Investigation of the interaction force between Cryptosporidium parvum oocysts and solid surfaces

Interaction force profiles between single Cryptosporidium parvum oocysts and positively charged, silane-coated silica particles were measured in aqueous solutions using an atomic force microscope. The oocysts were immobilized for the measurements by entrapment in Millipore polycarbonate membranes with 3 microm pore size. Experiments were performed in both NaCl and CaCl2 solutions at ionic strengths ranging from 1 to 100 mM. For both electrolytes, the decay length of the repulsive force profile was found to be nearly independent of the ionic strength and always much larger than the theoretical Debye length of the system. In addition, the magnitude of the force was found to be essentially the same for both electrolytes, suggesting that the long-range repulsive forces are primarily steric in nature. These results support the theory that the interaction force between oocysts and surfaces is controlled by an outer, weakly charged or uncharged carbohydrate layer. Measurements were also performed with oocysts that had been deactivated using either chemical (formalin) or heat treatment. The force profiles obtained with formalin-treated oocysts appear to be essentially the same as for the untreated oocysts, whereas the profiles measured with the heat-treated oocysts show a much stronger dependence on solution ionic strength. With either the heat-treated or formalin-treated oocysts, adhesion was observed much more frequently than with untreated oocysts, which is consistent with the increased deposition rate observed with treated oocysts by Kuznar and Elimelech (Kuznar, Z. A.; Elimelech, M. Langmuir 2005, 21, 710-716). These results also suggest that treated oocysts, especially ones that have been inactivated by heating, may not be good surrogates for viable oocysts in laboratory studies.     

Enumeration of Cryptosporidium spp. in water with US EPA method 1622, USA

The occurrence of Cryptosporidium parvum or other pathogenic Cryptosporidium species in water must be known in order to assess risk and determine the treatment needed to reduce Cryptosporidium oocysts to acceptable levels in finished drinking water. Because Cryptosporidium oocyst occurrence may be sparse, methods must concentrate a large volume of water and correctly identify oocysts in the concentrate. The U.S. Environmental Protection Agency Information Collection Rule (ICR) protozoan method gives low and variable recoveries of Cryptosporidium oocysts, making risk assessment difficult. Therefore, a method giving better oocyst recovery and more consistent results was needed. Method 1622 was developed with existing materials and procedures, and improvements were made in filtration, cleanup, and detection. Absolute porosity filters were used, with cleanup by immunomagnetic separation and detection by direct fluorescent antibody stain with 4',6-diamidino-2-phenylindole (DAPI) staining for additional cell structures. Both the level and consistency of oocyst recovery were improved compared to recovery with the ICR method.     

DLVO and steric contributions to bacterial deposition in media of different ionic strengths

The deposition of eight bacterial strains on Teflon and glass in aqueous media with ionic strengths varying between 0.0001 and 1 M was measured and interpreted. Two types of interactions were considered: (1) those described by the DLVO theory, which comprise van der Waals attraction and electrostatic repulsion (bacteria and surfaces are both negatively charged); and (2) steric interactions between the outer cell surface macromolecules and the substrata. As a trend, at low ionic strength (<0.001 M), deposition is inhibited by DLVO-type electrostatic repulsion, but at high ionic strength (≥0.1 M) it is dominated by steric interactions. The ionic strength at which the transition from the DLVO-controlled to the sterically controlled deposition occurs, is determined by the extension of the macromolecules into the surrounding medium, which varied between 5 and 100 nm among the bacterial strains studied. The steric interactions either promote deposition by bridging or inhibit it by steric repulsion. Between Teflon and hydrophobic bacteria, bridging is generally observed. The surface of one bacterial strain contains amphiphilic macromolecules that form bridges with Teflon but induce steric repulsion on glass. The presence of highly polar anionic polysaccharide coatings on the cell impedes attachment on both glass and Teflon. For practice, the general conclusion is that the deposition of most bacteria is: (1) strongly inhibited by DLVO-type electrostatic repulsion in aqueous environments of low ionic strength such as rain water, streams and lakes; (2) controlled by DLVO and/or steric interactions in systems as domestic waste waters and saliva; and (3) determined by steric interactions only in more saline environments as milk, urine, blood and sea water.     

Study of bioadhesion on a flat plate with a yeast/glass model system

The attachment of microorganisms to a surface is a critical first step of biofilm fouling in membrane processes. The shear-induced detachment of baker's yeast in adhesive contact with a plane glass surface was thus experimentally studied, using a specially designed shear stress flow chamber. The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast). A complete series of experiments for measuring the shear stress necessary to detach a given proportion of individual yeast cells of type I or II was performed under different environmental conditions (ionic strength, contact time). In parallel, the surface physicochemical properties of the cells (surface charge, hydrophobicity, and electron donor and electron acceptor components) were determined. For the first type of yeast cells, which were rather hydrophilic, adhesion to the glass plate was weak. This was due to both electrostatic effects and hydrophilic repulsion. Furthermore, adhesion was not sensitive to any variation of the ionic strength. For yeast of the second type, adhesion was drastically increased. This could be explained by their physicochemical surface properties and especially their hydrophobic and electron acceptor components, which caused strong attractive van der Waals and Lewis acid-base interactions, counterbalancing the electrostatic repulsion. For increasing ionic strengths, adhesion was greater, due to lower electrostatic repulsion. The results were quantified through the definition of a critical wall shear stress ( tau w 50% ) required to detach 50% of the yeast cells initially deposited on the glass surface. The influence of the contact time was also evaluated and it was shown that, whatever the type of yeast, macromolecules such as proteins were released into the extracellular medium due to cell lysis and could contribute to the formation of a conditioning film. As a result, the cells were more strongly stuck to the glass plate.     

Specific binding of different vesicle populations by the hybridization of membrane-anchored DNA

We demonstrate a method of heterogeneous vesicle binding using membrane-anchored, single-stranded DNA that can be used over several orders of magnitude in vesicle size, as demonstrated for large 100 nm vesicles and giant vesicles several microns in diameter. The aggregation behavior is studied for a range of DNA surface concentrations and solution ionic strengths. Three analogous states of aggregation are observed on both vesicle size scales. We explain the existence of these three regimes by a combination of DNA binding favorability, vesicle collision kinetics, and lateral diffusion of the DNA within the fluid membrane. The reversibility of the DNA hybridization allows dissociation of the structures formed and can be achieved either thermally or by a reduction in the ionic strength of the external aqueous environment. Difficulty is found in fully unbinding giant vesicles by thermal dehybridization, possibly frustrated by the attractive van der Waals minimum in the intermembrane potential when brought into close contact by DNA binding. This obstacle can be overcome by the isothermal reduction of the ionic strength of the solution: this reduces the Debye screening length, coupling the effects of DNA dehybridization and intermembrane repulsion due to the increased electrostatic repulsion between the highly charged DNA backbones.     

Multiscale-linking simulation of irreversible colloidal deposition in the presence of DLVO interactions

An efficient multiscale-linking algorithm, based on the self-consistent integration of Brownian dynamics simulation of particle trajectories with the solution of the continuum-level conservation equation for particle concentration subject to an adaptive Neumann boundary condition that accounts for the blocking effect of deposition, is developed. The algorithm has been already validated in the case of deposition of noninteracting hard spheres [R.V. Magan, R. Sureshkumar, Multiscale Model. Simul. 2 (2004) 475]. In this study, the above algorithm is extended to incorporate particle interactions modeled by the DLVO theory. The simulations are used to identify a time scale at which the deposition process transitions from a power-law to an asymptotic regime. Detailed characterization of the two regimes is provided for a wide range of ionic strength, particle surface charge density, bulk volume fraction, and substrate potential values. The radial distribution functions obtained for various ionic strengths can be collapsed into a master curve when the radial distance is normalized with respect to a characteristic length scale of inter-particle repulsion. Moreover, simulation results suggest a rescaled, uniformly valid soft random sequential adsorption (RSA) model. Simulation results for the kinetics and monolayers structure compare favorably with experimental data, without the use of adjustable parameters. Comparison with other dynamic simulation techniques shows that while their predictions are qualitatively similar, notable quantitative differences exist especially for small ionic strengths.     

Microelectrophoresis of Cryptosporidium parvum oocysts in aqueous solutions of inorganic and surfactant cations

Cryptosporidium parvum is a protozoan parasite associated with waterborne outbreaks of diarrhoeal disease. The life cycle of this parasite includes the production of a spheroidal oocyst that is of 4–6 microns in diameter. The thickness of the oocyst wall and its capacity to strongly adhere to both organic and inorganic surfaces are features of the oocysts which could be attributed to its survival in the environment for extended periods. Hence, the need to study their surface chemistry in the aqueous environment. The surface charging properties of the intact C. parvum oocysts were derived from microelectrophoresis measurements on these robust biological species. The ζ potentials of Cryptosporidium oocysts were measured in a range of inorganic electrolyte solutions and in solutions of a multivalent cationic surfactant. The surface potential of the oocyst was found to be pH dependent, with an isoelectric point in mM NaCl of ∼2, suggesting the presence of surface carboxylate groups associated with glycoproteins or phosphate groups. The area/charge for the fully ionised oocysts was found to be ∼80 nm², corresponding to a total maximum charge of 1.6×10⁻¹³ C per oocyst. The effect of a highly charged novel cage surfactant known as CS12 on the Cryptosporidium oocyst surface potential provided valuable insight into its uptake and possible surface activity. Uptake of CS12 was detected at concentrations as low as 2×10⁻⁸ M. At ∼2×10⁻⁵ M CS12 the oocyst surface was uncharged and became positively charged at higher concentrations. These findings suggest that there could be improvements to current concentration methods by manipulation of the surface charge.     

Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged-but otherwise apparently inert-surface alters the folding free energy of a simple biomolecule. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxylalkane-coated gold surface. We find that whereas surface attachment is destabilizing at low ionic strength, it becomes stabilizing at ionic strengths above ~130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis, we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate.     

Influence of natural organic matter on the deposition kinetics of extracellular polymeric substances (EPS) on silica

The influence of humic acid and alginate, two major components of natural organic matter (NOM), on deposition kinetics of extracellular polymeric substances (EPS) on silica was examined in both NaCl and CaCl(2) solutions over a wide range of environmentally relevant ionic strengths utilizing a quartz crystal microbalance with dissipation. Deposition kinetics of both soluble EPS and bound EPS extracted from four bacterial strains with different characteristics was investigated. EPS deposition on humic acid-coated silica surfaces was found to be much lower than that on bare silica surfaces under all examined conditions. In contrast, pre-coating the silica surfaces with alginate enhanced EPS deposition in both NaCl and CaCl(2) solutions. More repulsive electrostatic interaction between EPS and surface contributed to the reduced EPS deposition on humic acid-coated silica surface. The trapping effect induced by the rough alginate layer resulted in the greater EPS deposition on alginate-coated surfaces in NaCl solutions, whereas surface heterogeneities on alginate layer facilitated favorable interactions with EPS in CaCl(2) solutions. The presence of dissolved background humic acid and alginate in solutions both significantly retarded EPS deposition on silica surfaces due to the greater steric and electrostatics repulsion.     

Atomic force microscopy study of the adsorption and electrostatic self-organization of poly(amidoamine) dendrimers on mica

The adsorption behavior of poly(amidoamine) dendrimers to mica surfaces was investigated as a function of ionic strength and pH. The conformation and lateral distribution of the adsorbed dendrimers of generations G8 and G10 were obtained ex situ by tapping mode atomic force microscopy (AFM). The deposition kinetics of the dendrimers was found to follow a diffusion-limited process. Fractional surface coverage and pair correlation functions of the adsorbed dendrimers were obtained from the AFM images. The data are interpreted in terms of the random sequential adsorption (RSA) model, where electrostatic repulsion due to overlapping double layers is considered. Although the general trends typical for an RSA-determined process are well-reproduced, quantitative agreement is lacking at low ionic strengths.     

QCM study of the adsorption of polyelectrolyte covered mesoporous TiO2 nanocontainers on SAM modified Au surfaces

Mesoporous TiO(2) nanocontainers (NCs) covered with polyelectrolyte multilayers were adsorbed on self-assembled monolayer (SAM) modified gold substrates at different values of pH and ionic strength. The adsorption process was followed in situ by means of a quartz crystal microbalance (QCM) and the morphology of the adsorbate was investigated by means of FE-SEM images taken of the substrates after each adsorption process. Deposition could be achieved if either the particles and the surface had opposite charge, or if the salt concentration was sufficiently high, reducing the repulsion between the spheres and the surface. In the latter case the adsorption kinetics could be explained in the context of the DLVO-theory. Using conditions of like charges, one has a means to control the speed of deposition by means of ionic strength. However, interparticle aggregation and cluster deposition on the surface were observed at high ionic strength. Such conditions have to be avoided to obtain a uniform deposition of separated nanocontainers on the surface.     

Amphiphile disruption of pathogen attachment at the hematite (α-Fe2O3)-water interface

Prior studies have indicated that the subsurface transport of Cryptosporidium parvum oocysts is diminished in sediments containing iron oxides and that inner-sphere complexation of oocyst surficial carboxylate plays a role in the retardation. However, the impacts of natural organic matter (NOM) remain poorly understood. In this study, we used a model anionic surfactant, sodium dodecyl sulfate (SDS), as a surrogate for amphiphilic NOM components to examine the impacts of amphiphilic components on oocyst adhesion mechanisms. We employed in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to determine the effects of SDS on the molecular bonds that mediate interactions between oocyst surficial biomolecules and hematite (α-Fe(2)O(3)) surface functional groups over a wide range of solution pH. The results show that the presence of SDS significantly diminishes Fe-carboxylate complexation, as indicated by progressive decreases in intensity of asymmetric and symmetric stretching vibrations of carboxylate [ν(as)(COO(-)) and ν(s)(COO(-))] with reaction time. In addition, one of the ν(s)(COO(-)) bands shifted from 1370 to 1418 cm(-1) upon SDS introduction, suggesting that SDS also changed the complexation mode. The data indicate that competition from the sulfonate groups (OSO(3)(-)) of SDS at α-Fe(2)O(3) surface sites is a primary mechanism resulting in decreased Fe-carboxylate complexation. Sorptive competition from amphiphilic NOM components may therefore increase the mobility of C. parvum oocysts in the environment through disruption of interfacial pathogen-mineral surface bonds.     

Role of Cell Surface Lipopolysaccharides in Escherichia coli K12 adhesion and transport

The influence of bacterial surface lipopolysaccharides (LPS) on cell transport and adhesion has been examined by use of three mutants of Escherichia coli K12 with well-characterized LPS of different lengths and molecular composition. Two experimental techniques, a packed-bed column and a radial stagnation point flow system, were employed to investigate bacterial adhesion kinetics onto quartz surfaces over a wide range of solution ionic strengths. Although the two systems capture distinct deposition (adhesion) mechanisms because of their different hydrodynamics, similar deposition kinetics trends were observed for each bacterial strain. Bacterial deposition rates were directly related to the electrostatic double layer interaction between the bacteria and quartz surfaces, in qualitative agreement with classic Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. However, DLVO theory does not fully explain the deposition behavior for the bacterial strain with the lengthy, uncharged O-antigen portion of the LPS. Neither the length nor the charge characteristics of the LPS molecule directly correlated to deposition kinetics, suggesting a complex combination of cell surface charge heterogeneity and LPS composition controls the bacterial adhesive characteristics. It is further suggested that bacterial deposition behavior is determined by the combined influence of DLVO interactions, LPS-associated chemical interactions, and the hydrodynamics of the deposition system.     

Phase behavior of aqueous solutions containing dipolar proteins from second-order perturbation theory

Due to the interplay of Coulombic repulsion and attractive dipolar and van der Waals interactions, solutions of globular proteins display a rich variety of phase behavior featuring fluid-fluid and fluid-solid transitions that strongly depend on solution pH and salt concentration. Using a simple model for charge, dispersion and dipole-related contributions to the interprotein potential, we calculate phase diagrams for protein solutions within the framework of second-order perturbation theory. For each phase, we determine the Helmholtz energy as the sum of a hard-sphere reference term and a perturbation term that reflects both the electrostatic and dispersion interactions. Dipolar effects can induce fluid-fluid phase separation or crystallization even in the absence of any significant dispersion attraction. Because dissolved electrolytes screen the charge-charge repulsion more strongly than the dipolar attraction, the ionic strength dependence of the potential of mean force can feature a minimum at intermediate ionic strengths offering an explanation for the observed nonmonotonic dependence of the phase behavior on salt concentration. Inclusion of correlations between charge-dipole and dipole-dipole interactions is essential for a reliable calculation of phase diagrams for systems containing charged dipolar proteins and colloids.     

Energetics of lysozyme adsorption on mesostructured cellular foam silica: effect of salt concentration

The heat of lysozyme adsorption on mesostructured cellular foam (MCF) silica was measured using flow microcalorimetry (FMC) to investigate the influence of a neutral salt, sodium sulfate. At concentrations up to 0.5 M sodium sulfate, a complex initial exotherm was followed by an endotherm. Protein surface coverage, the magnitudes of the exothermic heat signals and the magnitudes of the net heat of adsorption increased with sodium sulfate concentration. These observations suggest that electrostatic interactions are the principal driving force at low ionic strengths; van der Waals interactions become dominant at higher salt concentrations. Each exotherm could be deconvoluted into two exotherms, indicating multiple modes of lysozyme attachment to the silica surface. The endothermic peak, associated with protein desorption, disappeared at the highest sodium sulfate concentration (1.0 M), indicating irreversible adsorption of the protein on the MCF silica surface. The data are consistent with an adsorption mechanism in which the initial attachment of lysozyme to the surface is followed by a reorientation and formation of a secondary or stronger attachment to the surface.     

Amphiphilic derivatives of dextran: adsorption at air/water and oil/water interfaces

Ionic amphiphilic dextran derivatives were synthesized by the attachment of sodium sulfopropyl and phenoxy groups on the native polysaccharide. A family of dextran derivatives was thus obtained with varying hydrophobic content and charge density in the polymer chains. The surface-active properties of polymers were studied at the air-water and dodecane-water interfaces using dynamic surface/interfacial tension measurements. The adsorption was shown to begin in a diffusion-limited regime at low polymer concentrations, that is to say, with the diffusion of macromolecules in the bulk solution. In contrast, at long times the interfacial adsorption is limited by interfacial phenomena: adsorption kinetics or transfer into the adsorbed layer. A semiempirical equation developed by Filippov was shown to correctly fit the experimental curves over the whole time range. The presence of ionic groups in the chains strongly lowers the adsorption kinetics. This effect can be interpreted by electrostatic interactions between the free molecules and the already adsorbed ones. The adsorption kinetics at air-water and oil-water interfaces are compared.     

Adsorption of poly(amido amine) (PAMAM) dendrimers on silica: importance of electrostatic three-body attraction

Adsorption of poly(amido amine) (PAMAM) dendrimers to silicon oxide surfaces was studied as a function of pH, ionic strength, and dendrimer generation. By combining optical reflectometry and atomic force microscopy (AFM), the adsorbed layers can be fully characterized and an unequivocal determination of the adsorbed mass becomes possible. For early stages, the adsorption process is transport limited and of first order with respect to the dendrimer solution concentration. For later stages, the surface saturates and the adsorbed dendrimers form loose but correlated liquidlike surface structures. This correlation is evidenced by a peak in the pair correlation function determined by AFM. The maximum adsorbed amount increases with increasing ionic strength and pH. The increase with the ionic strength is explained by the random sequential adsorption (RSA) model and electrostatic repulsion between the dendrimers. The adsorbing dendrimers interact by the repulsive screened Coulomb potential, whose range decreases with increasing ionic strength and thus leads to increasing adsorbed densities. The pH increase is interpreted as an effect of the substrate and is quantitatively explained by the extended three-body RSA model. This model stipulates the importance of a three-body interaction acting between two adsorbing dendrimers and the charged substrate. The presence of the charged substrate weakens the repulsion between the adsorbing dendrimers and thus leads to higher surface densities. This effect can be interpreted as an additional attractive three-body interaction, which acts in addition to the usual two-body repulsion and originates from the additional screening of the Coulomb repulsion by the counterions accumulating in the diffuse layer.     

Bacterial attachment over a wide range of ionic strengths

The number of bacterial cells adhered on a glass surface was counted over a wide range of ionic strengths. The counted number increased linearly with the square root of time. The rate of attachment increased with the increase in ionic strength and then plateaued. The rate of attachment was analyzed on the basis of the potential barrier between the surface of the bacterial cell and that of the substratum. An equation for formulating the dependence of the attachment rate on the ionic strength was proposed, which seems to be useful for the systematic understanding of bacterial attachment in various environments, from terrestrial to marine.