to an important cause of enterocolitis and diarrhea in humans.2-6 Cryptosporidium is widespread in the environment and has a worldwide distribution.7 Oocysts have been found in rivers and streams, lakes and reservoirs, raw and treated sewage, and treated surface waters. Several waterborne outbreaks of cryptosporidiosis have been documented. Outbreaks have occurred in the United Kingdom8 as well as right across the United States,9,10 most notably of which was the outbreak in Milwaukee where up to 400 000 people were infected and 100 deaths of immunocompromised patients were attributed to the disease.11 Occasional outbreaks have occurred with no * Author to whom correspondence should be addressed at CSIRO. Phone: 61 3 9545 2618. Fax: 61 3 9545 2515. E-mail: C.Drummond@ molsci.csiro.au. (1) Tyzzer, E. E. Proc. Soc. Exp. Biol. Med. 1907, 5, 12-13. (2) DuPont, H. L.; Chappell, C. L.; Sterling, C. R.; Okhuysen, P. C.; Rose, J. B.; Jakubowski, W. N. Engl. J. Med. 1995, 332, 855-859. (3) Okhuysen, P. C.; Chappell, C. L.; Sterling, C. R.; Jakubowski, W.; DuPont, H. L. Infect. Immun. 1998, 66, 441-443. (4) O’Donoghue, P. J. Int. J. Parasitol. 1995, 25, 139-165. (5) Jelinek, T.; Lotze, M.; Eichenlaub, S.; Loscher, T.; Nothdurft, H. D. Gut 1997, 41, 801-804. (6) Frost, F. J.; Craun, G. F. Infect. Immun. 1998, 66, 4008-4009. (7) Packham, R. F. J. Inst. Water. Environ. Manage. 1990, 4, 578580. (8) The National Cryptosporidium Survey Group. J. Inst. Water. Environ. Manage. 1992, 6, 697-703. (9) Solo-Gabriele, H.; Neumeister, S. J. Am. Water Works Assoc. 1996, 88, 76-86. (10) Kramer, M. H.; Herwaldt, B. L.; Craun, G. F.; Calderon, R. L.; Juranek, D. D. Morbidity Mortality Weekly Rep. 1996, 45, 1-33.

noted changes in source water quality or treatment processes.12 Such freak episodes, combined with the potential devastation of large-scale cryptosporidiosis epidemics, have necessitated research on reliable processes to eliminate Cryptosporidium parvum oocysts from drinking water supplies. Transmission of cryptosporidiosis occurs via the fecaloral route and involves the ingestion of a quantity of oocysts.2,3 Oocysts are biological microspheres that are 3-7 µm in diameter and consist of a biologically active core (upon excystation) surrounded by a thick, environmentally stable coating. Contact with oocysts can occur from any fecally contaminated medium, including environmental surfaces and contaminated food or water supplies.13 Of importance to municipal drinking water suppliers, the oocysts are resistant to conventional disinfection processes,14,15 and this has focused attention on the need for physical removal of oocysts from water supplies by sand filtration.16,17 Here we report a study of the interaction of individual Cryptosporidium parvum oocysts and a sandlike surface. Specifically, we have employed an atomic force microscope (AFM) to directly measure the force of interaction between a C. parvum oocyst and an amorphous silica surface (hydrolyzed AFM tip). The heterogeneity of the oocyst is (11) Eisenber, J. N. S.; Seto, E. Y. W.; Colford, J. M.; Olivieri, A.; Spear, R. Epidemiology 1998, 9, 255-263. (12) Roefer, P. A.; Monscvitz, J. T.; Rexing, D. J. J. Am. Water Works Assoc. 1996, 88, 95-106. (13) Donnelly, J. K.; Stentifold, E. I. Lebensmittel-Wissenscharft Technol. 1997, 30, 111-120. (14) Campbell, I.; Tzipori, S.; Hutchison, G.; Angus, K. W. Vet. Rec. 1982, 111, 414-415. (15) Korich, D. G.; Mead, J. R.; Madore, M. S.; Sinclair, N. A.; Sterling, C. R. Appl. Environ. Microbiol. 1990, 56, 1423-1428. (16) Clancy, J.; Fricker, C. Water Qual. Int. 1998, July/August, 3741. (17) Fogel, D.; Isaac-Renton, J.; Guasparini, R.; Moorehead, W.; Ongerth, J. J. Am. Water Works Assoc. 1993, 85, 77-84.

10.1021/la990205p CCC: $19.00 © 2000 American Chemical Society Published on Web 01/13/2000

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assessed by both topographical and lateral-array force measurements. The effect of solution pH and ionic strength is assessed, and comparisons with electrophoretic mobility measurements and electrical double-layer theory are made. Experimental Procedure Materials. All electrolyte solutions were made up with water obtained from a Milli-Q system. The potassium nitrate was obtained from BDH Chemicals and was of analytical reagent grade. The nitric acid and potassium hydroxide used in pH adjustment were also obtained from BDH Chemicals. The potassium hydroxide was obtained as pellets, and both the nitric acid and potassium hydroxide were of analytical reagent grade and used without further purification. Oocysts of Cryptosporidium parvum. The oocysts of C. parvum were isolated from a bovine source by Macquarie Research Limited (Australia) and supplied as a dispersion of 1.006 × 107 oocysts per 0.5 mL of PBS (phosphate buffered saline). The oocysts were surface sterilized by exposure to 70% ice-cold ethanol for 20 min and stored at 4 °C. The oocysts (that from electrophoresis we know to be negatively charged) were deposited onto a positively charged, pH stable, surface. The surface was prepared by plasma deposition (power ) 20 W, frequency ) 200 kHz) of 3-amino-propylene (time ) 25 s, pressure ) 0.125 mmHg)18 onto a smooth silicon wafer (Silica Source Technology Corp). The prepared surface was then incubated with a 0.5% w/w solution of glutaric dialdehyde (Aldrich Chemical Co., Inc., obtained as 25% w/w glutaric dialdehyde in water) for approximately 1 h. Excess glutaric dialdehyde was removed by rinsing with copious amounts of Milli-Q water. A drop of the oocyst PBS dispersion was then placed on the glutaric dialdehyde surface and left to dry in an evacuated environment (25 mmHg), and the dried oocysts were then washed with Milli-Q water. It is important to note that desiccation may affect the surface characteristics of the oocysts, but the fixative process is required for the AFM measurement procedure and has also been used in other ultrastructural works published on oocysts (e.g. Reduker et al.19,20). Comparison of glutaric dialdehyde/tip and oocyst/tip force interactions confirmed that the glutaric dialdehyde film did not contaminate the scanned region of the oocyst. Microelectrophoresis. A RANK Bros Mark II was used to obtain the microelectrophoretic mobility data. The velocity of particles in an applied electric field (50-100 V/cm) was measured. At each pH, the particle velocity was measured at both stationary planes and with the electric field reversed, involving an excess of 40 measurements, from which the times were averaged. Mobilities were converted to the electrophoretic (ζ) potential by the Helmholtz-Smolouchowski equation, which is valid for large values of κR (where κ-1 is the Debye length and R is the particle radius). A 150 µL aliquot of the oocyst dispersion (2.0127 × 107/ mL of PBS) was transferred to a 25 mL volumetric flask and diluted with the appropriate solution. Prior to the addition of particles, all KNO3 solutions were filtered through a 450 nm membrane prior to the dropwise addition of a similarly filtered potassium hydroxide solution (pH ) 11) or a nitric acid solution (pH ) 2), depending on pH requirements. Atomic Force Microscopy. A Nanoscope III (Digital Instruments, CA) was used to measure topographical features and force measurements in force-volume mode. In this mode, force curves are measured at a series of points as the AFM tip is scanned laterally across the surface. Accordingly, a two-dimensional matrix of force versus distance curves can be acquired. Each respective curve can be analyzed and then characterized by various parameters (for electrostatic forces these may be the Debye length and the fitted diffuse layer potential) which can then be mapped across the scan. In our work, we have measured force curves (each of 1024 data points) to a resolution of 64 data points (8 × 8 matrix of force curves) across the surface. Forcevolume files were analyzed for both height and force data using (18) Thissen, H. Article in preparation. (19) Reduker, D. W.; Speer, C. A.; Blixt, J. A. Can. J. Zool. 1985, 63, 1892-1896. (20) Reduker, D. W.; Speer, C. A.; Blixt, J. A. J. Protozool. 1985, 32, 708-711.

Figure 1. ζ-potential, derived from mobility data for the oocysts of C. parvum as a function of pH in 1 mM KNO3 and 0.2 mM KNO3 solutions. a combination of AFM Analysis V2.0 (Prof. D. Y. C. Chan, The University of Melbourne, Australia) and basic spreadsheet operations. All AFM force measurements reported here correspond to one set of measurements (all with the same tip). Repeat measurements were made with a total of four oocysts and three tips; in all cases the measurements obtained with these other oocyst/tip configurations were found to be within the limits of variation discussed below. All measurements were made in a fluid cell and with standard v-shaped silicon nitride cantilevers (nominal spring constant 0.06 N/m) with integrated tips. Spring constants were determined by measuring a loaded and unloaded frequency,21 and were found to be 0.095 ( 0.01 N/m. The piezoelectric ceramic was calibrated by the laser interferometry method.22 Prior to an interaction measurement, the tip was hydrolyzed to silanol functionality by exposure to a water plasma (Harrick, PDC-329: at medium power setting and 0.05 mmHg) for at least 20 min. The hydrolyzed tip/oxidized silicon wafer pH titrations were performed before and after the oocyst/tip experiments. The diffuse layer potential of the tip was found to vary by a maximum of (5 mV at particular pHs, and no measurable change in the point of zero charge was observed. This observation is consistent with a surface of stable silanol functionality and supports the notion that minimal irreversible adsorption of the oocyst surface proteins onto the AFM tip occurred (since this would alter the measured tip/wafer interaction).

Results and Discussion Microelectrophoresis. The microelectrophoretic mobility of the oocysts was measured as a function of pH at the ionic strengths 1 and 0.2 mM KNO3 (Figure 1 has the mobility converted to a ζ-potential). In common with earlier reports in the literature,23-25 the oocysts exhibit an isoelectric point < pH 3 and a potential at high pH of around -(35-45) mV. The general agreement with earlier reports indicates that the surface sterilization procedure used here has probably not altered the net surface charge density. Establishing the molecular origin of the surface charge of the oocysts is made difficult by the relatively small number of investigations into its structure but is aided by the application of the polymerase chain reaction (PCR) to the detection of C. parvum in environmental samples.26 DNA sequencing27 of C. parvum oocyst surface proteins suggests a surface-adherent molecule rich in cysteine, proline, and histidine and capable of forming multiple disulfide bonds.28 Other studies have shown that the outer wall of C. parvum oocysts contains galactose/ (21) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403-405. (22) Jaschke, M.; Butt, H.-J. Rev. Sci Instrum. 1995, 66, 1258-1259. (23) Ongerth, J. E.; Pecoraro, J. P. J. Environ. Eng. 1996, 122, 228231. (24) Drozd, C.; Schwartzbrod, J. Appl. Environ. Microbiol. 1996, 62, 1227-1232. (25) Karaman, M. E.; Pashley, R. M.; Bustamante, H.; Shanker, S. R.Colloids Surf., A 1999, 146, 213-216.

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galactosamine and glucose/glucosamine residues, possibly with both N- and O-linked glycosylation.29-31 Atomic Force Microscopy. Evaluation of the Effective Radius (Reff) and the Diffuse-Layer Potential of the AFM Tip. The first steps employed in the deconvolution of AFM force data were to (i) obtain the effective tip radius in order to normalize the force of interaction, (ii) ensure that the tip had been hydrolyzed to predominantly silanol functionality, and (iii) determine the magnitude of the diffuse layer potential of the hydrolyzed tip. In the theory of Derjaguin, Landau, Verwey, and Overbeek (DLVO) it is assumed that two additive forces, electrical double-layer and van der Waals forces, govern the overall interaction between two surfaces. In this work, we have assumed that the electrical double-layer interaction can be computed as a function of separation by employing the nonlinearized Poisson-Boltzmann equation. We have assumed that the van der Waals interaction can be described by

F/R )

-AH 6H2

where AH is the nonretarded Hamaker constant (ca. 1 × 10-20 J for the oocyst/tip interaction) and H is the intersurface separation. It should be noted that, at the long-range separations that we are primarily concerned with, the van der Waals component to the overall interaction is negligible. In the case of tip geometry estimation, it is the range of the forces acting between the tip and the underlying surface that will determine the most appropriate effective tip geometry. In the case of the long-range surface forces measured in the present study, the effective tip radius can be calculated by employing the procedure of Drummond and Senden.32 In this method, the interaction force F between a spherical particle of known radius R and a planar surface, both coated with a maximum surface coverage of an adsorbed surfactant bilayer in aqueous solution, is measured and plotted as F/R versus the separation of closest approach. This standard sphere/flat plate interaction is then compared with the force of interaction between AFM tips and planar surfaces possessing the same adsorbed surfactant coatings. A DLVO fit can be obtained for the standard surfactant-coated silica sphere/flat plate system, and the theoretical fit can then be compared with the experimental tip/flat plate data. The effective radius Reff required to normalize the tip data can in turn be determined. A surface potential of 90 mV (derived from the DLVO fit of the standard surfactantcoated sphere/flat plate system) is required in the DLVO fit of the surfactant-coated tip/wafer interaction.33,34 The (26) Laxer, M. A.; Timblin, B. K.; Patel, R. J. Am. J. Trop. Med. Hyg. 1991, 45, 688-694. (27) Ranucci, L.; Muller, H.-M.; Rosa, G. L.; Reckmann, I.; Morales, M. A. G.; Spano, F.; Pozio, E. Infect. Immun. 1993, 61, 2347-2356. (28) Tilley, M.; Upton, S. J. In Cryptosporidium and Cryptosporidiosis; Fayer, R., Ed.; CRC Press: Boca Raton, FL, 1997; pp 163-180. (29) Nichols, G. L.; McLauchlin, L.; Samuel, D. J. Protozool. 1991, 38, 237s. (30) Llovo, J.; Lopez, A.; Fabreges, J.; Munoz, A. J. Infect. Dis. 1993 167, 1477-1480. (31) Gut, J.; Nelson, R. G. Proceedings of the 47th Annual Meeting of the Society of Protozoolology; Allen Press: Cleveland State University, 1994. (32) Drummond, C. J.; Senden, T. J. Colloids Surf., A 1994, 87, 217234. (33) Johnson, S. B.; Drummond, C. J.; Scales, P. J.; Nishimura, S. Langmuir 1995, 11, 2367-2375. (34) Senden, T. J.; Drummond, C. J. Colloids Surf., A 1995, 94, 2951.

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Figure 2. Normalized force versus separation (Reff ) 700 nm) curve for a silica tip interacting with a silica flat plate (thermally oxidized silicon wafer) in an aqueous 1 mM KNO3 solution at pH 3.81 (diamonds) and pH 8.88 (circles). The electrical doublelayer interaction has been calculated for constant potential (lower limit) and constant charge (upper limit) with diffuse layer potentials of 60 mV and 90 mV for pH 3.81 and 8.88, respectively.

value of Reff required to normalize the tip data in the present study was found to be 700 nm, and the experimental tip/wafer data, along with the DLVO fit, has been supplied in the Supporting Information (Figure S1). The derived Reff value is comparable to other values for square pyramidal silicon nitride tips that have been derived from an analysis of either known electrical double-layer interactions34-36 or van der Waals interaction.37 It is important to note that the effective radius of cuvature measured by this method is not the nominal tip radius of curvature (reported by the manufacturer to be 20-60 nm) but is the effective radius of curvature measured at large separations by surface force measurement. The experiment to determine the effective tip radius was always performed after all the other experiments had been completed. The force of interaction between a tip and a thermally oxidized silicon wafer can be used to determine the diffuse layer potential of the tip. The method involves fitting a DLVO fit to the experimental data to calculate the potential. The process serves the dual purpose of verifying the tip hydrolysis (silylamine has a higher isoelectric point than that of silanol, which is around a pH of 2-3)34 and determining the tip potential as a function of pH for later interpretation of the tip/oocyst interaction. A pair of tip/ silicon wafer (thermally oxidized) interactions on surface approach has been fitted by DLVO theory in Figure 2. The fitted diffuse layer potential, as a function of pH, has been plotted for an ionic strength of 0.2 mM (Figure 3a) and 1 mM (Figure 3b) KNO3. For comparison, microelectrophoresis data for 3-10 µm glass beads (Polysciences Inc.) have been provided. The point of zero charge (pzc) of native silicon nitride tips is around pH 5-7, and the pzc of bare silica tips is around pH 2-3.34 The results contained in the figure thus confirm that the predominant tip functionality is silanol, since the measured pzc is around 2-3. Cryptosporidium Oocyst Topography. The surface features of the Cryptosporidium oocysts were measured in contact mode in situ by the tip that was later used in the force measurements. The presence of a “suture” (or fold) in the oocyst surface, and its purpose in the protozoan (35) Senden, T. J.; Drummond, C. J.; Ke´kicheff, P. Langmuir 1994, 10, 358-362. (36) Biggs, S.; Mulvaney, P. J. Chem. Phys. 1994, 100, 8501-8505. (37) Drummond, C. J.; Vasic, Z. R.; Geddes, N.; Jurich, M. C.; Chatelier, R. C.; Gengenbach, T. R.; Griesser, H. J. Colloids Surf., A 1997, 129-130, 117-129.

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Figure 3. Diffuse layer potentials, derived from the silica tip/ flat plate force versus separation curves (filled circles) and ζ-potentials, determined from microelectrophoretic mobility measurements of glass beads (open circles), as a function of pH in (a) 0.2 mM and (b) 1 mM aqueous KNO3 solution. The error bars correspond to the measured standard deviation for at least 10 measurements.

lifecycle, has received some attention in earlier transmission electron and optical microscopy studies.19,20,33 Occasionally the oocyst sutures were clearly visible, although this was not always the case. This may indicate that the oocyst deposition is random and not oocyst surface site specific (i.e. oocysts attach to the glutaric dialdehyde suture-side-up as readily as suture-side-down). A contact mode AFM scan of the oocyst has been presented (Figure 4). The z range of the figure is 2 µm. The biological microspheres cannot be described as smooth. The largest asperities were found to be around 200 nm in diameter and around 30 nm high. Occasionally, tip-edge effects were apparent along the oocyst edge (for instance, the grooves along the right-hand edge in Figure 4b). The rootmean-square roughness of an x,y plane-fitted 2 µm scan has been determined to be around 17 nm with a peak to valley height of around 50 nm across the scan. Cryptosporidium Oocyst/Silica Tip Interaction. A typical height scan measured during force measurement has been presented in Figure 5a. The force of interaction between the hydrolyzed tip (Reff ) 700 nm) and the oocyst surface was measured at each point on the 8 × 8 matrix. Example force curves measured at points that roughly correspond to locations A4, D4, E2, and H7 on the oocyst surface have been presented in Figure 5b. The force traces of Figure 5b were measured at pH 6.09 and an ionic strength of 1 mM. For comparison, the tip/glutaric dialdehyde interaction (included in Supporting Information Figure S2) is characterized by a short-range attraction on surface approach and a relatively large adhesion on separation. This is in contrast to the oocyst/tip interactions

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Figure 4. Contact mode AFM image of a typical oocyst. The image was obtained with the same type of v-shaped cantilever as was used in the force measurements. The total z-range of the figure is 2000 nm, and the background contains no data because of the low load applied: (a) surface plot; (b) top view.

where an approximately exponential force of repulsion exists between the oocyst and the tip on surface approach and where the force is generally mirrored on separation but for occasional adhesion “spikes”. Oocyst Force Maps. The force-volume mode was used to measure force curves and height across the oocyst surface simultaneously. All oocyst force-volume maps have been obtained over fixed scans of 5 µm with the oocyst centered in the scan; this results in a lateral resolution of the force curve array at around 625 nm. Given that the force measurements were obtained with a tip that had a measured effective surface force radius of 700 nm, this is a satisfactory lateral resolution for the system under investigation. The approximately exponential repulsive force on surface approach (Figure 5b) was present in every force trace measured on all the oocysts, under all the measured conditions. Therefore, the force curves on approach could be analyzed for the repulsive force decay length and magnitude in addition to the constant compliance, and force maps measured in 1 mM KNO3 are presented in Figures 6 and 7. (Force maps for the 0.2 mM KNO3 system are included as Supporting Information Figures S3 and S4.) Constant Compliance. The constant compliance, or sensitivity, is a measure of the compressibility of the sample, since it corresponds to the measured deflection per piezo movement when the tip and sample are in contact. The units of constant compliance are thus

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Figure 5. (a) Height scan of an oocyst at the resolution of the force maps. (b) Representative oocyst/tip force-separation curves (Reff ) 700 nm) on surface approach (triangles) and on surface retraction (squares) measured at pH 6.09 and an ionic strength of 1 mM. The labels A4, D4, E2, and H7 correspond to locations on the oocyst force map grid presented in the height scan of part a. For each of the four pairs of force curves, the retraction curves have been displaced one division on the force axis for clarity.

photodiode position (V) per piezo distance (nm). Typical values of hard inorganic surfaces such as silica were measured to be around 0.11 ( 0.01 V/nm. Values reported in the compliance maps range from a minimum of 0.09 V/nm to a maximum of 0.14 V/nm although most values were around 0.11 ( 0.02 V/nm. Thus, the measured values of the constant compliance across the oocyst surface are quite similar to those measured for silica. The measured variation is slightly larger than that for silica, and this may reflect regions of variable compressibility across the oocyst surface. The presence of surface features (surface roughness) may also contribute to the measured variation in the constant compliance by evoking lateral (notmeasured) deflection. Nonetheless, we can conclude that the zero separation on the force plots (position of constant compliance) corresponds to oocyst/tip hard-wall contact and that the oocyst wall is relatively hard (comparable in hardness to silica as assessed from the constant compliance region of the force curve). It is important to note that the

Figure 6. Force maps of an oocyst obtained with a silica tip at 1 mM KNO3 and pH 3.87. Maps correspond to height (a), constant compliance (b), the decay length of the repulsive force (c), and the extrapolated (to zero separation; see text) magnitude of the repulsive force (d).

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Figure 8. Schematic representation of the complication that can arise with tip/oocyst alignment. Interactions measured at the edge of the oocyst contain significant torsional effects. The oocyst profile (large hemicircle) has been obtained from a sectioned scan of the oocyst presented in Figure 4.

Figure 7. Force maps of an oocyst obtained with a silica tip at 1 mM KNO3 and pH 8.16. Maps correspond to height (a), constant compliance (b), the decay length of the repulsive force (c), and the extrapolated (to zero separation; see text) magnitude of the repulsive force (d).

definition of zero separation corresponds to the reference of hard-wall contact and is a relative separation not necessarily the separation of closest approach. Repulsive Force Decay Length. The repulsive force on surface approach has been characterized by its decay length or log linear (natural log) slope of the force at separations between 20 and 50 nm from hard-wall contact. The decay lengths appear to be greatest around the circumference of the oocyst, and this trend was repeatedly observed. We propose that the “doughnut” effect may be due to the differences in the alignment of the tip with the oocyst throughout the force map. For instance, in the height scan of Figure 5a, the white corresponds to the scenario most akin to coaxial alignment of the tip and occyst while the darker regions correspond to positions where the tip is far from coaxial alignment with the oocyst. A schematic representation of the alignment problem is presented in Figure 8, where the section of a scanned oocyst has been used and the effective radius of the tip drawn to the corresponding scale. In this figure the range of the repulsive force (dashed line) has been drawn to approximately 4 experimental decay lengths. It is apparent from the schematic representation that the force profiles obtained at the edge of the oocyst contain an additional torsional component. Given the complexity of the system (complex tip geometry and oocyst topography), it is not feasible to resolve the actual surface force component from the edge effects. A curious point to note is that in all of the decay length force maps there appears to be regions of contrasting decay lengths (always located around the circumference). The locations of long decay length were generally not found to be associated with topographical, compliance, or magnitude anomalies. Certain peaks (such as at B5 in Figure 6c, pH ) 3.87) exhibited a strong pH dependence (not present in Figure 7c, pH ) 8.16) while the pH dependence is less clear for others (such as E2 in Figures 6c and 7c). Random edge effects, as well as surface protein/tip interactions, probably contribute to the contrast in the decay length plots. However, a substantial statistical analysis of multiple force curves at each precise location on the 8 × 8 matrix would be required to distinguish the dependence of the contrast on pH and ionic strength affects from random edge affects. Nevertheless, the relative absence of such peaks for the condition of coaxial alignment further supports the notion that the force of interaction is strongly dependent on the alignment of the force probe with the sample curvature. Therefore, data contained within the central regions (D4, D5, E4, and E5) of the force maps have been used to assess the distribution of the repulsive force decay length and magnitude, as well as pH and ionic strength effects. The decay length has been plotted as a function of pH for two ionic strengths

Oocysts of Cryptosporidium Parvum

Figure 9. (a) Exponential repulsive force decay length as a function of pH in aqueous 1 mM (circles) and 0.2 mM (diamonds) KNO3 solutions. (b) Exponential repulsive force extrapolated (to zero separation; see text) magnitude as a function of pH in aqueous 1 mM (circles) and 0.2 mM (diamonds) KNO3 solutions. The error bars correspond to the standard deviation of the spread at locations D4, D5, E4, and E5.

in Figure 9a. The open symbols correspond to the average of the decay length for four centrally located points on the oocyst surface (D4, D5, E4, and E5) while the error bars correspond to the standard deviation across this region. The Debye lengths for 0.2 and 1 mM 1:1 electrolyte solutions are 21.6 and 9.7 nm, respectively. The experimental decay lengths measured at the respective ionic strengths have been found to be around 18 ( 2 and 14 ( 3 nm. Although the spread results in an overlapping error (around 15-20% of the mean), from close inspection of Figure 9a it is apparent that a decrease in the decay length occurs with an increase in ionic strength. This observation is generally consistent with conventional electrical doublelayer theory. Therefore, the measured decay length data suggest that the measured repulsive force arises, at least in part, from an electrical double-layer interaction. Repulsive Force Magnitude. The magnitude of the repulsive force has been defined as the log linear (natural log) intercept of the force extrapolated from separations between 20 and 50 nm from hard-wall contact, and this parameter has been plotted as a function of lateral position in Figures 6d and 7d. The distribution of these values across the oocyst surface does not, in general, follow the “doughnut” trend measured in the decay length. No apparent trends in distribution are immediately distinguishable. The contrast in the magnitude of the repulsive force across most of the scanned oocyst surface is large (values within the square of C3, C6, F3, and F6 vary by around 20-40% of the mean) while the values in the central region (D4, D5, E4, and E5) also vary significantly (similarly 20-40% of the mean). The effect of pH on the magnitude of the repulsive force has been plotted in Figure 9b. The open symbols correspond to the average of the values located at D4, D5, E4, and E5, and the error bars correspond to the standard deviation within this region.

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Figure 10. (a) Typical oocyst/tip normalized force versus separation curves measured in 0.2 mM KNO3 at pH 3.85 (diamonds) and pH 8.23 (circles). Solid lines correspond to the electrical double-layer repulsion (for the boundary condition of constant charge interaction) predicted by using the tip diffuse layer potential (Figure 3) and the oocyst ζ-potential (Figure 1) at pH 8.23 (upper solid line) and 3.85 (lower solid line). (b) Displacement of the electrical double-layer lines in Figure 10a by 40 nm (pH 3.85) and 35 nm (pH 8.23) in order to obtain “pseudo” fits.

Although the large spread of values apparent in Figure 9b across individual oocysts makes trends difficult to distinguish, it is apparent that the magnitude is a little larger at higher pH. This trend is reaffirmed by comparison of Figure 6d with Figure 7d. In line with the conclusions made upon analysis of the decay length, the qualitative behavior of the magnitude is generally consistent with conventional electrical double-layer theory (i.e. the surfaces may be “charging-up” with an increase in pH). However, as is discussed below, a significant quantitative difference exists between the magnitude predicted from the electrophoretic potential and the measured magnitude. Exponential Repulsive Force and Poison Boltzmann Equation. Force curves on surface approach at pH 3.85 and pH 8.23 in aqueous 0.2 mM KNO3 (Figure 10) have been plotted as log(F/R) versus separation (where hard-wall contact is defined as zero separation). For comparison with theory, the full nonlinearized PoissonBoltzmann equation for the interaction between dissimilar surfaces in a 1:1 electrolyte39 has been made by utilizing the determined corresponding tip diffuse layer potentials (Figure 3) and Cryptosporidium oocyst ζ-potentials (Figure 1). The calculated electrical double-layer force (for the surface condition of constant charge) has been plotted as (38) Robertson, L. J.; Campbell, A. T.; Smith, H. V. Appl. Environ. Microbiol. 1993, 59, 2638-2641. (39) McCormack, D.; Carnie, S. L.; Chan, D. Y. C. J. Colloid Interface Sci. 1995, 169, 177-196.

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the upper solid line (pH 8.23) and the lower solid line (pH 3.85) in the figure. It is apparent that the magnitude of the calculated electrical double-layer force is less than that of the measured force. Under the aqueous electrolyte conditions used in the present study, for metal oxides that have either no or a relatively thin organic coating ( 100 nm is evidence that the tip and oocyst may not actually be out of contact until they are well separated (>150 nm) from the oocyst surface. The “spikes” in the force-separation measurements may lend further support to the idea that the tip may experience an “electrosteric” force prior to hard-wall contact with the oocyst. Summary and Conclusions We propose that force mapping with the AFM is a useful tool in measuring the force of interaction between any two surfaces possessing a macroscopic radius of curvature, since the alignment is measured in addition to the standard force curve. Alignment of the force probe with the biological microsphere has been found to have a dramatic effect on the measured decay length for the interaction in aqueous solution. An exponential repulsive force has been found to exist between the oocysts of C. parvum and an amorphous silica surface. The decay length and magnitude of the exponential force have been mapped across the oocyst surface at various ionic strengths and pH values. Although the measured decay lengths (for coaxial alignment) are, in general, comparable to the Debye length, the observed values of the magnitude are much larger than would be predicted from independent measurements of the diffuse layer potential. Explanations, mainly relating to surface roughness and protein molecules acting as a steric layer, have been proposed to explain the difference. Analysis of the force curves upon separation further supports the idea that tethering (probably by protein molecules) occurs between the tip and the oocyst. In this work, we have reported the first direct measurement of the force of interaction between C. parvum oocysts and a siliceous (sandlike) surface. A better understanding of the surface interaction between C. parvum oocysts and siliceous materials can provide insights into how to manipulate solution conditions to improve oocyst detection and to develop better processes for removing the oocysts from source waters. Acknowledgment. We thank David Davey (University of South Australia) for discussions. R.C. is the recipient of an Australian Post-Graduate Research Award. We also thank The University of South Australia and the Australian Cooperative Research Centre for Water Quality and Treatment for support. Supporting Information Available: Figure S1, normalized force versus separation curve for a silica tip interacting with a silica flat plate in an aqueous CTAB [1.6 mM] solution; Figure S2, Typical normalized force of interaction for the glutaric dialdehyde/tip system on approach and upon separation measured in Milli-Q water at natural pH; Figure S3, force maps of an oocyst obtained with a silica tip at 0.2 mM KNO3 and pH 3.85; Figure S4, force maps of an oocyst obtained with a silica tip at 0.2 mM KNO3 and pH 8.23. This material is available free of charge via the Internet at http://pubs.acs.org. LA990205P (43) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Guntherodt, H.-J.; Misevic, G. N. Science 1995, 267, 1173-1175.