Biomedical subjects

Paul R Van Tassel

Publications and source records attributed to Paul R Van Tassel.

13 recordsLinked to original sources

PubMed2007

Continuous polyelectrolyte adsorption under an applied electric potential.

Interactions between charged macromolecules (e.g., proteins, nucleic acids, polyelectrolytes) and charged surfaces govern many natural and industrial processes. We investigate here the influence of an applied electric potential on the adsorption of charged polymers, and report the following significant result: the adsorption of certain amine side chain-containing polycations may become continuous, i.e., asymptotically linear (or nearly linear) in time over hours, upon the application of a modest anodic potential. Employing optical waveguide lightmode spectroscopy (OWLS) and an indium tin oxide (ITO) substrate, we show that asymptotic kinetics, and the adsorbed mass at the onset of the asymptotic regime, depend sensitively on polymer chemistry (in particular, side chain volume and charge location), increase with applied potential and ionic strength (conditions favoring a thicker initial layer), and are independent of bulk polymer concentration (suggesting postadsorption events to be rate limiting). X-ray photoelectron spectra reveal a suppressed polymer charge within layers formed via continuous adsorption, but no evidence of electrochemical reactions. We propose a mechanism based on polymer-polymer binding within the adsorbed layer, enabled by suppressed electrostatic repulsion and/or enhanced ionic correlations near the conducting surface, and stabilized by short-range attractive interactions. Continuous adsorption under an applied electric potential offers the possibility of nanoscale films of tailored polymer content realized in a single step.

Adsorption↗

PubMed2006

Fibronectin terminated multilayer films: protein adsorption and cell attachment studies.

Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10nm, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(L-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn)--a matrix protein known to promote cell adhesion--onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL-terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS-terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase-contrast optical microscopy to investigate the time-dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. Positively charged PLL-terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS-terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS-terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL-terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell area and circularity with time) for films without Fn, and for DS-terminated films with Fn. Overall, the Fn-coated PLL monolayer and the Fn-coated PLL-terminated multilayer are the best performing films in promoting cell spreading. We conclude the presence of Fn to be an important factor (more so than film charge or layer number) in controlling the interaction between multilayer films and living cells, and thus to represent a promising strategy toward in vivo applications such as tissue engineering.

Adsorption↗

PubMed2006

Structuring of macroions confined between like-charged surfaces.

We investigate the structuring of charged spherical nanoparticles and micelles (i.e., "macroions") between two surfaces as a function of bulk macroion concentration. Structuring is deduced from measured force profiles between a silica particle and a silica plate in the presence of an aqueous macroion (Ludox silica nanoparticle or sodium dodecyl sulfate micelle) solution, obtained with an atomic force microscope. We observe oscillatory force profiles that decay with separation. We find that the wavelength of the force profiles scales with the bulk number density as rho(-)(1/3), rather than with the effective macroion size. Only at very high silica nanoparticle concentration (above 10 vol %) in a low ionic strength solution does the wavelength become smaller than that predicted by the simple rho(-)(1/3) scaling; however, the original scaling is recovered upon the addition of a small amount of electrolyte. A comparison between the measured wavelength and the predicted spacing between the macroions in the bulk shows that the two variables differ in both magnitude and bulk density scaling. This finding suggests that confined macroions are more ordered than those in the bulk and the nature of this ordering is maintained over a relatively wide range of bulk concentration.

Journal Article↗

PubMed2005

Adsorbed layers of oriented fibronectin: a strategy to control cell-surface interactions.

Fibronectin (Fn) is a matrix protein known to induce cell attachment and spreading through its cell binding site and related synergy sites. Fn-coated surfaces are therefore useful in tissue engineering and other cell contacting applications, but a problem with many immobilization strategies is a random distribution of molecular orientations. We sought to control Fn orientation, and thus enhance the availability of its cell binding site, by immobilizing Fn via a carboxymethyl dextran layer onto which are chemically attached monoclonal antibodies specific to a region near to Fn's C terminus (and thus away from the cell binding site). Using optical waveguide lightmode spectroscopy, we show the presence of chemically coupled antibodies to yield a considerably denser and thicker Fn layer, consistent with a more vertically aligned protein. Human umbilical vein endothelial cells spread significantly faster, and in a more spherically symmetric way, on an oriented Fn layer (i.e., in the presence of immobilized monoclonal antibodies) as compared with a control Fn layer (i.e., in the absence of bound antibodies). However, we observe human umbilical vein endothelial cell spreading on the oriented Fn layer to be similar to that on a Fn layer in the absence of a carboxymethyl dextran layer, suggesting that although orienting Fn is a promising strategy, coupling strategies using linkers other than dextran may be needed.

Adsorption↗

PubMed2005

Replica Ornstein-Zernike theory of adsorption in a templated porous material: interaction site systems.

Molecular templating offers the possibility of porous materials whose selectivity rivals the molecular recognition observed in nature. The design of templated materials requires a molecular understanding of the templating effect on the material structure and performance. We present here a theoretical description of adsorption in a model templated porous material. Our model material is a quenched, equilibrated mixture of template and matrix molecular species where the template component has been subsequently removed. We propose a set of site-site [i.e., reference interaction site model (RISM)] replica Ornstein-Zernike equations relating the correlation functions of template, matrix, and adsorbing fluid molecules. To test this approach, we focus here on systems interacting via hard-sphere site-site potentials and employ a Percus-Yevick closure. We consider chain and cluster species composed of up to five spheres and observe a range of effects associated with template structure, including higher affinity toward, and enhanced templating by, compact cluster molecules. We assess these effects by grand canonical Monte Carlo simulation and discuss their implication to the design of templated molecular recognition materials.

Adsorption↗

PubMed2005

In situ layer-by-layer film formation kinetics under an applied voltage measured by optical waveguide lightmode spectroscopy.

Layer-by-layer (LbL) thin film assembly occurs via the alternate adsorption of positively and negatively charged macromolecular species. We investigate here the control of LbL film growth through the electric potential of the underlying substrate. We employ optical waveguide lightmode spectroscopy (OWLS) to obtain in situ kinetic measurements of poly(allylamine hydrochloride)/poly(sodium 4-styrenesulfonate) (PAH/PSS) and poly(L-lysine)/dextran sulfate (PLL/DXS) multilayer film formation in the presence of an applied voltage difference (deltaV) between the adsorbing substrate, an indium tin oxide- (ITO-) coated waveguiding sensor chip, and a parallel platinum counterelectrode. We find initial layer adsorption to be significantly enhanced by an applied potential for both polyelectrolyte systems: the mass and thickness of (positively charged) PAH and PLL layers on ITO are about 60% and 500% larger, respectively, at deltaV = 2 V than at open circuit potential (OCP), in apparent violation of electrostatics. A kinetic analysis reveals the initial attachment rate constant to decrease with voltage, in agreement with electrostatics. To reconcile these results, we propose a more coiled and loosely bound adsorbed polymer conformation at higher applied potential. Following 10 adsorption steps, the mass and thickness of a PAH/PSS film grown under deltaV = 2 V are about 15% less than those of a comparable film grown under OCP, reflecting a lower degree of complexation between adsorbing polyanions and more highly coiled adsorbed polycations. Following 14 adsorption steps, the mass and thickness of a PLL/DXS film grown under deltaV = 2 V are about 70% greater than those of a comparable film grown under OCP, reflecting the increased charge overcompensation in the initial layer. We find the scaling of film mass () with the number of adsorption steps (n) to be linear in the PAH/PSS system and exponential (i.e., approximately eyn) in the PLL/DXS system, irrespective of applied voltage. We observe to decrease with applied voltage and to exhibit a crossover to a smaller value around n = 5. Extrapolation reveals PLL/DXS multilayer films to be suppressed by increased voltage in the limit of large n: the mass of films grown at OCP and deltaV = 1 V would surpass that of a film grown under deltaV = 2 V at about the 23rd and 18th adsorption steps, respectively. The formation kinetics of PLL/DXS, but not PAH/PSS, change qualitatively under voltage: PLL adsorption is slow to reach a plateau, possibly due to the formation of secondary structure, and a decrease in film mass occurs toward the end of each DXS adsorption step, suggesting spontaneous removal of some PLL/DXS complexes from the film.

Journal Article↗

PubMed2005

Conformational transition free energy profiles of an adsorbed, lattice model protein by multicanonical Monte Carlo simulation.

Proteins often undergo changes in internal conformation upon interacting with a surface. We investigate the thermodynamics of surface induced conformational change in a lattice model protein using a multicanonical Monte Carlo method. The protein is a linear heteropolymer of 27 segments (of types A and B) confined to a cubic lattice. The segmental order and nearest neighbor contact energies are chosen to yield, in the absence of an adsorbing surface, a unique 3x3x3 folded structure. The surface is a plane of sites interacting either equally with A and B segments (equal affinity surface) or more strongly with the A segments (A affinity surface). We use a multicanonical Monte Carlo algorithm, with configuration bias and jump walking moves, featuring an iteratively updated sampling function that converges to the reciprocal of the density of states 1/Omega(E), E being the potential energy. We find inflection points in the configurational entropy, S(E)=k ln Omega(E), for all but a strongly adsorbing equal affinity surface, indicating the presence of free energy barriers to transition. When protein-surface interactions are weak, the free energy profiles F(E)=E-TS(E) qualitatively resemble those of a protein in the absence of a surface: a free energy barrier separates a folded, lowest energy state from globular, higher energy states. The surface acts in this case to stabilize the globular states relative to the folded state. When the protein surface interactions are stronger, the situation differs markedly: the folded state no longer occurs at the lowest energy and free energy barriers may be absent altogether.

Adsorption↗

PubMed2004

Probing adsorbed fibronectin layer structure by kinetic analysis of monoclonal antibody binding.

Adsorbed protein layers are often away from equilibrium and thus exhibit history dependent structures. We use the kinetics of monoclonal antibody binding, as measured using optical waveguide lightmode spectroscopy (OWLS), to investigate the structure of adsorbed fibronectin (Fn) layers formed under different kinetic paths. For all of the layers investigated, we find no difference between the apparent adsorption rate constants of (i) monoclonal antibodies specific to Fn's cell binding site (alpha-Fn) and (ii) monoclonal antibodies specific to cytochrome c (alpha-CC, as a control), indicating initial adsorption of antibodies to be non-specific. For certain layers, the saturation density and the initial projected area per antibody differ significantly between alpha-Fn and alpha-CC, suggesting specific binding to follow the initial non-specific attachment. The fraction of antibodies binding specifically to the Fn layer, and the number of Fn binding sites per specific binding event, are estimated in terms of the difference in initial projected areas between alpha-Fn and alpha-CC. For a Fn layer formed at a bulk concentration of 2 microg/mL, we find a decrease in specific binding with an increase in Fn layer formation time, suggesting post-adsorption structural changes of a lower density adsorbed layer diminish binding site availability. Conversely, for a Fn layer formed at a bulk concentration of 40 microg/mL, we find an increase in specific binding with an increase in the aging time of the Fn layer, implying post-adsorption structural changes reveal binding sites for a higher density adsorbed layer.

Adsorption↗

PubMed2004

Probing macromolecular adsorbed layer structure and history dependence via the interfacial cavity function.

Adsorbed layers of proteins and other macromolecules often relax structurally more slowly than they form, rendering layer growth an out-of-equilibrium process. We show here how the interfacial cavity function, Phi (the average Boltzmann factor for a single probe molecule), may be determined, using kinetic data available from optical waveguide lightmode spectroscopy, and used as a continuous, in situ measure of history dependent adsorbed layer structure. The increase of Phi observed with residence time for fibronectin and lysozyme layers suggests post-adsorption clustering on a time scale longer than that predicted by a surface diffusion model.

Adsorption↗

PubMed2004

Fibronectin adsorption onto polyelectrolyte multilayer films.

The Layer-by-layer deposition of positively and negatively charged macromolecular species is an ideal method for constructing thin films incorporating biological molecules. We investigate the adsorption of fibronectin onto polyelectrolyte multilayer (PEM) films using optical waveguide lightmode spectroscopy (OWLS) and atomic force microscopy (AFM). PEM films are formed by adsorption onto Si(Ti)O2 from alternately introduced flowing solutions of anionic poly(sodium 4-styrenesulfonate) (PSS) and cationic poly(allylamine hydrochloride) (PAH). Using OWLS, we find the initial rate and overall extent offibronectin adsorption to be greatest on PEM films terminated with a PAH layer. The polarizability density of the adsorbed protein layer, as measured by its refractive index, is virtually identical on both PAH- and PSS-terminated films; the higher adsorbed density on the PAH-terminated film is due to an adsorbed layer of roughly twice the thickness. The binding of monoclonal antibodies specific to the protein's cell binding site is considerably enhanced to fibronectin adsorbed to the PSS layer, indicating a more accessible adsorbed layer. With increased salt concentration, we find thicker PEM films but considerably thinner adsorbed fibronectin layers, owing to increased electrostatic screening. Using AFM, we find adsorbed fibronectin layers to contain clusters; these are more numerous and symmetric on the PSS-terminated film. By considering the electrostatic binding of a segmental model fibronectin molecule, we propose a picture of fibronectin adsorbed primarily in an end-on-oriented monolayer on a PAH-terminated film and as clusters plus side-on-oriented isolated molecules onto a PSS-terminated film.

Adsorption↗

PubMed2003

Protein adsorption: kinetics and history dependence.

The adsorption of protein and other macromolecular species is often strongly history dependent owing to the slow relaxation of nonequilibrium structures. The rate at which molecules adsorb is exquisitely sensitive to, and thus a sensitive measure of, interfacial structure; adsorption kinetics may therefore serve both to identify and to quantify history dependence. Optical waveguide lightmode spectroscopy (OWLS) in multistep mode, where an adsorbing surface is alternately exposed to a protein solution and one free of protein, enables the comparison of adsorption rates onto interfacial layers of identical composition but different formation histories. Contributions to the overall kinetics from the apparent adsorption rate constant, the desorption rate constants of molecules in different adsorbed states, and the interfacial one-body cavity function (a measure of the probability of a "cavity" on the surface free of protein) may be isolated from OWLS kinetic data. We find the rate of adsorption and the corresponding cavity function to increase significantly-for systems of fibronectin, cytochrome c, and lysozyme adsorbing onto Si(Ti)O(2)-from a first to a subsequent adsorption step and attribute this to a clustering transition among adsorbed protein molecules. From the kinetic data, we determine important structural and temporal information on this event.

Absorption↗

PubMed2003

Biosensing under an applied voltage using optical waveguide lightmode spectroscopy.

An applied dc voltage offers a means of controlling immobilization during biosensor fabrication and detection during biosensing application. We present a method to directly and continuously measure the adsorption of biomacromolecules or other polyelectrolytes, under an applied potential difference, based on optical waveguide lightmode spectroscopy (OWLS). An indium tin oxide (ITO) film of thickness ca. 10 nm coated onto a silicon titanium oxide (STO) waveguiding film serves as the working (sensing) electrode. We observe the effective refractive index of the 0th transverse electric guided mode to increase significantly in the presence of an applied potential due to charging of the interfacial double layer and, possibly, modest electrochemical oxidation. Adsorption from solution onto the ITO electrode is detected by a further increase in the effective refractive index. We achieve accurate detection by employing an optical model in which the STO and ITO layers are combined into a single waveguiding film. No improvement is found using models treating the ITO as a separate layer, either dielectric or conducting. Using this method, we find the adsorption of human serum albumin and horse heart cytochrome c to be considerably enhanced in the presence of an applied potential exceeding 1 V. We attribute this behavior to adsorption at positions on the protein molecules of complementary charge.

Biosensing Techniques↗

PubMed2002

Surface-induced conformational changes in lattice model proteins by Monte Carlo simulation.

We present Monte Carlo simulations of thermal, structural, and dynamic properties of a 27-segment lattice model protein adsorbed to a solid surface. The protein consists of a sequence of A and B segments whose order and topological contact energy values are chosen so that a unique (3x3x3 cubic) folded state occurs in the absence of an adsorbing surface [E. I. Shakhnovich and M. Gutin, Proc. Natl. Acad. Sci. USA 90, 7195 (1993)]. The surface consists of a plane of sites that interact either (i) equally with all contacting protein segments (an equal affinity surface) or (ii) more strongly with type A contacting segments (an A affinity surface). For both surfaces, we find the conformational change of an initially folded protein to begin with a continuous transition to a structure where all segments contact the surface. This is followed by a partial refolding to a low energy state; this step is continuous and results in full surface contact for the equal affinity surface and is activated and results in significant loss of surface contact for the A affinity surface. We also observe a lesser (greater) degree of average surface contact in the equal (A) affinity surface with an increase in temperature.

Monte Carlo Method↗