Force of Interaction between a Biocolloid and an Inorganic Oxide

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Force of Interaction between a Biocolloid and an Inorganic Oxide: Complexity of Surface Deformation, Roughness, and Brushlike Behavior Robert F. Considine,† Calum J. Drummond,*,‡ and David R. Dixon 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 October 18, 2000. In Final Form: February 21, 2001 Force-separation measurements between a deformable, rough, biological surface (Cryptosporidium parvum) and an inorganic oxide (silica) have been obtained using the atomic force microscope. The system was chosen because oocysts of C. parvum have been associated with waterborne outbreaks of disease, and one of the main barriers to oocyst contamination of drinking waters is provided by sand-bed filtration. The oocysts are shown to be significantly rough on the scale of Derjaguin-Landau-Verwey-Overbeek forces and have been found to be compressible on the scale of the loads applied during force measurement. The surface compressibility is reported in terms of an interfacial spring constant. The force of interaction prior to this Hookean region is long-range and repulsive. The long-range force has been compared to models of the electrical double layer force (based on the measured ζ-potentials and bulk electrolyte concentration) as well as an electrosteric force (treating the surface as a polyelectrolyte brush). The comparison has led to the conclusion that the surface can be described as a polyelectrolyte brush at intermediate separations (≈10-30 nm from linear compliance) with an electrical double layer often observed at greater separations. The dependence of the force on surface separation suggests that tethering between the oocyst and silica can occur. The variation of the interaction with pH and upon subtle changes in the ionic strength, compared to the variation from oocyst to oocyst, has also been assessed.

* Author for whom correspondence should be addressed. Ph: 61 2 9420 0663. Fax: 61 2 9420 0692. E-mail: [email protected]. † Present Address: Melbourne Water Corporation, Australia. ‡ Present Address: cap-XX Pty. Ltd. Units 9 & 10, 12 Mars Road, Lane Cove, NSW 2066 Australia.

conventional AFM is that the separation is inferred rather than directly measured. The process of obtaining forceseparation data from the AFM has been well documented8-10 and verified for hard, incompressible surfaces under specific electrolyte conditions. However, with the increasing application of the AFM to biological surfaces11 the consequence of surface deformation (as opposed to indentation, see below) during measurement has received relatively little attention. Many studies by surface force measurement suggest that tethered biomolecules, such as collagen, poly-L-lysine, and gelatin, may adopt brushlike structures when absorbed at interfaces.12,13 Recently, Likos et al.14 showed, with small-angle neutron scattering (SANS) experiments, that enhanced stabilization of colloidal particles can be obtained with adsorbed gelatin. Results of the SANS spectra were interpreted in terms of a stabilization mechanism that involved a simple combination of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory (van der Waals plus electrical double layer interaction) and a steric force described by a model of an adsorbed polymer brush. It is not surprising that tethered proteins may be well approximated as a polyelectrolyte brush, given that various acidic and basic amino acid groups may be distributed along the polypeptide backbone, similar to the charged functional groups distributed along the polymer backbone of a synthetic polyelectrolyte. Therefore, the

(1) Israelachvili, J. N.; Adams, G. E. J. Chem. Soc., Faraday Trans. 1978, 1, 975. (2) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930. (3) Wong, J. Y.; Kuhl, T. L.; Israelachvili, J. N.; Mullah, N.; Zalipsky, S. Science 1997, 275, 820. (4) Koehler, J. A.; Ulbricht, M.; Belfort, G. Langmuir 1997, 13, 4162. (5) Radmacher, M.; Fritz, M.; Kacher, C. M.; Cleveland, J. P.; Hansma, P. K. Biophys. J. 1996, 70, 556. (6) Considine, R. F.; Dixon, D. R.; Drummond, C. J. Langmuir 2000, 16, 1323. (7) Bowen, W. R.; Hilal, N.; Lovitt, R. W.; Wright, C. J. Colloids Surf., A 1998, 136, 231.

(8) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353, 239. (9) Larson, I.; Drummond, C. J.; Chan, D. Y. C.; Grieser, F. Langmuir 1997, 13, 2109. (10) Considine, R. F.; Drummond, C. J. Langmuir, submitted. (11) Shao, Z.; Mou, J.; Czajkpwsky, D. M.; Yang, J.; Yuan, J.-Y. Adv. Phys. 1996, 45, 1. (12) Komiyama, Y.; Israelachvili, J. Macromolecules 1992, 25, 5081. (13) Klein, J.; Luckham, P. F. Colloids Surf. 1984, 10, 65. (14) Likos, C. N.; Vaynberg, K. A.; Lowen, H.; Wagner, N. J. Langmuir 2000, 16, 4100.

Introduction Knowledge of the interaction of biological surfaces with inorganic oxides is crucial to many biotechnologies, from bioprocessing to the development of biomedical devices. Over the past thirty years, extensive effort has been applied to the direct measurement of the interaction of biological surfaces with inorganic oxides using a variety of techniques, including the surface force apparatus (SFA)1 and the atomic force microscope (AFM).2 The main advantage of the SFA is its ability to measure the force of interaction as a function of separation directly, typically by interferometry. The main drawback of previous reports of the SFA experiments of biological surfaces, however, is that they have been largely limited to idealized surfaces, that is, arrays of biomolecules anchored to atomically smooth substrates such as mica.3,4 On the other hand, the AFM has enabled the direct measurement of the interaction between biological surfaces (including human platelets,5 protozoa,6 and yeast cells7) and inorganic oxides. A possible drawback of force-separation measurements by

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knowledge gained by modeling the behavior of charged polymers at interfaces15-22 ought to, at least qualitatively, be useful in understanding the interaction of biomolecules with surfaces. The interaction of Cryptosporidium parvum oocysts with inorganic oxide surfaces is a critical component in ensuring the safe distribution of drinking water.23-25 Some conflict exists in the literature regarding the effectiveness of various disinfection methodologies such as UV exposure26,27 and ozonation,28,29 owing largely to the ambiguity regarding definitions of oocyst viability and infectivity. Consequently, many regulators require water suppliers to establish physical barriers (i.e., filtration) to help prevent distribution of oocysts in public drinking water.30 Sand-bed filtration is widely applied as a treatment technology for C. parvum.31 Here, we report a study of the interaction of individual C. parvum oocysts and a sandlike surface. This builds on the foundations of an earlier study,6 where the force of interaction between pyramidal AFM silica tips and oocysts was reported. An atomic force microscope has been used to measure the force of interaction between a C. parvum oocyst and an inorganic oxide (silica). The surface topography and compressibility are assessed, and the role of surface deformation in the force of an interaction is quantified in terms of an interfacial spring constant. The effect of solution pH, ionic strength, and oocyst to oocyst variability is examined, and the surface is described by a combination of electrical double layer and polyelectrolyte brush models. Finally, the adhesion between the two surfaces on separation is consistent with oocyst proteins frequently tethering the two surfaces together. Experimental Procedure Materials. All electrolyte solutions were made up with water obtained from a Milli-Q system. The potassium nitrate, nitric acid, and potassium hydroxide were all obtained from BDH Chemicals, were of analytical reagent grade, and were used without further purification. Oocysts of C. parvum. The oocysts of C. parvum (Iowa isolate) were isolated and supplied by Waterborne, Inc. (LA). The oocysts were obtained from the faeces of experimentally infected calves and were purified by sucrose and Percoll density gradient centrifugation, after initial extraction from the faeces with diethyl ether. The identification methods used were direct immunofluorescence microscopy with genus-specific monoclonal antibodies and also phase microscopy. Comparison of the measured elec(15) Alexander, S. J. J. Phys. (Paris) 1977, 38, 983. (16) De Gennes, P. G. Macromolecules 1982, 15, 492. (17) Pincus, P.; Witten, T. Europhys. Lett. 1987, 3, 315. (18) Miklavic, S. J.; Marcelja, S. J. Phys. Chem. 1988, 93, 6718. (19) Milner, S. T.; Witten, T. A.; Cates, M. E. Macromolecules 1989, 22, 853. (20) Pincus, P. Macromolecules 1991, 24, 2912. (21) Zhulina, E. B.; Borisov, O. V.; Birshtein, T. M. J. Phys. II 1992, 2, 63. (22) von Goeler, F.; Muthukumar, M. Macromolecules 1995, 28, 6608. (23) Harter, T.; Wagner, S.; Atwill, E. R. Environ. Sci. Technol. 2000, 34, 62-70. (24) Weber-Shirk, M. L.; Dick, R. I. J.sAm. Water Works Assoc. 1997, 89, 87-100. (25) Fogel, D.; Isaac-Renton, J.; Guasparini, R.; Moorehead, W.; Ongerth, J. J.sAm. Water Works Assoc. 1993, 85, 77-84. (26) Gyurek, L. L.; Li, H.; Belosevic, M.; Finch, G. R. J. Environ. Eng. 1999, 125, 913. (27) Peeters, J. E.; Mazas, E. A.; Masschelein, W. J.; de Maturana, I. V. M.; Debacker, E. Appl. Environ. Microbiol. 1989, 55, 1519-1522. (28) Clancy, J. L.; Hargy, T. M.; Marshall, M. M.; Dyksen, J. E. J.s Am. Water Works Assoc. 1998, 90, 92-102. (29) Neumann, N. F.; Gyurek, L. L.; Gammie, L.; Finch, G. R.; Belosevic, M. Appl. Environ. Microbiol. 2000, 66, 406-412. (30) Fogel, D.; Isaac-Renton, J.; Guasparini, R.; Moorehead, W.; Ongerth, J. J.sAm. Water Works Assoc. 1993, 85, 77. (31) Clancy, J.; Fricker, C. Water Qual. Int. 1998, July/August, 37.

Considine et al. trophoretic mobility of the oocysts with genotyped oocysts32 reveal the charge behavior of the oocyst surface to be representative of C. parvum. The oocysts were used as supplied (viable, 1 × 106 oocysts/mL in PBS with penicillin, streptomycin, and gentamicin) without surface modification or inactivation and were typically used within 6-8 weeks of supply. Oocysts were stored at 4 °C. The procedure for anchoring oocysts has been outlined in a previous study.6 Briefly, the underlying surface of the oocyst was covalently anchored to a coated silicon wafer, with a localized fixative process. The smooth silicon wafer (Silica Source Technology Corp.) was exposed to a plasma deposition (power ) 20 W, frequency ) 200 kHz) of 3-amino-propylene (time ) 25 s, pressure ) 0.125 mmHg).33 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 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. The minimum drying time (12 h) was used, and comparison of the oocyst-oxide force of interaction showed no significant change over 40 h, suggesting that the surface is stable over the course of an experiment. Comparison of glutaric dialdehyde/ silica and oocyst/silica force curves confirmed that the glutaric dialdehyde film did not contaminate the scanned region of the oocyst. 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).34 The isoelectric point and ζ-potentials as a function of pH (see below) are consistent with earlier reports of silica surfaces.35 Therefore, it has been assumed that the surface is predominantly composed of silanol functionality and can thus serve as a suitable model of a sand surface. The silica particles were mounted on the AFM cantilever springs according to the method of Ducker et al.8 Microelectrophoresis. A RANK Bros Mark II was used to obtain the microelectrophoretic mobility data. The velocity of particles in an applied electric field E (50-100 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 measurements, from which the times were averaged. Mobilities, u, 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) and relates ζ to u by taking into account the bulk electrolyte viscosity η and permittivity ,

u)

v ζ ) E η

Approximately 250 µL of the oocyst dispersion was transferred to a 50 mL flask and diluted with approximately 25 mL of the appropriate solution. The pH adjustment was conducted with dropwise addition of either potassium hydroxide solution (pH ) 11) or nitric acid solution (pH ) 2). Atomic Force Microscopy. A Nanoscope III (Digital Instruments, CA) was used to measure topographical features and force-separation curves. Electrical double layer mode36 (cf. softcontact mode37) with simultaneous deflection mode was used for obtaining topographical data. Before imaging in electrical double layer mode, the lateral scan axes were disengaged, and the forceseparation curve was measured. As evident in the force measurements (see below), a repulsive force was found to exist between the oocyst and an oxide surface. Therefore, the setpoint was adjusted to minimize the force of interaction between the (32) Considine, R. F.; Dixon, D. R.; Drummond, C. J. Water Res., submitted. (33) Thissen, H. Article in preparation. (34) CRC Handbook of Chemistry and Physics, 60th ed.; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1981; B-51. (35) Hartley, P. G.; Larson, I.; Scales, P. J. Langmuir 1997, 13, 2207. (36) Senden, T. J.; Drummond, C. J.; Kekicheff, P. Langmuir 1994, 10, 358. (37) Fleming, B. D.; Wanless, E. J. Microsc. Microanal. 2000, 6, 104.

Biocolloid and Inorganic Oxide Interaction Force

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tip and sample, thus enabling the contact force (and therefore deformation) to be reduced from Born repulsion to the electrical double layer force between the tip and sample. Typical forces of 0.5-1 nN were used. Commercially available silicon nitride oxidesharpened tips (Nanoprobes, Digital Instruments, CA) with quoted spring constants of 0.58 N m-1 were used for imaging in electrical double layer mode. It will be shown in a forthcoming publication that the isoelectric point of these tips is in the range of pH 5-7.38 Therefore, all electrical double layer mode imaging has been performed in 1 mM KNO3 at pH 8.5, to obtain strong electrostatic repulsion between the tip and oocyst. In an earlier report,6 we outlined the importance of obtaining coaxial alignment for force measurements between curved surfaces. In that study, we used force-volume mode39 to measure an array of force curves across the oocyst surface in order to ensure the surfaces were centrally aligned, and it was found that noncoaxial alignment led to edge artifacts. Therefore, in the present study, only forces measured for coaxial alignment have been reported. The surfaces were aligned according to the protocol of Larson et al.,40 where contact mode imaging of the underlying particle is used to align the surfaces, immediately prior to force curve data acquisition. Occasionally, full force-volume matrices over the oocyst were collected and it was found that, for any given oocyst, the coaxially aligned force curves were reproducible whether the force-volume or image-aligned force curve acquisition modes were used. Once the force-separation data had been acquired, each respective curve was analyzed and then characterized by a number of parameters (see below). All measurements were made in a fluid cell (cleaned by UV exposure, 30 min). Spring constants for force curve measurements were determined by measuring a loaded and unloaded frequency41 and were found to be 0.25 ( 0.02 N m-1. The piezoelectric ceramic was calibrated by the laser interferometry method.42 Prior to an interaction measurement, the silica colloid probe assembly was cleaned by exposure to an air/water plasma (Harrick, PDC-329; at medium power setting and 0.05 mmHg) for approximately 30 s.

Results and Discussion Surface Topography. The topographical features of both the silica spheres and oocysts were measured in aqueous electrolyte in electrical double layer mode. In this mode, the force of contact is minimized, and the usefulness of the technique for imaging soft surfaces has been evidenced by the acquisition of images of surfactant micelles37 and individual protein molecules.43 Images of the silica spheres will be presented in a forthcoming publication,10 and images of the oocysts are presented in Figure 1. The silica spheres have a root-mean-square (rms) roughness of approximately 1-2 nm for a 2 × 2 µm, xy plane fitted scan. The roughness is distributed as gentle undulations of a few hundred nanometers in the lateral dimensions and approximately 10-20 nm in the vertical dimension. In contrast, the surface topography of the oocysts is substantially rougher. The rms roughness of a 2 × 2 µm, xy plane fitted scan has been determined to be approximately 5 nm with a peak to valley height of approximately 50 nm across the scan. Inspection of the 1.25 × 1.25 µm zoom (Figure 1b) shows that rather than gentle undulations, the roughness appears as discrete asperities, ranging in size from approximately 50 to 250 nm in the lateral dimensions. The observation that the (38) Ruohola, A. M.; Considine, R. F.; Drummond, C. J.; Dixon, D. R. Langmuir, article in preparation. (39) Radmacher, M.; Cleveland, J. P.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Biophys. J. 1994, 66, 2159. (40) Larson, I.; Drummond, C. J.; Chan, D. Y. C.; Grieser, F. J. Phys. Chem. 1995, 99, 2114. (41) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403. (42) Jaschke, M.; Butt, H.-J. Rev. Sci. Instrum. 1995, 66, 1258. (43) Najbar, L. V.; Considine, R. F.; Drummond, C. J. Biophys. J., submitted.

Figure 1. AFM image of a typical oocyst obtained in electrical double layer mode. The image was obtained with a sharpened silicon nitride cantilever at pH 8.5 and at 1 mM KNO3. (a) 3.4 µm scan; the total z-range of the figure is 1000 nm, and the edge of a second oocyst can be observed in the top left corner of the image. (b) 1.25 µm scan showing the roughness of the oocyst surface.

oocysts are rough is consistent with an earlier report,6 where the oocysts were found to have an rms of 17 nm across a 2 × 2 µm, xy plane fitted scan. The variation in precise roughness values reflects the variation among oocysts. Microelectrophoresis. The microelectrophoretic mobility of the silica spheres and oocysts was measured as a function of pH at ionic strengths of 1, 2, and 5 mM KNO3 (Figure 2 has the mobility converted to a ζ-potential). In common with earlier reports in the literature, the silica spheres exhibit an isoelectric point (IEP) lower than pH 3 and a plateau ζ-potential of approximately -(80-90) mV at high pH.35 For most inorganic oxides, increasing the ionic strength reduces the absolute values of the ζ-potential, and neither K+ or NO3- is anticipated to be a potential determining ion (i.e., result in a shift in the IEP).44 The large standard deviation among the total of (44) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: New York, 1981; Chapter 7.

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Figure 2. ζ-potential, derived from mobility data for the silica particles (4) and oocysts of C. parvum (O) as a function of pH in 1, 2, and 5 mM KNO3 solutions (open, gray, and filled symbols, respectively). The error bars correspond to the standard deviation for the total of 40 velocities, measured under an applied field, at each solution condition.

40 measurements for each pH (error bars in Figure 2) makes trends in the ζ-potential resulting from the influence of ionic strength difficult to distinguish, other than that the IEP appears stationary as the ionic strength is increased. On the other hand, although the oocysts also exhibit an IEP less than pH 3, the potential at high pH is only approximately -(15-18) mV. The values in the ζ-potential have been found to be in close agreement (within the standard deviation) with earlier reports in the literature.45-47 Increasing the ionic strength has been found to decrease the ζ-potential without altering the IEP. However, the surface force measurements (see below) indicate that the surface of the oocysts of C. parvum cannot be described purely in terms of electrical double layer theory, which may have implications for the conversion of mobility to ζ-potential. Nevertheless, the measured ζ-potential maintains the consistent definition as the potential at the plane of shear. The origin of the surface charge of the oocysts is likely to be in the form of ionizable (acidic) amino acid groups, viz., dissociated carboxylic acid functionality. Other workers have characterized the oocyst surface by amino acid sequencing (polymerase chain reaction), and these investigations suggest a surfaceadherent molecule rich in cysteine, proline, and histidine.48 Calibration of Force-Separation Curves. The raw data for the interaction (on surface approach) between an oocyst of C. parvum and a silica sphere are presented in Figure 3a. The raw data have the typical form for AFM force measurements, viz., cantilever deflection measured as volts on a photodiode detector versus piezoelectric ceramic movement in nanometers. The data consists of three distinct regions: at very long range the cantilever experiences zero force and so does not deflect with piezo movement, at intermediate range the cantilever experiences repulsive surface forces and deflects nonlinearly with piezo movement, and at close range the cantilever deflects linearly with piezo movement. (45) Lytle, D. A.; Rice, E. W.; Johnson, C. H.; Owens, J. H.; Marshall, M. M. Systematic Study on the Surface Charge of Microorganisms in Drinking Water; U.S. Environmental Protection Agency, U.S. Government Printing Office: Washington, DC, 1999. (46) Drozd, C.; Schwartzbrod, J. Appl. Environ. Microbiol. 1996, 62, 1227. (47) Karaman, M. E.; Pashley, R. M.; Bustamante, H.; Shanker, S. R. Colloids Surf., A 1999, 146, 213. (48) Ranucci, L.; Muller, H.-M.; Rosa, G. L.; Reckmann, I.; Morales, M. A. G.; Spano, F.; Pozio, E. Infect. Immun. 1993, 61, 2347.

Figure 3. Typical force-separation data for the interaction between an oocyst of C. parvum (oocyst 4) and a silica particle in 1 mM KNO3 at pH ) 8.86: (a) raw experimental data, (b) normalized force data calibrated by the internal linear compliance, and (c) normalized force data calibrated by a reference hard sample. See text for discussion.

Critical to the deconvolution of the raw data to normalized force versus separation is the calibration of the deflection from volts to nanometers and the definition of zero separation. The calibration of the cantilever deflection is critical because the normalized force is obtained via Hooke’s law (F ) -K∆x) from the nanometer deflection and also because the separation axis is obtained from the sum of the piezo movement and the deflection of the cantilever. For hard, incompressible surfaces, the region of linear deflection (also known as compliance) is generally taken to be a region where the two surfaces are coupled


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such that the separation is assumed to be zero.6,8,9,36,40 Under these circumstances, the gradient of linear compliance (V nm-1) can be utilized to obtain nanometer deflection and so normalized force versus separation. The result of performing this operation on the raw data of Figure 3a is presented in Figure 3b. Note that the deconvoluted data appear to have a well-defined region of zero separation. However, on inspection of the value of the gradient of linear compliance against an oocyst (0.096 V nm-1) compared to the gradient against a silicon wafer (- 0.152 V nm-1) it becomes apparent that the deconvolution operation is inappropriate. The reduced absolute magnitude of the gradient of linear compliance against the oocyst implies that the oocyst surface may be deforming during an interaction measurement. Further, reporting normalized force as a function of separation, without reference to the gradient of linear compliance, reduces the quantitative certainty of consequent conclusions drawn from such measurements. Ducker et al.49 proposed that the cantilever deflection (in that case, for a bubble-particle interaction) could be calibrated by comparing the measured gradient of linear compliance to that of a hard surface and showed that the surface compressibility can be readily accounted for in terms of a measured interfacial spring constant by

Kint )

(

Ks

Ch -1 Cint

)

where Kint represents the effective spring constant of the interface, 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. Recently, Hartley et al.50 correlated the effective spring constant with the interfacial tension of an oil droplet interface, illustrating the usefulness of the parameter in quantifying surface compressibility. Significant error (up to 30%) was found to occur by varying the location of the laser on the back of the cantilever. Therefore, in the present study the location of the laser has been held constant (by not removing the cantilever from the AFM assembly) while the underlying surface was changed. The remaining obstacle in obtaining accurate normalized force-separation data from the experimental raw data is the definition of zero separation. For many AFM studies of biological surfaces, nanomechanical properties are studied using cantilevers of relatively high spring constant.51-53 In such studies, the “point of contact” is typically defined by overlaying the deflection data of a reference hard, incompressible surface and back calculating the indentation (and consequently the tip-sample separation). However, to sense molecular level forces, cantilevers with spring constants of approximately an order of magnitude lower than those for indentation studies must be used, and defining a single point of contact becomes difficult. In the present study, we have defined the onset of linear compliance as our reference zero (49) Ducker, W. A.; Xu, Z.; Israelachvili, J. N. Langmuir 1994, 10, 3279. (50) Hartley, P. G.; Grieser, F.; Mulvaney, P.; Stevens, G. W. Langmuir 1999, 15, 7282. (51) Braet, F.; Rotsch, C.; Wisse, E.; Radmacher, M. Appl. Phys. A 1998, 66, S1. (52) Hofmann, U. G.; Rotsch, C.; Parak, W. J.; Radmecher, M. J. Struct. Biol. 1997, 119, 84. (53) Burnham, N. A.; Colton, R. J. J. Vac. Sci. Technol., A 1989, 7, 2906.

Figure 4. Interfacial spring constant as a function of pH (a) for five different oocysts at 1 mM KNO3 and (b) for oocyst 4 at three ionic strengths.

separation. This provides a consistent definition and permits the comparison of force measurements made between surfaces of different compressibility. Therefore, to summarize, in the present study the normalized force versus separation has been obtained in the following way. The deflection (volts) has been calibrated to nanometers via the gradient of linear compliance against an external standard (silicon wafer, without moving the laser on the back of the cantilever). The normalized force has then been obtained from the nanometer deflection in the conventional manner, that is, multiplication of the measured cantilever spring constant and division by the measured harmonic mean of the two radii (oocyst and colloid probe). The separation has been obtained as the sum of the piezo movement and the nanometer cantilever deflection (calibrated as above) and zeroed according to the onset of linear compliance. A forceseparation curve for the interaction of an oocyst of C. parvum and a silica particle deconvoluted in this way is presented in Figure 3c. Interfacial Spring Constant. The interfacial spring constant for five different oocysts is presented in Figure 4a. The five oocysts have been selected at random by moving the colloid probe assembly around the wafer loaded with attached oocysts and selecting oocysts for measurement. The five oocysts have been numbered for reference and retain their designated nomenclature for subsequent discussion of the decay length, magnitude, and adhesion. Four out of the five oocysts show no systematic trend with pH, and the spring constants are below 1 N m-1 throughout the measured pH range (values for oocyst 5 varied up to

6330


Figure 5. Force-separation curve on surface approach of a C. parvum oocyst (oocyst 4 in 2 mM KNO3 at pH 8.88) and a silica particle exhibiting multiple regions of linear compliance. Solid lines are to guide the eye only.

Figure 6. Force-separation curve on surface approach of a C. parvum oocyst (oocyst 5 in 1 mM KNO3 at pH 8.86) and a silica particle exhibiting two regions of near hard-wall contact (boxed data) separated by a region of surface collapse; see text for details.

5 N m-1). The interfacial spring constant, as a function of ionic strength, for a single oocyst (oocyst 4) is presented in Figure 4b. At first glance, it appears that the influence of ionic strength is to increase the interfacial spring constant. However, the large variation in Kint for the data obtained at 5 mM KNO3 makes it difficult to make significant conclusions regarding the influence of ionic strength on the measured spring constant. Further, large variation has recently been observed upon addition of calcium nitrate.32 The variation in the interfacial spring constant may, at least in part, be due to the significant roughness of the oocyst surface. The oocyst surface is known to possess surface asperities ranging in size from approximately 50 to 250 nm in the lateral dimensions and having a vertical range of up to 50 nm (see Figure 1b). The consequence of these asperities during linear compliance may be the following: as the oocyst surface undergoes deformation, various asperities will be compressed and contribute to the macroscopic quantity Kint to varying extents depending on their geometry and individual compressibility. A series of discrete compression events would result in discontinuities in the linear compliance. Force-separation curves exemplifying such discontinuities are presented in Figures 5 and 6. The discontinuities in Figure 5 are expressed as two regions of linear compliance but of distinctly different gradients. In the boxed regions of Figure 6, it is apparent that the two surfaces approach hard-wall contact on two separate

Considine et al.

occasions, but having gone into hard-wall contact the surface temporarily collapses before returning to hardwall contact. The features of Figure 6 are similar to the force-separation data obtained for the interaction between surfactant bilayers, absorbed at hard-wall interfaces such as silica,36,54 where prior to penetration of the surfactant film hard-wall contact was obtained at the surfactant interface. To obtain a consistent basis for comparison, the gradient of linear compliance used to calculate the interfacial spring constant has been measured between 10 and 15 mN m-1 on the F/R axis. This means that the Kint values are all derived from results obtained in the same range of applied load. Interestingly, in our earlier work6 where we investigated the AFM tipoocyst interaction, only hard-wall linear compliance was observed. Presumably, the smaller radius of curvature of the tip (relative to the silica particles) resulted in sufficient pressure to penetrate the “soft” protein covering on the oocyst surface. Repulsive Surface Force: Origins. All forceseparation curves between oocysts of C. parvum and silica exhibited a repulsive force at separations prior to linear compliance, on surface approach. To investigate the origin of the repulsive force, a conventional approach has been to evoke theoretical models and obtain fits with experimental data.55 It has been shown elsewhere that the surface of C. parvum is not well approximated by DLVO theory alone and that a steric force coexists with the electrical double layer force.6 Therefore, we have attempted to fit the repulsive force with DLVO theory combined with a model of a polylectrolyte brush based principally on the developments of Pincus.17,20 In DLVO theory,56,57 it is assumed that two additive forces, electrical double layer and van der Waals, 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 oocyst-silica interaction) and H is the intersurface separation. At the long-range separations that we are primarily concerned with, the van der Waals component to the overall interaction is negligible. Many theoretical models of the force of interaction between polymer-bearing surfaces have been proposed.15-22 Calculations for charged polymers adsorbed at interfaces reveal a brushlike conformation that extends further with increasing charge density or decreasing salt concentration.18,19 To calculate the force of interaction between surfaces bearing a polyelectrolyte brush, we have utilized the theory of Pincus.17,20 Neglecting chain stiffening and excluded volume effects, Pincus quantified the interaction forces in terms of the counterion osmotic pressure as a function of separation distance H to be (54) Johnson, S. B.; Drummond, C. J.; Scales, P. J.; Nishimura, S. Langmuir 1995, 11, 2367. (55) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1992. (56) Deryagin, B. V.; Landau, L. Acta Physicochim. URSS 1941, 14, 633. (57) Verwey, E. J. W.; Overbeek, J. Th. G. Theory of the Stability of Lyophobic Colloids; Elsevier: New York, 1948.


Π≈

Langmuir, Vol. 17, No. 20, 2001 6331

2fNBkT d2H

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 is the temperature. Abraham et al.58 showed that, upon integration of this equation and use of the Derjaguin approximation, the energy of interaction between two polyelectrolyte brushes of uniform monomer concentration profile throughout the brush of width LB can be given by

F/R )

4πfkTNB 2

d

[ ]

ln

2LB H

The above equation corresponds to the interaction of two surfaces, both bearing adsorbed polymer. However, the experimental situation is that the silica surface is well described by electrical double layer theory35 and the brush must therefore be entirely located on the oocyst surface. Therefore, the parameter 2LB has been taken to be the brush width on the oocyst surface. Although the grafted interchain distance d is unknown, it has been fixed at 2.31 nm for all calculations. This value was chosen because the oocyst wall has been shown to consist of predominantly proline, cystine, and histidine;48 the largest of these amino acids is histidine, measuring 2.31 nm in the longest linear dimension predicted from the van der Waals radius of the constituent atoms. Although the value of d has been arbitrarily fixed, it is unlikely to vary as a function of pH and does permit the investigation of other parameters that describe the steric force of interaction. The value of the temperature T has also been fixed in these calculations at 298 K. The remaining terms correspond to the fraction of ionicity, f, and the number of monomers within the hydrophilic block, NB. Both of these dimensionless terms describe the chemical nature of the extended brush and are expected to be related; that is, as f increases NB will probably also increase, given the increase in charge on the polypeptide backbone. Therefore, NB and f have been combined to form NBf, where large values correspond to a brush that is composed of molecules that are highly charged and predominantly hydrophilic. The measured normalized force versus separation data for the interaction between an oocyst of C. parvum and a silica sphere, in 1 mM KNO3 at pH ) 8.86, has been presented in Figure 7. The DLVO fit has been presented as thin solid lines for the case of constant potential (lower limit) and constant charge (upper limit). The calculation has been made for the interaction of two surfaces governed by a Hamaker constant of 1 × 10-20 J and diffuse layer potentials corresponding to the measured ζ-potentials, immersed in the bulk electrolyte concentration of 1 mM KNO3. To obtain an order of magnitude fit, the origin of the plane of charge in the DLVO calculation has had to be shifted approximately 35 nm from the onset of linear compliance. The data are seen to be well described by DLVO theory at separations >35 nm with close agreement with the predicted decay length (9.5 nm). At separations 10 nm, the force of interaction is well described by the theory of Pincus (thick line). The calculation has been based on a brush width (2LB) of 50 nm with a NBf of approximately 0.3 and all other parameters fixed. For comparison, the fraction of ionicity (58) Abraham, T.; Giasson, S.; Gohy, J. F.; Je´roˆme, R. Langmuir 2000, 16, 4286.

Figure 7. Normalized force of interaction between an oocyst of C. parvum (oocyst 4 in 1 mM KNO3 at pH 8.86) and a silica sphere (O). The thin solid lines correspond to the result of the DLVO calculation based on an approximated Hamaker constant of 1 × 10-20 J, the corresponding ζ-potentials, and the bulk electrolyte concentration, with the DLVO plane of charge origin being shifted approximately 35 nm to obtain an order of magnitude fit. The solid line corresponds to the result of the force predicted from a Pincus model of a polyelectrolyte brush of width 50 nm; see text for further information. Table 1. Fitting Parameters for the DLVO and Pincus Models, for the Interaction of a C. parvum Oocyst and a Silica Particle in 1 mM KNO3 as a Function of pH, Where H∆ Denotes the DLVO Shift in Origin, 2LB Denotes the Pincus Brush Width, and NBf Denotes a Dimensionless Term Describing the Fraction of Ionicity and Relative Hydrophilicity pH

H∆ (nm)

2LB (nm)

8.86 7.25 6.76 5.81

35 ( 1 43 ( 3 34 ( 2 34 ( 3

50 ( 2 50 ( 2 44 ( 2 40 ( 2

NBf

pH

H∆ (nm)

2LB (nm)

NBf

0.30 ( 0.02 5.79 40 ( 4 46 ( 3 0.25 ( 0.01 0.28 ( 0.04 4.44 43 ( 2 42 ( 2 0.26 ( 0.02 0.29 ( 0.01 3.78 33 ( 3 30 ( 2 0.25 ( 0.07 0.26 ( 0.03

and number of monomers in the hydrophilic block have been estimated from the amino acid sequence provided by Ranucci et al.48 The constituent amino acids have been designated as ionizable (acidic or basic) and nonionizable (polar or nonpolar) according to conventional biochemical definitions.59 Grouping the total number of characterized amino acids (1252) in this way, we found that 273 were ionizable (f ) 0.2) and that the molecule was predominantly hydrophilic (58%). The overlay of the experimental data and theoretical curves presented in Figure 7 permit the diagnosis of the origin of the exponential repulsive force. At separations greater than 35 nm, the interaction is well described by electrical double layer theory. At separations between 10 and 35 nm, the interaction is well described as the collapse of a polyelectrolyte brush of width 50 nm. The implication from the theoretical modeling is that protein “hairs” extend into solution from the surface of C. parvum and act as a significant repulsive barrier. The comparison of the experimental data with the DLVO and Pincus predictions has been performed as a function of pH at 1 mM KNO3 for the interaction between a C. parvum oocyst and a silica particle. The results of the fit, in terms of the DLVO shift in origin (H∆), the Pincus brush width (2LB), and NBf, are presented in Table 1. It has been found that both the brush width and NBf parameters used to fit the Pincus model to (59) Brock, T. D. Biology of Microorganisms, 8th ed.; Madigan, M. T., Martinko, J. M., Parker, J., Eds.; Prentice Hall International: London, 1997.

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Figure 8. Normalized force of interaction between an oocyst of C. parvum (oocyst 1 in 1 mM KNO3 at pH 8.16) and a silica sphere (O). The thin solid lines correspond to the result of the DLVO calculation described in the caption of Figure 7 (only with the DLVO plane of charge origin being shifted approximately 75 nm to obtain an order of magnitude fit). The solid line corresponds to the result of the force predicted from a Pincus model of a polyelectrolyte brush of width 85 nm; see text for further information.

the experimental data decrease with pH, consistent with the hairs decharging as the surface approaches its macroscopic isoelectric point. On comparison of H∆ and 2LB, it is apparent that the values diverge at high pH. This observation is consistent with penetration of surface proteins into the electrical double layer at high pH, disrupting the well-defined regions of steric and electrical double layer interactions evidenced at low pH (i.e., for the situation where H∆ ) 2LB). Not all oocysts exhibited force-separation curves that could be fit with DLVO and Pincus predictions in the same manner as for Figure 7 and Table 1. A representative force-separation curve which could not be well described by DLVO theory is presented in Figure 8, where the origin of the plane of charge in the DLVO calculation has been shifted by 75 nm. The inability to obtain agreement with DLVO theory is probably a combined result of the significant surface roughness and extension of the oocyst surface proteins into the electrical double layer. Indeed, it is somewhat surprising that frequent agreement with DLVO theory can be obtained at all, given the complexity of the surface. To make comparisons between all oocysts, the range of the repulsive force has been analyzed in terms of the decay length (Figure 9) and magnitude (Figure 10). Repulsive Surface 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 the onset of linear compliance. The values of the measured decay length for the interaction between five different oocysts of C. parvum and a silica particle, as a function of pH, are presented in Figure 9a. Values range from approximately 35 nm (oocyst 3) to less than 10 nm (oocyst 5). It has already been shown that the origin of the repulsive force (analyzed for less than 50 nm from linear compliance) is probably a result of the compression of surface molecules extending into solution because of charge repulsion of ionizable surface groups along the molecules. The variation from oocyst to oocyst would be consistent with the surface molecules having different charge densities and steric layer thicknesses. Of all the oocysts, the most systematic trend with pH (i.e., decrease with lower pH) was exhibited by oocyst 3, which also exhibited the longest decay length at high pH. The dependence on pH may be interpreted in

Figure 9. Repulsive surface force decay length as a function of pH (a) for five different oocysts at 1 mM KNO3 and (b) for oocyst 4 at three ionic strengths.

terms of the ionizable carboxylic acid surface groups. Consistent with this interpretation is the behavior of oocyst 4 with ionic strength (1, 2, 5 mM KNO3, see Figure 9b). It is apparent that a systematic decrease in the measured decay length occurs with an increase in ionic strength. We have recently observed similar behavior of the decay length with added calcium nitrate for a number of oocysts, which will be reported in a forthcoming publication.32 For comparison with the DLVO and Pincus theories, the power law dependence of the experimentally measured decay length (1/κexp) on increasing electrolyte concentration (CS) can be obtained from the slope of a log-log plot of the decay length versus electrolyte concentration. Although only three electrolyte concentrations were assessed, the quality of the power law fit was found to be quite high (well within the error associated with the decay length) with an r2 value of 0.998 and gave the following power law dependence:

1/κexp ∝ CS-0.23 The measured exponent is significantly smaller than anticipated from DLVO theory according to the nonlinearized Poisson-Boltzmann equation, which predicts a dependence of the Debye length (1/κDebye) on the electrolyte concentration according to 1/κDebye ∝ CS-0.5. Pincus20 predicted the force between adsorbed polyelectrolyte layers to be less sensitive to Debye screening. This prediction is consistent with most of the experimental findings of the present study, where the measured decay lengths of four out of the five oocysts (oocyst 5 exhibited the Debye length)


Langmuir, Vol. 17, No. 20, 2001 6333

Figure 11. A selection of force-separation measurements on separation showing the variation of the adhesion between oocysts of C. parvum and silica: main plot, oocyst 2 at pH 6.17; inset upper curve, oocyst 4 at pH 8.86; inset lower curve, oocyst 5 at pH 9.07, all in 1 mM KNO3.

Figure 10. Repulsive surface force magnitude as a function of pH (a) for five different oocysts at 1 mM KNO3 and (b) for oocyst 4 at three ionic strengths.

have been significantly longer than the corresponding Debye length (cf. regions 35 nm for Figure 7). The measured exponent is closer to that predicted by Pincus, who calculated a dependence of the brush width on the electrolyte concentration of LB ∝ CS-0.33. The slight discrepancy in exponents may have arisen because of the complexity of the surface (e.g., heterogeneity and roughness). The behavior of the repulsive force decay length with pH and added salt yields a generalized picture of the oocyst surface. That is, oocyst surface proteins often extend into solution because of charge repulsion between ionizable groups distributed along surface molecules. As the pH is reduced, both intermolecular and intramolecular electrostatic interactions decrease, therefore reducing the range of interactions. Also, as the electrolyte concentration is increased, the intermolecular and intramolecular electrostatic interactions are screened, which may also result in a reduction in the range of interactions. Repulsive Surface Force: Magnitude. The magnitude of the repulsive force has been defined as the log linear (natural log) intercept of the force at zero separation, extrapolated from separations between 20 and 50 nm from the onset of linear compliance. The magnitude of interaction for five different oocysts is presented in Figure 10a. The values vary from approximately 5 to 10 mN m-1, and unlike the values for the interfacial spring constant and decay length, the variation from oocyst to oocyst is no larger than the variation for the standard deviation of single oocysts. Although oocysts 4 and 5 may show some slight dependence of the magnitude on pH, it is unclear

as to the significance of this trend relative to the substantial standard deviation. The magnitude has been presented as a function of ionic strength for oocyst 4 in Figure 10b. Whereas the slight dependence of the magnitude on pH for the data obtained at 1 mM KNO3 is apparent on close inspection, no such trend is distinguishable for data obtained at 2 and 5 mM KNO3. Therefore, like the interfacial spring constant, it may be that the significant surface roughness contributes to the variation in the measured magnitude of the force of interaction. Nonetheless, a comparison can be made between the experimental magnitude and the magnitude predicted for the electrical double layer interaction between an oocyst of C. parvum and a silica particle, of corresponding ζ-potential. For example, for the interaction of two surfaces having diffuse layer potentials -80 and -20 mV and interacting under the boundary condition of constant charge while immersed in a bulk electrolyte concentration of 1 mM KNO3, the resulting interaction has a corresponding magnitude value of ≈1.5 mN m-1. Therefore, although there is significant standard deviation in the measured values of the magnitude of interaction, the magnitude is always very large compared to DLVO predictions. Adhesion on Separation. Despite the repulsive surface force on approach, most oocysts of C. parvum were found to adhere to silica to varying extents. The origin of the adhesion is likely to be quite different from the origin of the repulsion observed on surface approach. As has already been discussed, the silica surface experiences a repulsive force prior to the onset of linear compliance, probably because of the compression of charged surface molecules that extend into solution. However, it has been shown that surface specific interactions are largely responsible for the adsorption of proteins from solution onto inorganic oxides.60,61 Therefore, it is likely that similar specific surface interactions may be responsible for the adhesion between oocyst surface molecules and silica, once the surfaces are in contact. Force curves exemplifying the range of adhesive behavior on separation are presented in Figure 11. The adhesion varied from negligible, in the case of oocyst 5, to attractive energy minima over 1 µm in width, in the case of oocyst 2. The gentle downturn prior to a snap to zero force (oocyst 4) is consistent with (60) Radmacher, M.; Fritz, M.; Cleveland, J. P.; Walters, D. A.; Hanmsa, P. K. Langmuir 1994, 10, 3809. (61) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368.

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molecular tethering by oocyst surface molecules to the silica surface. Quite similar behavior was observed for the oocyst-silica tip measurements reported earlier6 and also in numerous other studies of tip-sample adhesion measurements explained in terms of molecular bridging by macromolecules.60-64 The large energy minimum evident in the main plot, however, is unlikely to correspond to bridging of individual surface molecules between the two surfaces, given the relatively enormous extension of the adhesion from hard-wall contact. Therefore, it is likely that macroscopic features (most likely oocyst surface asperities) bridge the surfaces to such long separations from linear compliance. To compare the force of adhesion for various oocysts and ionic strengths, the adhesion has been analyzed in terms of the maximum extension from linear compliance HJump-out (e.g., location of arrow in Figure 11) and the corresponding force of adhesion given by

(RF)

) Adhesion

-KSHJump-out R

where Ks corresponds to the measured cantilever spring constant and R is the harmonic mean of the radii of the two surfaces. The surface forces on separation have been analyzed using the gradient of linear compliance obtained against the reference 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 HJump-out and (F/R)Adhesion are presented for five oocysts and for oocyst 4 at three ionic strengths (1, 2, and 5 mM KNO3) in Figure 12. The values of the force of adhesion apparent in Figure 12a vary widely from a few mN m-1 (oocyst 5) to hundreds of mN m-1 (oocyst 2). The variation from oocyst to oocyst is substantially larger than the variation for a single oocyst as a function of pH. Low adhesion values were obtained for oocyst 5, an oocyst which also exhibited the stiffest interfacial spring (Figure 4a) and lowest decay length (Figure 9a). On the other hand, the particularly strong adhesion exhibited by oocyst 2 is coupled with a particularly low interfacial spring constant and relatively high decay length. These observations are consistent with the hypothesis that the adhesion between the oocysts of C. parvum and the silica surface may be a result of bridging by oocyst surface molecules (either in the molecular form or incorporated as macroscopic asperities). However, on inspection of Figure 12b little correlation of the measured adhesion with ionic strength is apparent, as would be anticipated from the decay length behavior. It may be that although there is some general correlation of the adhesion, interfacial spring constant, and decay length, localized hetergeneities induce significant variability masking the subtle effects of the range of electrolyte concentrations varied here. Summary and Conclusions The experimental findings reported herein can be utilized to gain a fundamental understanding of the surface of oocysts of C. parvum and how oocysts interact with silica surfaces. The interaction is dominated by a strong repulsion on surface approach and frequently adhesion on separation, both of which are consistent with (62) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Guntherodt, H.-J.; Misevic, G. N. Science 1995, 267, 1173. (63) Chen, X.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 4106. (64) Ortiz, C.; Hadziioannou, G. Macromolecules 1999, 32, 780.

Figure 12. Normalized force of adhesion, derived from corresponding jump-out distances, as a function of pH (a) for five different oocysts at 1 mM KNO3 and (b) for oocyst 4 at three ionic strengths.

a surface that is molecularly hairy. It is known that the surface of a C. parvum oocyst generally consists of a complex protein matrix. Amino acid sequencing of a predominant surface protein reveals a predominantly hydrophilic molecule that possesses a reasonable population of ionizable surface groups. Therefore, oocyst adherent molecules may be “stretched” into solution by electrostatic repulsion between charges distributed along the molecule. Comparison with DLVO theory based on the microelectrophoresis data has revealed that the location of the ζ-potential lies either at the edge of or within the hairy layer. Comparison with the theory of Pincus reveals that the surface can be described as a polyelectrolyte brush, consistent with the notion that charged surface molecules extend into solution from the oocyst surface. As the surfaces are brought into intimate contact, the surfaces undergo compression, typically (but not always) failing to obtain hard-wall contact. The compressibility of the surface has been analyzed in terms of an interfacial spring constant, which has been found to vary from oocyst to oocyst. As the surfaces are pulled apart, oocyst surface molecules can tether the two surfaces together, presumably a result of specific surface interactions that were formed during contact. In this work, we have reported the first direct measurement of the force of interaction between C. parvum oocysts and a silica particle of well-defined geometry. The results suggest that there is a repulsive force of interaction between the surfaces and that the magnitude of the repulsive force is substantially greater than predicted from DLVO theory based on the ζ-potentials. The range of the repulsive force of interaction has been found to be shorter


in solutions of higher ionic strength, although substantial variation in the force range has been observed from oocyst to oocyst. Adhesion between the oocyst and silica surface has been measured and has been found to be little influenced by ionic strength. The findings of the present study have been used to arrive at a new fundamental understanding of the oocyst-silica interaction and also the quantitative variation that exists among oocysts. A better understanding of the surface interaction between C. parvum oocysts and siliceous materials may provide insights into how to manipulate solution conditions to

Langmuir, Vol. 17, No. 20, 2001 6335

improve oocyst detection and to develop better processes for removing the oocysts from source waters. Acknowledgment. R.C. gratefully acknowledges the support and friendship of S. Gillies. R.C. is the recipient of an Australian Post-Graduate Research Award. We also thank the University of South Australia and the Cooperative Research Centre for Water Quality and Treatment for support. LA001459+

Force of Interaction between a Biocolloid and an Inorganic Oxide

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