Specific and nonspecific interaction forces between Escherichia coli and silicon nitride, determined by poisson statistical analysis

The nature of the physical interactions between Escherichia coli JM109 and a model surface (silicon nitride) was investigated in water via atomic force microscopy (AFM). AFM force measurements on bacteria can represent the combined effects of van der Waals and electrostatic forces, hydrogen bonding, steric interactions, and perhaps ligand-receptor type bonds. It can be difficult to decouple these forces into their individual components since both specific (chemical or short-range forces such as hydrogen bonding) and nonspecific (long-range colloidal) forces may be present in the overall profiles. An analysis is presented based on the application of Poisson statistics to AFM adhesion data, to decouple the specific and nonspecific interactions. Comparisons with classical DLVO theory and a modified form of a van der Waals expression for rough surfaces were made in order to help explain the nature of the interactions. The only specific forces in the system were due to hydrogen bonding, which from the Poisson analysis were found to be -0.125 nN. The nonspecific forces of 0.155 nN represent an overall repulsive interaction. These nonspecific forces are comparable to the forces calculated from DLVO theory, in which electrostatic-double layer interactions are added to van der Waals attractions calculated at the distance of closest approach, as long as the van der Waals model for "rough" spherical surfaces is used. Calculated electrostatic-double layer and van der Waals interactions summed to 0.116 nN. In contrast, if the classic (i.e., smooth) sphere-sphere model was used to predict the van der Waals forces, the sum of electrostatic and van der Waals forces was -7.11 nN, which appears to be a large overprediction. The Poisson statistical analysis of adhesion forces may be very useful in applications of bacterial adhesion, because it represents an easy way to determine the magnitude of hydrogen bonding in a given system and it allows the fundamental forces to be easily broken into their components.     

The role of growth temperature in the adhesion and mechanics of pathogenic L. monocytogenes: an AFM study

The adhesion strengths of pathogenic L. monocytogenes EGDe to a model surface of silicon nitride were quantified using atomic force microscopy (AFM) in water for cells grown under five different temperatures (10, 20, 30, 37, and 40 °C). The temperature range investigated was chosen to bracket the thermal conditions in which L. monocytogenes survive in the environment. Our results indicated that adhesion force and energy quantified were at their maximum when the bacteria were grown at 30 °C. The higher adhesion observed at 30 °C compared to the adhesion quantified for bacterial cells grown at 37, 40, 20, and 10 °C was associated with longer and denser bacterial surface biopolymer brushes as predicted from fitting a model of steric repulsion to the approach distance-force data as well from the results of protein colorimetric assays. Theoretically predicted adhesion energies based on soft-particle DLVO theory agreed well with the adhesion energies computed from AFM force-distance retraction data (r(2) = 0.94); showing a minimum energy barrier to adhesion at 30 °C.     

Analysis of bacterial adhesion using a gradient force analysis method and colloid probe atomic force microscopy

The atomic force microscope (AFM) has been used to examine the stickiness of bacteria on the basis of the analysis of approach and retraction force curves between the AFM tip and the bacterial surface. One difficulty in analyzing approach curve data is that the distance between the AFM tip and the surface of the bacterium is difficult to define. The exact distances are difficult to determine because the surface of the bacterium deforms during force imaging, producing a highly nonlinear region in the approach curve. In this study, AFM approach and retraction curves were obtained using a colloid probe AFM for three strains of Escherichia coli (D21, D21f2, and JM109). These strains differed in their relative adhesion to glass surfaces, on the basis of measurements of sticking coefficients in packed bed flow through column tests. A gradient force curve analysis method was developed to model the interactions between the colloid probe and a surface. Gradient analysis of the approach curve revealed four different regions of colloid-surface interactions during the approach and contact of the probe with the bacterial surface: a noninteraction region, a noncontact phase, a contact phase, and a constant compliance region. The noncontact phase, which ranged from 28 to 59 nm for the three bacterial strains, was hypothesized to arise primarily from steric repulsion of the colloid by extracellular polymers on the bacterial surface. The contact phase, spanning 59-113 nm, was believed to arise from the initial pressure of the colloid on the outer membrane of the cell. The constant compliance region likely reflected the response of the colloid probe to the stiff peptidoglycan layer that confers strength and rigidity to gram negative bacteria. It was shown that the sticking coefficients reported for the three E. coli strains were correlated with the length of the noncontact phase but not the properties of the other phases. Sticking coefficients were also not correlated with any parameters determined from retraction force curves such as pull-off distances or separation energies. These results show that gradient analysis is useful for studying the contribution of the length of the exopolymers on the cell surface to bacterial adhesion to glass surfaces.     

Determination of interaction strength between corrole and phenol derivatives in aqueous media using atomic force microscopy

Atomic force microscopy (AFM) was used to measure single interaction forces between corrole (host) and phenol derivatives (guests) in aqueous media. A gold tip was modified with thiol derivatives of corrole via the Au–S covalent bond. Such a tip was used to measure adhesion forces with a planar gold substrate modified with thiol derivatives of phenol and ortho-nitrophenol in aqueous solutions. The mean force between the corrole and ortho-nitrophenol was higher than that between corrole and phenol, probably reflecting stronger hydrogen bond interaction in the former complex. In the presence of a supporting electrolyte (0.1 M K₂SO₄), the mean force increased, suggesting that electrostatic and π–π interactions play an essential role in the adhesion force. In addition, the adhesion force measured at pH 6.0 was larger than that at pH 10, reflecting the electrostatic repulsion at the higher pH. These behaviours are consistent with the potentiometric responses of a liquid membrane based on corrole to phenolic compounds. Also, the values of forces for the interaction between corrole and phenol derivatives showed the same tendency as energy calculated for these complexes. The Poisson method was used for the calculation of the single force of the chemical bond between the corrole host and the phenolic guests.     

Importance of LPS structure on protein interactions with Pseudomonas aeruginosa

Atomic force microscopy (AFM) was used to quantify the adhesion forces between Pseudomonas aeruginosa PAO1 and AK1401, and a representative model protein, bovine serum albumin (BSA). The two bacteria strains differ in terms of the structure of their lipopolysaccharide (LPS) layers. While PAO1 is the wild-type expressing a complete LPS and two types of saccharide units in the O-antigen (A(+) B(+)), the mutant AK1401 expresses only a single unit of the A-band saccharide (A(+) B(-)). The mean adhesion force (F(adh)) between BSA and AK1401 was 1.12 nN, compared to 0.40 nN for F(adh) between BSA and PAO1. In order to better understand the fundamental forces that would control bacterial-protein interactions at equilibrium conditions, we calculated interfacial free energies using the van Oss-Chaudhury-Good (VCG) thermodynamic modeling approach. The hydrogen bond strength was also calculated using a Poisson statistical analysis. AK1401 has a higher ability to participate in hydrogen bonding with BSA than does PAO1, which may be because the short A-band and absence of B-band polymer allowed the core oligosaccharides and lipid A regions to be more exposed and to participate in hydrogen and chemical bonding. Interactions between PAO1 and BSA were weak due to the dominance of neutral and hydrophilic sugars of the A-band polymer. These results show that bacterial interactions with protein-coated surfaces will depend on the types of bonds that can form between bacterial surface macromolecules and the protein. We suggest that strategies to prevent bacterial colonization of biomaterials can focus on inhibiting these bonds.     

Force measurements of bacterial adhesion on metals using a cell probe atomic force microscope

The adhesion of microbial cells to metal surfaces in aqueous media is an important phenomenon in both the natural environment and engineering systems. The adhesion of two anaerobic sulfate-reducing bacteria (Desulfovibrio desulfuricans and a local marine isolate) and an aerobe (Pseudomonas sp.) to four polished metal surfaces (i.e., stainless steel 316, mild steel, aluminum, and copper) was examined using a force spectroscopy technique with an atomic force microscope (AFM). Using a modified bacterial tip, the attraction and repulsion forces (in the nano-Newton range) between the bacterial cell and the metal surface in aqueous media were quantified. Results show that the bacterial adhesion force to aluminum is the highest among the metals investigated, whereas the one to copper is the lowest. The bacterial adhesion forces to metals are influenced by both the electrostatic force and metal surface hydrophobicity. It is also found that the physiological properties of the bacterium, namely the bacterial surface charges and hydrophobicity, also have influence on the bacteria-metal interaction. The adhesion to the metals by Pseudomonas sp. and D. desulfuricans was greater than by the marine SRB isolate. The cell-cell interactions show that there are strong electrostatic repulsion forces between bacterial cells. Cell probe atomic force microscopy has provided some useful insight into the interactions of bacterial cells with the metal surfaces.     

Streptococcus mutans and Streptococcus intermedius adhesion to fibronectin films are oppositely influenced by ionic strength

Bacterial adhesion to protein-coated surfaces is mediated by an interplay of specific and nonspecific interactions. Although nonspecific interactions are ubiquitously present, little is known about the physicochemical mechanisms of specific interactions. The aim of this paper is to determine the influence of ionic strength on the adhesion of two streptococcal strains to fibronectin films. Streptococcus mutans LT11 and Streptococcus intermedius NCTC11324 both possess antigen I/II with the ability to bind fibronectin from solution, but S. intermedius binds approximately 20x less fibronectin than does the S. mutans strain under identical conditions. Both strains as well as fibronectin films are negatively charged in low ionic strength phosphate buffered saline (PBS, 10x diluted), but bacteria appear uncharged in high ionic strength PBS. Physicochemical modeling on the basis of overall cell surface properties (cell surface hydrophobicity and zeta potentials) demonstrates that both strains should favor adhesion to fibronectin films in a high ionic strength environment as compared to in a low ionic strength environment, where electrostatic repulsion between equally charged surfaces is dominant. Adhesion of S. intermedius to fibronectin films in a parallel plate flow chamber was completely in line with this modeling, while in addition atomic force microscopy (AFM) indicated stronger adhesion forces upon retraction between fibronectin-coated tips and the cell surfaces in high ionic strength PBS than in low ionic strength PBS. Thus, the dependence of the interaction on ionic strength is dominated by the overall negative charge on the interacting surfaces. Adhesion of S. mutans to fibronectin films, however, was completely at odds with theoretical modeling, and the strain adhered best in low ionic strength PBS. Moreover, AFM indicated weaker repulsive forces upon approach between fibronectin-coated tips and the cell surfaces in low ionic strength PBS than in high ionic strength PBS. This indicated that the dependence of the interaction on ionic strength is dominated by electrostatic attraction between oppositely charged, localized domains on the interacting surfaces, despite their overall negative charge. In summary, this study shows that physicochemical modeling of bacterial adhesion to protein-coated surfaces is only valid provided the number of specific interaction sites on the cell surfaces is low, such as on S. intermedius NCTC11324. Nonspecific interactions are dominated by specific interactions if the number of specific interaction sites is large, such as on S. mutans LT11. Its ionic strength dependence indicates that the specific interaction is electrostatic in nature and operative between oppositely charged domains on the interacting surfaces, despite the generally overall negatively charged character of the surfaces.     

Simple test system for single molecule recognition force microscopy

We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG₈₀₀ diamine was glutarylated, the mono-adduct NH₂-PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy.     

Direct Quantification of Aspergillus niger Spore Adhesion in Liquid Using an Atomic Force Microscope

An atomic force microscope has been used to quantify directly the adhesion between single Aspergillus niger spores and freshly cleaved mica surfaces. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. Adhesion was quantified from force-distance data for the retraction of the spore from the surface. Studies in NaCl solutions over a range of pH and electrolyte concentration showed that the decrease of long-range electrostatic repulsion with decreasing pH provided a contribution in increasing the overall adhesion, but the variation of such repulsion with ionic strength did not correlate with changes in the magnitude of adhesion. Specific interactions between appendages and protusions on the spore surface must play an important role in adhesion. The AFM spore probe technique provides a useful new method for evaluating the interactions of spores and surfaces. It has the potential to become a powerful asset for both fundamental studies and the assessment of new materials with low adhesion properties. Copyright 2000 Academic Press.     

Impact of ionic strength of growth on the physiochemical properties, structure, and adhesion of Listeriamonocytogenes polyelectrolyte brushes to a silicon nitride surface in water

The adhesion energies between pathogenic Listeriamonocytogenes EGDe to a model surface of silicon nitride were quantified using atomic force microscopy (AFM) in water for cells grown in pure media (as the control) and in media of four different ionic strengths of added NaCl (IS of 0.05 M, 0.1 M, 0.3 M and 0.5 M NaCl). The physiochemical properties of L. monocytogenes EGDe surface brushes were shown to have a strong influence on the adhesion of the microbe to the silicon nitride surface. The transitions in the adhesion energies, physiochemical properties, and the structure of bacterial surface polyelectrolyte brushes were observed for the cells grown in the media of 0.1 M added NaCl. Our results suggested that the highest long-range electrostatic repulsion which was partially balanced by the Liftshitz-van der Waals attraction for the cells grown at 0.1 M was responsible for the highest energy barrier to adhesion for these cells as predicted by the soft-particle analysis of DLVO theory and the lower adhesion measured by AFM.     

Thermodynamic Investigation of Staphylococcus epidermidis interactions with protein-coated substrata

We evaluated self-assembled monolayers (SAMs) as potential coatings to prevent bacterial adhesion to biomaterials. Bacterial retention experiments were conducted on SAMs, some of which were coated with the model proteins fetal bovine serum (FBS) and fibronectin (FN). A thermodynamic approach was applied to calculate the Gibbs free energy changes of adhesion (DeltaG(adh)) of Staphylococcus epidermidis interacting with the substrates. When only nonspecific interactions controlled bacterial attachment, such as for the non-protein-coated substrates or the FBS substrates, the correlation between the thermodynamic predictions and measured values of bacterial retention was strong. However, when FN was adsorbed to the surfaces, the thermodynamic modeling underestimated bacterial adhesion, presumably since specific interactions between proteins of S. epidermidis and FN led to stronger attachment. Bacterial viability on the substrates was correlated with thermodynamic properties. For example, although bacteria attached more to surfaces having negative DeltaG(adh) values, these cells experienced the greatest loss of viability, presumably since strongly attached bacteria were unable to divide and grow. When the DeltaG(adh) values were decoupled into their components, we saw that acid-base interactions due to hydrogen bonding dominated the interactions of bacteria and proteins with each other and with the substrates in aqueous media. Finally, we discuss concerns regarding the use of the thermodynamic model to predict bacterial adhesion behavior in biomaterials systems.     

A method for calculating the Gibbs energy of nonspecific solvation

A method for calculating the Gibbs energy of nonspecific solvation of nonelectrolytes was suggested. The new equation for the Gibbs energy of nonspecific solvation contains one solvent parameter that characterize nonspecific solvent-solute interactions and two experimental Gibbs energies of solvation in two standard solvents. The method is applicable to a wide range of solutes and solvents. It was successfully used to describe some 800 Gibbs energies of solvation for systems without specific solvent-solute interactions.     

The role of polymer compatibility in the adhesion between surfaces saturated with modified dextrans

Wet and dry adhesion between dextran-coated surfaces were measured aiming to understand the influence of polymer compatibility. The wet adhesion measurements were performed using the atomic force microscope (AFM) colloidal probe technique whereas the dry adhesion measurements were performed using the micro adhesion measurement apparatus (MAMA). Two types of dextrans were used, one cationically modified dextran (DEX) and one that was both cationically and hydrophobically modified (HDEX), leading to three different combinations of polymer-coated surfaces; (1) DEX:DEX, (2) HDEX:DEX, and (3) HDEX:HDEX. DEX increased dry adhesion more than HDEX did, which likely is due to differences in the ability to form specific interactions, especially hydrogen bonding. HDEX gave strong wet adhesion, probably due to its poorer solvency, while DEX contributed to reducing the wet adhesion due to its hydrophilicity. All combinations showed a steric repulsion on approach in aqueous media. Furthermore, when HDEX was adsorbed on either or both surfaces a long range attractive force between the surfaces was detected outside this steric regime.     

Adsorption of ions onto polymer surfaces and its influence on zeta potential and adhesion phenomena

 The adhesion behavior that governs many technologically and biologically relevant polymer properties can be investigated by zeta potential measurements with varied electrolyte concentration or pH. In a previous work [1] it was found that the difference of the adsorption free energies of Cl^- and K⁺ ions correlates with the adhesion force caused by van der Waals interactions, and that the decrease of adhesion strength by adsorption layers can be elucidated by zeta potential measurements. In order to confirm these interrelations, zeta potential measurements were combined with atomic force microscopy (AFM) measurements. Force–distance curves between poly(ether ether ketone) and fluorpolymers, respectively, and the Si₃N₄ tip of the AFM device in different electrolyte solutions were measured and analysed. The adsorption free energy of anions calculated from the Stern model correlates with their ability to prevent the adhesion between the polymer surface and the Si₃N₄ tip of the AFM device. These results demonstrate the influence of adsorption phenomena on the adhesion behavior of solids. The results obtained by AFM confirm the thesis that the electrical double layer of solid polymers in electrolyte solutions is governed by ion adsorption probably due to van der Waals interactions and that therefore van der Waals forces can be detected by zeta potential measurements. Received: 18 November 1997 Accepted: 19 January 1998     

Colloidal properties and specific interactions of bacterial surfaces

The research field of bacterial interactions, which is characterized by the apparent dichotomy between colloidal surface properties and specific interactions, has seen significant recent progress along both lines. In the colloidal approach, the main emphasis has been on the quantification of bacterial interaction forces, including a comparison with physico-chemical theory, the probing of bacterial surface heterogeneity using atomic force microscopy (AFM) and a focus on irreversible interactions and bond aging. In parallel, the biophysical aspects of specific interactions have been partially elucidated using force spectroscopy.     

Evaluating protein attraction and adhesion to biomaterials with the atomic force microscope

Failure of implanted biomaterials is commonly due to nonspecific protein adsorption, which in turn causes adverse reactions such as the formation of fibrous capsules, blood clots, or bacterial biofilm infections. Current research efforts have focused on modifying the biomaterial interface to control protein reactions. Designing biomaterial interfaces at the molecular level, however, requires an experimental technique that provides detailed, dynamic information on the forces involved in protein adhesion. The goal of this study was to develop an atomic force microscope (AFM)-based technique to evaluate protein adhesion on biomaterial surfaces. In this study, the AFM was used to evaluate (i) protein-protein, (ii) protein-substrate, and (iii) protein-dextran interactions. The AFM was first used to measure the pull-off forces between bovine serum albumin (BSA) tips/BSA surfaces and BSA tips/anti-BSA surfaces. Results from these protein-protein studies were consistent with the literature. More importantly, the successful measurement of antibody-antigen binding interactions demonstrates that both the BSA and anti-BSA proteins retain their folded conformation and remain functional following our immobilization protocol. The AFM was also used to quantify the physiochemical interactions of proteins during adhesion to various self-assembled monolayers (SAMs) and dextran-coated substrates representative of potential biomaterial interface modifications. Dextran, which renders surfaces very hydrophilic, was the only surface coating that BSA protein did not adhere to. Hydrophobic interactions were not found to play a significant role in BSA adhesion. Therefore, the dextran molecules may resist protein adhesion by repulsive steric effects or hydration pressure. Moreover, the AFM-based methodology provides dynamic, quantitative information about protein adhesion at the nanoscale level.     

Interactions between silica particles and poly(2-vinylpyridine) brushes in aqueous solutions of monovalent and multivalent salts

AFM colloidal probe technique, scratch tests, and spectroscopic ellipsometry are employed to study the conformation of a poly(2-vinyl pyridine) brush grafted to a planar surface and its interaction with microsized silica spheres in solutions containing monovalent (Cl^?) and multivalent counterions (SO₄ ^2? and PO₄ ^3?) at pH 2.5. During approach of the sphere, steric repulsion is observed with all salts at any concentration. The approach force-distance curves are fitted according to the Alexander-de Gennes model in order to calculate the equilibrium brush thickness L. These data are compared to the brush thickness determined by ellipsometry and AFM scratch tests. Different values are obtained but all of them decrease with increasing salt concentration. This effect is enhanced by counterions of higher valence because they have a stronger screening effect and ion correlation due to their greater charge per unit volume. With NaCl solutions, a reswelling of diluted P2VP coils is observed at Cl^? concentrations >1 M. When the sphere is retracted, weak adhesion forces occur at Cl^? concentrations >1.3?×?10^?2 M and at all concentrations of SO₄ ^2? and PO₄ ^3?.     

Surface thermodynamics and adhesion forces governing bacterial transmission in contact lens related microbial keratitis

Contact lens induced microbial keratitis results from bacterial transmission from one surface to another. We investigated the adhesion forces of Pseudomonas aeruginosa, Staphylococci and Serratia to different contact lenses, lens cases and corneal surfaces using AFM, and applied a Weibull analysis on these adhesion forces to calculate bacterial transmission probabilities from lens case to corneas with a contact lens as an intermediate. Also a new surface thermodynamic parameter was introduced, the interfacial free energy of transmission, which in essence compares the interfacial free energies of bacterial adhesion, calculated from measured contact angles with liquids on the donating and receiving surfaces in the transmission process. Bacterial adhesion forces were generally strongest among all eight strains for the lens case (-6.5 to -12.0 nN) and corneas (-3.5 to -11.5 nN), while contact lenses (-0.6 to -13.1 nN) exerted slightly smaller adhesion forces. Consequently, bacterial transmission from lens case to contact lens yielded a smaller contribution in the final transmission than from contact lens to cornea. Bacterial transmission probabilities as derived from force analyses were higher when the interfacial free energies of transmission were more negative, which is in line with surface thermodynamic principles. Therewith this parameter could provide useful in analyzing other bacterial transmission phenomena between donating and receiving surfaces as well.     

Identifying nonspecific ligand binding in electrospray ionization mass spectrometry using the reporter molecule method

The application of the reporter molecule (M_rep) method for identifying nonspecific complexes in the ES-MS analysis of protein-ligand and DNA-ligand interactions in vitro is described. To test the reliability of the method, it was applied to the ES-MS analysis of protein-carbohydrate complexes originating from specific interactions in solution and from nonspecific interactions in the ES process. These control experiments confirm the basic assumptions underlying the M_rep method, namely that nonspecific ligand binding is a random process, and that the ES droplet histories for specific and nonspecific complexes are distinct. The application of the M_rep method to the ES-MS analysis of the sequential binding of the ethidium cation, a DNA intercalator, to single and double strand oligodeoxynucleotides is also described, and highlights the general utility of the method.     

Thermodynamics of saturated and unsaturated ketones C<Subscript>13</Subscript> and C<Subscript>18</Subscript> and the energy of specific intermolecular interactions

P-T dependences of the saturated vapor of pseudoionone, hexahydropseudoionone, and saturated and unsaturated ketones C₁₈ were studied using the statistical approach, and the enthalpies and entropies of its vaporization were calculated. The presence of monomer forms of the molecules of these compounds was established by studying the unsaturated vapor pressure. It was demonstrated that the role of isostructural methyl group in ketones is not related to the all-explaining steric effect concealing the real nature of the specific interaction. The energies of specific intermolecular interactions were determined in liquid symmetrical and unsymmetrical ketones.