Human serum albumin self-assembly on weak polyelectrolyte multilayer films structurally modified by pH changes

Adsorption of proteins onto film surfaces built up layer by layer from oppositely charged polyelectrolytes is a complex phenomenon, governed by electrostatic forces, hydrogen bonds, and hydrophobic interactions. The amounts of the interacting charges, however, both in polyelectrolytes and in proteins adsorbed on such films are a function of the pH of the solution. In addition, the number and the accessibility of free charges in proteins depend on the secondary structure of the protein. The subtle interplay of all these factors determines the adsorption of the proteins onto the polyelectrolyte film surfaces. We investigated the effect of these parameters for polyelectrolyte films built up from weak "protein-like" polyelectrolytes (i.e., polypeptides), poly(L-lysine) (PLL), and poly(glutamic acid) (PGA) and for the adsorption of human serum albumin (HSA) onto these films in the pH range 3.0-10.5. It was found that the buildup of the polyelectrolyte films is not a simple function of the pure charges of the individual polyelectrolytes, as estimated from their respective pKa values. The adsorption of HSA onto (PLL/PGA)n films depended strongly on the polyelectrolyte terminating the film. For PLL-terminated polyelectrolyte films, at low pH, repulsion, as expected, is limiting the adsorption of HSA (having net positive charge below pH 4.6) since PLL is also positively charged here. At high pH values, an unexpected HSA uptake was found on the PGA-ending films, even when both PGA and HSA were negatively charged. It is suggested that the higher surface rugosity and the decrease of the alpha-helix content at basic pH values (making accessible certain charged groups of the protein for interactions with the polyelectrolyte film) could explain this behavior.     

A nanochannel array based device for determination of the isoelectric point of confined proteins

A nanochannel array based nanodevice can mimic the biological environments and thus unveil the natural properties, conformation and recognition information of biomolecules such as proteins and DNA in confined spaces. Here we report that porous anodic alumina (PAA) of a highly parallel nanochannel array covalently modified with proteins significantly modulates the transport of a negatively charged probe of ferricyanide due to the electrostatic interactions between the probes and modified nanochannel inner surface. Results show that such electrostatic interaction exists in a wide range of ionic strength from 1 mM to 100 mM in 20 nm nanochannels modified with proteins (hemoglobin, bovine serum albumin, and goat anti-rabbit IgG secondary antibody). In addition, the maximal steady-state flux of the charged probe through the modified nanochannel array is directly related to the ionic strength which determines the electric double layer thickness and solution pH which modulates the nanochannel surface charge. Thus, the modulated mass transport of the probe by solution pH can be used to study the charge properties of the immobilized proteins in nanochannel confined conditions, leading us to obtain the isoelectric point (pI) of the proteins confined in nanochannels. The determined pI values of two known proteins of hemoglobin and bovine serum albumin are close to the ones of the same proteins covalently modified on a 3-mercaptopropionic acid self-assembled monolayer/gold electrode. In addition, the pI of an unknown protein of goat anti-rabbit IgG secondary antibody confined in nanochannels was determined to be 6.3. Finally, the confinement effect of nanochannels on the charge properties of immobilized proteins has been discussed.     

A theoretical investigation on the pH-induced switching of mixed polyelectrolyte brushes

A theoretical investigation on the pH-induced switching of mixed polyelectrolyte brushes was performed by using a molecular theory. The results indicate that the switching properties of mixed polyelectrolyte brushes are dependent on the pH values. At low pH, negatively charged chains adopt a compact conformation on the bottom of the brush while positively charged chains are highly stretched away from the surface. At high pH values, the inverse transformation takes place. The role of pH determining the polymer chains conformation and charge behavior of mixed polyelectrolyte brushes was analyzed. It is found that there exists a mechanism for reducing strong electrostatic repulsions： stretching of the chains. The H＋ and OH- units play a more important role as counterions of the charged polymers do. The collapse of the polyelectrolyte chains for different pH values could be attributed to the screening of the electrostatic interactions and the counterion-mediated attractive interaction along the chains.     

Electrostatic free energy of weakly charged macromolecules in solution and intermacromolecular complexes consisting of oppositely charged polymers

When oppositely charged polyelectrolytes are mixed in water, attraction between oppositely charged groups may lead to the formation of polyelectrolyte complexes (associative phase separation, complex coacervation, interpolymer complexes). Theory is presented to describe the electrostatic free energy change when ionizable (annealed) (macro-)molecules form a macroscopic polyelectrolyte complex. The electrostatic free energy includes an electric term as well as a chemical term that is related to the dissociation of the ionic groups in the polymer. An example calculation for complexation of polyacid with polybase uses a cylindrical diffuse double layer model for free polymer in solution and electroneutrality within the complex and calculates the free energy of the system when the polymer is in solution or in a polyelectrolyte complex. Combined with a term for the nonelectrostatic free energy change upon complexation, a theoretical stability diagram is constructed that relates pH, salt concentration, and mixing ratio, which is in qualitative agreement with an experimental diagram obtained by Bungenberg de Jong (1949) for complex coacervation of arabic gum and gelatin. The theory furthermore explains the increased tendency toward phase separation when the polymer becomes more strongly charged and suggests that complexation of polyacid or polybase with zwitterionic polymer (e.g., protein) of the same charge sign (at the "wrong side" of the iso-electric point) may be due (in part) to an induced charge reversal of the protein.     

A minimalist model for exploring conformational effects on the electrospray charge state distribution of proteins

The electrospray ionization (ESI) charge state distribution of proteins is highly sensitive to the protein structure in solution. Unfolded conformations generally form higher charge states than tightly folded structures. The current study employs a minimalist molecular dynamics model for simulating the final stages of the ESI process in order to gain insights into the physical reasons underlying this empirical relationship. The protein is described as a string of 27 beads ("residues"), 9 of which are negatively charged and represent possible protonation sites. The unfolded state of this bead string is a random coil, whereas the native conformation adopts a compact fold. The ESI process is simulated by placing the protein inside a solvent droplet with a 2.5 nm radius consisting of 1600 Lennard-Jones particles. In addition, the droplet contains 14 protons which are modeled as highly mobile point charges. Disintegration of the droplet rapidly releases the protein into the gas phase, resulting in average charge states of 4.8+ and 7.4+ for the folded and unfolded conformation, respectively. The protonation probabilities of individual residues in the folded state reveal a characteristic pattern, with values ranging from 0.2 to 0.8. In contrast, the protonation probabilities of the unfolded protein are more uniform and cover the range from 0.8 to 1.0. The origin of these differences can be traced back to a combination of steric and electrostatic effects. Residues exhibiting a small accessible surface area are less likely to capture a proton, an effect that is exacerbated by partial electrostatic shielding from nearby positive residues. Conversely, sites that are sterically exposed are associated with electrostatic funnels that greatly increase the likelihood of protonation. Unfolding enhances the steric and electrostatic exposure of protonation sites, thereby causing the protein to capture a greater number of protons during the droplet disintegration process.     

Defect formation on surfaces bombarded by energetic multiply charged proteins: Implications for the conformation of gas-phase electrosprayed ions

Indirect information on the conformation of highly charged molecular ions may be obtained by monitoring their collisional cross sections and the course of simple gas-phase reactions such as hydrogen-deuterium exchange. In this work, another indirect but more visually oriented approach is explored: electrosprayed protein ions are accelerated toward a highly oriented pyrolytic graphite surface and the resulting single-ion defects are imaged by scanning force and tunneling microscopy. All protein impacts generated shallow hillocks: the shapes depended on the identity and charge state of the incident protein. Lysozyme and myoglobin, both compact, globular proteins in the native state, produced compact, almost circular hillocks. However, hillocks generated by myoglobin that had been denatured in the solution phase were elongated, and the elongation was positively correlated with the charge state of the ion. It appears that structural information about gas-phase multiply charged proteins can be derived from imprints generated by energetic protein impacts on surfaces.     

Adsorption of branched-linear polyethyleneimine-ethylene oxide conjugate on hydrophilic silica investigated by ellipsometry and Monte Carlo simulations

The adsorption of and conformation adopted by a branched-linear polymer conjugate to the hydrophilic silica-aqueous solution interface have been studied by in situ null ellipsometry and Monte Carlo simulations. The conjugate is a highly branched polyethyleneimine structure with ethyleneoxide chains grafted to its primary and secondary amino groups. In situ null ellipsometry demonstrated that the polymer conjugate adsorbs to the silica surface from water and aqueous solution of 1 mM asymmetric divalent salt (calcium and magnesium chloride to emulate hard water) over a large pH range. The adsorbed amount is hardly affected by pH and large charge reversal on the negatively charged silica surface occurred at pH = 4.0, due to the adsorption of the cationic polyelectrolyte. The Monte Carlo simulations using an appropriate coarse-grained model of the polymer in solution predicted a core-shell structure with no sharp boundary between the ethyleneimine and ethyleneoxide moieties. The structure at the interface is similar to that in solution when the polymer degree of protonation is low or moderate while at high degree of protonation the strong electrostatic attraction between the ethyleneimine core and oppositely charged silica surface distorts the ethyleneoxide shell so that an "anemone"-like configuration is adopted. The adsorption of alkyl benzene sulfonic acid (LAS) to a preadsorbed polymer layer was also investigated by null ellipsometry. The adsorption data brought additional support for the existence of a strong polymer adsorption and showed the presence of a binding which was further enhanced by the decreased solvency of the surfactant in the salt solution and confirmed the surface charge reversal by the polymer adsorption at pH = 4.0.     

Reversible switching of protein uptake and release at polyelectrolyte multilayers detected by ATR-FTIR spectroscopy

The reversible switching of uptake and release of the proteins lysozyme (LYZ, IEP = 11.1) and human serum albumin (HSA, IEP = 4.8) at the surface attached polyelectrolyte multilayer (PEM) consisting of poly(ethylene-imine) (PEI) and poly(acrylic acid) (PAC) is shown. Protein adsorption could be switched by pH setting due to electrostatic interaction. Adsorption of positively charged LYZ at PEM-6 took place at pH = 7.3, where the outermost PAC layer was negatively charged. Complete desorption was obtained at pH = 4, where the outermost PAC layer was neutral. Additionally the charge state of the last adsorbed PAC layer in dependence of the pH of the medium could be determined in the ATR-FTIR difference spectra by the ν(COO⁻) and ν(C=O) band due to carboxylate and carboxylic acid groups. Adsorption of negatively charged HSA at PEM-7 was achieved at pH = 7.3, where the outermost PEI layer was positively charged. Part desorption was obtained at pH = 10, where the outermost PEI layer was neutral. PEM of PEI/PAC may be used for the development of bioactive and bionert materials and protein sensors.     

Polysaccharide/Surfactant complexes at the air-water interface - effect of the charge density on interfacial and foaming behaviors

The binding of a cationic surfactant (hexadecyltrimethylammonium bromide, CTAB) to a negatively charged natural polysaccharide (pectin) at air-solution interfaces was investigated on single interfaces and in foams, versus the linear charge densities of the polysaccharide. Besides classical methods to investigate polymer/surfactant systems, we applied, for the first time concerning these systems, the analogy between the small angle neutron scattering by foams and the neutron reflectivity of films to measure in situ film thicknesses of foams. CTAB/pectin foam films are much thicker than the pure surfactant foam film but similar for high- and low-charged pectin/CTAB systems despite the difference in structure of complexes at interfaces. The improvement of the foam properties of CTAB bound to pectin is shown to be directly related to the formation of pectin-CTAB complexes at the air-water interface. However, in opposition to surface activity, there is no specific behavior for the highly charged pectin: foam properties depend mainly upon the bulk charge concentration, while the interfacial behavior is mainly governed by the charge density of pectin. For the highly charged pectin, specific cooperative effects between neighboring charged sites along the chain are thought to be involved in the higher surface activity of pectin/CTAB complexes. A more general behavior can be obtained at lower charge density either by using a low-charged pectin or by neutralizing the highly charged pectin in decreasing pH.     

The model Lysozyme-PSSNa system for electrostatic complexation: Similarities and differences with complex coacervation

We review, based on structural information, the mechanisms involved when putting in contact two nano-objects of opposite electrical charge, in the case of one negatively charged polyion, and a compact charged one. The central case is mixtures of PSS, a strong flexible polyanion (the salt of a strong acid, and with high linear charge density), and Lysozyme, a globular protein with a global positive charge. A wide accurate and consistent set of information in different situations is available on the structure at local scales (5-1000?), due to the possibility of matching, the reproducibility of the system, its well-defined electrostatics features, and the well-defined structures obtained. We have related these structures to the observations at macroscopic scale of the phase behavior, and to the expected mechanisms of coacervation. On the one hand, PSS/Lysozyme mixtures show accurately many of what is expected in PEL/protein complexation, and phase separation, as reviewed by de Kruif: under certain conditions some well-defined complexes are formed before any phase separation, they are close to neutral; even in excess of one species, complexes are only modestly charged (surface charges in PEL excess). Neutral cores are attracting each other, to form larger objects responsible for large turbidity. They should lead the system to phase separation; this is observed in the more dilute samples, while in more concentrated ones the lack of separation in turbid samples is explained by locking effects between fractal aggregates. On the other hand, although some of the features just listed are the same required for coacervation, this phase transition is not really obtained. The phase separation has all the macroscopic aspects of a fluid (undifferentiated liquid/gas phase) - solid transition, not of a fluid-fluid (liquid-liquid) one, which would correspond to real coacervation). The origin of this can be found in the interaction potential between primary complexes formed (globules), which agrees qualitatively with a potential shape of the type repulsive long range attractive very short range. Finally we have considered two other systems with accurate structural information, to see whether other situations can be found. For Pectin, the same situation as PSS can be found, as well as other states, without solid precipitation, but possibly with incomplete coacervation, corresponding to differences in the globular structure. It is understandable that these systems show smoother interaction potential between the complexes (globules) likely to produce liquid-liquid transition. Finally, we briefly recall new results on Hyaluronan/Lysozyme, which present clear signs of coacervation in two liquid phases, and at the same time the existence of non-globular complexes, of specific geometry (thin rods) before any phase separation. These mixtures fulfill many of the requirements for complex coacervation, while other theories should also be checked like the one of Shklovskii et al.     

Effect of gelatin molecular charge heterogeneity on formation of intermolecular complexes and coacervation transition

We investigate the molecular charge heterogeneity of gelatin molecules at three different pHs: isoelectric pH = 5 (pI), and at pHs = pI ± 1 by measuring the zeta potential distributions. Its effect on the formation of soluble intermolecular complexes induced by the presence of a nonsolvent, ethanol, was explored. The charge distributions were found to be symmetric at pH = 5, where the onset of binding, and formation of soluble complexes were observed to be facilitated by the presence of a small net charge (close to zero) with the molecules exhibiting polyampholyte (PA) behavior. These distributions turned asymmetric at pH = 6, yet complex formation and coacervation occurred. On the other hand, for pH = 4 samples, these distributions were found to be strongly asymmetric with the molecules possessing very high net positive charge, such a system did not yield coacervation. The PA to polyelectrolyte transition and its repercussion on coacervation has been discussed in the light of the experimental results obtained from electrophoresis, turbidimetry, atomic force microscopy, and nanoindentation studies. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 1511–1520, 2007     

Interaction and structure of surfaces coated by poly(vinyl amines) of different line charge densities

Interactions between preadsorbed films of poly(vinyl amine) (PVA) of two different line charge densities on silica substrates were studied with the colloidal probe technique based on the atomic force microscope (AFM). The preadsorbed films were prepared by adsorption of PVA from a pH 4 solution without any added salt. The highly charged PVA adsorbs in a flat configuration and in laterally heterogeneous layers, while the more weakly charged PVA analog adsorbs in thicker and more homogeneous films. As revealed by reflectivity measurements, such preadsorbed PVA films are stable in polyelectrolyte-free solutions. However, force measurements with the colloidal probe reveal that their interactions depend strongly on the ionic strength. Upon approach, interactions are dominated by electrostatic diffuse layer overlap forces. Both PVA films have very similar diffuse layer charge densities of about 1.5 mC/m2. Since these values are substantially lower than what would be expected from the total charge of the adsorbed polyelectrolytes measured by reflectivity, we infer that coadsorption of anions represents the principal mechanism in charge neutralization. Upon retraction, the adhesion between the films is dominated by bridging forces due to single polymer chains. Such bridging adhesion becomes progressively important with increasing ionic strength, whereby their range and frequency increase. The work of adhesion due to bridging is about 0.3 mN/m. At low ionic strengths, the films behave differently. While the highly charged PVA shows unspecific adhesion at small distances, the more weakly charged PVA analog shows few adhesion events occurring at long distances.     

Uncovering the Contributions of Charge Regulation to the Stability of Single Alpha Helices**

The single alpha helix (SAH) is a recurring motif in biology. The consensus sequence has a di-block architecture that includes repeats of four consecutive glutamate residues followed by four consecutive lysine residues. Measurements show that the overall helicity of sequences with consensus E₄K₄ repeats is insensitive to a wide range of pH values. Here, we use the recently introduced q-canonical ensemble, which allows us to decouple measurements of charge state and conformation, to explain the observed insensitivity of SAH helicity to pH. We couple the outputs from separate measurements of charge and conformation with atomistic simulations to derive residue-specific quantifications of preferences for being in an alpha helix and for the ionizable residues to be charged vs. uncharged. We find a clear preference for accommodating uncharged Glu residues within internal positions of SAH-forming sequences. The stabilities of alpha helical conformations increase with the number of E₄K₄ repeats and so do the numbers of accessible charge states that are compatible with forming conformations of high helical content. There is conformational buffering whereby charge state heterogeneity buffers against large-scale conformational changes thus making the overall helicity insensitive to large changes in pH. Further, the results clearly argue against a single, rod-like alpha helical conformation being the only or even dominant conformation in the ensembles of so-called SAH sequences.     

Layer-by-layer assemblies of chitosan and heparin: effect of solution ionic strength and pH

The growth of polysaccharide multilayers consisting of positively charged chitosan (CH) and negatively charged heparin (HEP) was monitored in situ by employing a quartz crystal microbalance (QCM-D) and dual-polarization interferometry (DPI). The main focus was on how the physicochemical properties of the solution affect the growth and structure of the resulting multilayer film. These results showed that when increasing the ionic strength of the polysaccharide solutions at a fixed pH, both the "dry" (optical) (DPI) mass and wet (QCM) mass of the adsorbed multilayer film increased. The same effect was found when increasing the pH while keeping the ionic strength constant. Furthermore, the growth of multilayers showed an exponential-like behavior independent of the solution conditions that were used in this study. It was also established that chitosan was the predominant species present in the chitosan-heparin multilayer film. We discuss the viscoelastic properties of the adsorbed layers and their variation during the multilayer buildup. Interestingly and contrary to common interpretation of the QCM-D results, we found that under one particular solution condition (pH 4.2 and 30 mM NaCl) the increase in the dissipation of oscillation energy from the adsorbed layer was a consequence of layer stiffening rather than indicating a more hydrated and viscous film. On the basis of the widely used Voigt viscoelastic model for an adsorbed layer, we show that it is the film viscosity and shear that define the layer viscoelasticity (structure) of the film and not the absolute value of energy dissipation, which in fact can be very misleading.     

Numerical study of the interplay of monomer-surface electrostatic and non-electrostatic interactions in the adsorption of weak polyelectrolytes on oppositely charged surfaces

The adsorption of weak polybase on oppositely charged planar surfaces has been investigated numerically by using the self-consistent field theory (SCFT). Particular attention was paid to the interplay of monomer-surface electrostatic and non-electrostatic interactions in the adsorption behaviors of weak polybase. In this study, the strength of monomer-surface non-electrostatic interactions was set to be no more than the thermal energy k _B T. It was found from the numerical study that in the regime of low surface charge density of the substrate and low pH or high bulk degree of ionization, both the screening-enhanced and screening-reduced salt effects emerge. On the contrary, in the opposite regime, only the screening-reduced salt effect was observed. Moreover, the overall charge neutrality inside the adsorption layer was analyzed. The underlying mechanism governing the adsorption behaviors of weak polybase on oppositely charged surfaces was elucidated.     

Electrostatic interactions between amphoteric latex particles and proteins

The electrostatic interactions between amphoteric polymethyl methacrylate latex particles and proteins with different pI values were investigated. These latex particles possess a net positive charge at low pH, but they become negatively charged at high pH. The nature and degree of interactions between these polymer particles and proteins are primarily controlled by the electrostatic characteristics of the particles and proteins under the experimental conditions. The self-promoting adsorption process from the charge neutralization of latex particles by the proteins, which have the opposite net charge to that of the particles, leads to a rapid reduction in the zeta potential of the particles (in other words colloidal stability), and so strong flocculation occurs. On the other hand, the electrostatic repulsion forces between similarly charged latex particles and the proteins retard the adsorption of protein molecules onto the surfaces of the particles. Therefore, latex particles exhibit excellent colloidal stability over a wide range of protein concentrations. A transition from net negative charge to net positive charge, and vice versa (charge reversal), was observed when the particle surface charge density was not high enough to be predominant in the protein adsorption process.     

Stimulation and binding properties of polyelectrolyte multilayers verified by ATR‐FTIR spectroscopy

We report on the deposition and properties of multilayers composed of reactive polymers on planar surfaces. As reactive polymers the poly cations poly(ethylene‐imine), poly(L‐lysine) (PLL), poly(allylamime) (PAA) and the polyanions poly(acrylate) (PAC), poly(vinylsulfate), poly(maleate‐co‐olefines) were used. ATR‐FTIR Spectroscopy was adopted to study deposition, binding and stimulation properties of polymer multilayers. The binding of charged species of different molecular size such as rhodanide anions and sodium oleate from solution was examined, whereby binding was found to be dependent on the charge of the outermost layer. For these two analytes a selectivity parameter Q, defined as the ratio between the adsorbed amount obtained at the negatively charged and that at the positively charged surface, respectively, was determined. Furthermore, swelling experiments on multilayer assemblies of PLL and PAC exposed to mixtures of ethanol/water (10–70% EtOH content) were carried out. Our experiments gave evidence, that the PLL layers showed a more significant increase in density than the PAC layers. The conformation of PLL incorporated into multilayers could be changed by pH variation.     

Adsorption kinetics of cationic polyelectrolytes studied with stagnation point adsorption reflectometry and quartz crystal microgravimetry

The effects of charge density, pH, and salt concentration on polyelectrolyte adsorption onto the oxidized surface of silicon wafers were studied using stagnation point adsorption reflectometry and quartz crystal microgravimetry. Five different polyelectrolytescationic polyacrylamides of four charge densities and one cationic dextranwere examined. The adsorption kinetics was characterized using each technique, and the adsorption kinetics observed was in line with the impinging jet theory and the theory for one-dimensional diffusion, respectively. The polyelectrolyte adsorption increased with pH as an effect of the increased silica surface charge. A maximum in the saturation adsorption for both types of polyelectrolytes was found at 10 mM NaCl concentration. A significant adsorption also occurred at 1 M NaCl, which indicated a significant nonionic contribution to the adsorption mechanism. The fraction of solvent in the adsorbed layer was determined to be 70-80% by combining the two analysis techniques. This indicated a loose structure of the adsorbed layer and an extended conformation at the surface, favoring loops and tails. However, considering the solution structure with a hydrodynamic diameter larger than 100 nm for the CPAM and a thickness of the adsorbed layer on the order of 10 nm, the results showed that the adsorption is accompanied by a drastic change in polymer conformation. Furthermore, this conformation change takes place on a time scale far shorter than seconds.     

All-cellulose multilayers: long nanofibrils assembled with short nanocrystals

Self-organized multilayer films were formed by sequential addition of oppositely charged cellulose I nanoparticles. The all-cellulosic multilayers were prepared via adsorption of cationicially modified cellulose nanofibrils (cat NFC) and anionic short crystalline cellulose (CNC) at pH 4.5 and pH 8.3. The properties and build-up behavior of layer-by-layer-constructed films were studied with microgravimetry (QCM-D) and the direct surface forces in these systems were explored with colloidal probe microscopy to gain information about the fundamental interplay between cat NFC and anionic CNC. The importance of the first layer on the adsorption of the consecutive layers was demonstrated by comparing pure in situ adsorption in the QCM-D with multilayer films made by spin coating the first cationic NFC layer and then subsequently adsorbing the following layers in situ in the QCM-D chamber. Differences in adsorbed amount and viscoelastic behavior were observed between those two systems. In addition, a significant pH dependence of cat NFC charge was found for both direct surface interactions and layer properties. Moreover the underlying cellulose layer in multilayer film was established to influence the surface forces especially at lower pH, where the cat NFC chains extensions were facilitated and overall charge was affected by the cationic counterpart within the layers. This enhanced understanding the effect of charge and structure on the interaction between these renewable nanoparticles is valuable when designing novel materials based on nanocellulose.     

Study of a Solvated Adsorbed Gelatin Layer Using a Modified Force Microscope

The interaction energies between gelatin-coated surfaces at various electrolyte and pH conditions are reported. The surfaces are of glass and are negatively charged under all conditions used here. Gelatin is a polyampholyte, with an isoelectric pH (IEP) of approximately 4.9. At low pH the gelatin molecules have a net positive charge, and thus the polyampholyte tends to adsorb with a relatively flat conformation. As the pH is increased the strong attractive interaction between the surface and the polyampholyte decreases as more negative charges and then fewer positive charges appear on the polyampholyte, and so the gelatin extends away from the surface. On changing electrolyte concentrations after adsorption no effect was seen at the IEP, but the layer was observed to swell at more alkaline pHs. This is consistent with the net minimum charge situation on the polymer under these conditions. Changing the adsorption conditions was seen to have an effect, and this is attributed to the different affinities of the gelatin chain to the surface depending on the solution chemistry. Results obtained when the gelatin was initially adsorbed on one surface or two were similar, suggesting that the gelatin transfers rapidly from one surface to another. The importance of adsorption conditions as well as current conditions is discussed. Copyright 1999 Academic Press.