Joanna Raczkowska

Breath figures in polymer and polymer blend films spin-coated in dry and humid ambience.

We investigate effects of two spin-coating parameters, relative humidity (5% < or = RH < or = 80%) in ambient atmosphere and water content (3 wt % < or = f(H2O) < or = 20 wt %) in solution (rich in tetrahydrofuran), on the structure of breath figures (BF) formed in spin-cast films of polar poly(methyl methacrylate) (PMMA) and PMMA mixed with nonpolar polystyrene (PS). Film morphologies, examined with atomic and lateral force microscopy, are analyzed with integral geometry analysis to yield morphological BF measures. In PMMA, water added to solution has much stronger impact than that from moisture on formed BFs, which could be ordered (with conformational entropy S approximately 0.9-1.0). In PMMA/PS, BFs decorate exclusively polar PMMA domains, resulting in morphologies with two length scales (sub-micrometer BFs and domains >10 microm). This suggests a novel strategy for herarchic structure formation in multicomponent polymer films. In PS/PMMA, BFs are better developed than in pure PMMA spin-coated in identical conditions. These observations show that the air boundary layer facing the spin-cast polymer film (region) is more important than the ambient atmosphere.

Selective protein adsorption on polymer patterns formed by self-organization and soft lithography.

Thin films, with both isotropic and ordered patterns of polymer domains, are used as substrates to study selective adsorption of two proteins (concanavalin A and lentil lectin) and to test reconstruction of polymer patterns by these proteins. Integral geometry approach is used to compare quantitatively fluorescence micrographs of protein patches with AFM images of original isotropic patterns, formed during blend casting of polystyrene/poly(methyl methacrylate) and PS/poly(ethylene oxide). Preferential adsorption of both lectins to PMMA phase domains, enhanced for PS/PMMA interfaces is concluded. In turn, protein binding to PS phase regions of PS/PEO blends is highly selective. Ordered protein grouping is obtained as a result of selective adsorption to alternating stripes of polystyrene (partly brominated to enable identification) and cross-linked PEO, prepared with solvent-assisted micromolding applied to PBrS/PEO bilayers. Biological activity test, performed with concanavalin A, confirms preserved functionality of a complementary protein, carboxypeptidase Y, adsorbed to polymer patterns.

PDMS substrate stiffness affects the morphology and growth profiles of cancerous prostate and melanoma cells.

A deep understanding of the interaction between cancerous cells and surfaces is particularly important for the design of lab-on-chip devices involving the use of polydimethylsiloxane (PDMS). In our studies, the effect of PDMS substrate stiffness on mechanical properties of cancerous cells was investigated in conditions where the PDMS substrate is not covered with any of extracellular matrix proteins. Two human prostate cancer (Du145 and PC-3) and two melanoma (WM115 and WM266-4) cell lines were cultured on two groups of PDMS substrates that were characterized by distinct stiffness, i.e. 0.75 ± 0.06 MPa and 2.92 ± 0.12 MPa. The results showed the strong effect on cellular behavior and morphology. The detailed analysis of chemical and physical properties of substrates revealed that cellular behavior occurs only due to substrate elasticity.

Temperature and pH dual-responsive POEGMA-based coatings for protein adsorption

Poly(oligo(ethylene glycol)ethyl ether methacrylate (POEGMA246) coatings were successfully fabricated using novel approach via polymerization from oligoperoxide grafted to premodified glass substrate. Wettability, content and composition of coatings fabricated with different polymerization times were determined using contact angle measurements, ellipsometry and Time of Flight-Secondary Ion Mass Spectrometry (TOF-SIMS). Thermo- and pH-responsive properties of POEGMA246 coatings were found to depend significantly on concentration of the grafted POEGMA246. Coatings fabricated with polymerization time 30 h exhibit not only temperature- but also pH-dependence of wettability. Thermal response of wettability, measured between 20 and 32°C, was prominent at pH 9 and 7 and diminished or was absent at pH 5 and 3, indicating a transition between hydrated loose coils and hydrophobic collapsed chains, blocked at low pH. Protein adsorption, observed by fluorescence microscopy and analyzed semi-quantitatively using integral geometry approach, decreased dramatically for model protein (lentil lectin labeled with fluorescein isothiocyanate) at transition from pH 5 to pH 9, showing only very weak thermal-dependence. Strong protein adsorption response to pH and very weak one to temperature was confirmed by TOF-SIMS and Principal Component Analysis.

Protein coverage on silicon surfaces modified with amino-organic films: A study by AFM and angle-resolved XPS

An approach to determine structural features, such as surface fractional coverage F and thickness d of protein layers immobilized on silicon substrates coated with amino-organic films is presented. To demonstrate the proposed approach rabbit gamma globulins (RgG) are adsorbed from a 0.66muM solution onto SiO(2) and Si(3)N(4) modified with (3-aminopropyl)triethoxysilane (APTES). Atomic force microscopy data are analyzed by applying an integral geometry approach to yield average coverage values for silanized Si(3)N(4) and SiO(2) coated with RgG, F=0.99+/-0.01 and 0.76+/-0.08, respectively. To determine the RgG thickness d from angle-resolved X-ray photoelectron spectroscopy (ARXPS), a model of amino-organic bilayer with non-homogeneous top lamellae is introduced. For an APTES layer thickness of 1.0+/-0.1nm, calculated from independent ARXPS measurements, and for fractional surface RgG coverage determined from AFM analysis, this model yields d=1.0+/-0.2nm for the proteins on both silanized substrates. This value, confirmed by an evaluation (1.0+/-0.2nm) from integral geometry analysis of AFM images, is lower than the RgG thickness expected for monomolecular film ( approximately 4nm). Structures visible in phase contrast AFM micrographs support the suggested sparse molecular packing in the studied RgG layers. XPS data, compared for bulk and adsorbed RgG, suggest preferential localization of oxygen- and nitrogen-containing carbon groups at silanized silicon substrates. These results demonstrate the potential of the developed AFM/ARXPS approach as a method for the evaluation of surface-protein coverage homogeneity and estimation of adsorbed proteins conformation on silane-modified silicon substrates used in bioanalytical applications.

Integral Geometry Analysis of Fluorescence Micrographs for Quantitative Relative Comparison of Protein Adsorption onto Polymer Surfaces

Most methods developed to study protein binding to distinct surfaces can only determine the average amount of adsorbed protein or merely provide (qualitative) information on its spatial distribution. Both these features can be characterized rigorously by integral geometry analysis of fluorescence micrographs. This approach is introduced here to compare the relative protein adsorption onto various polymer surfaces: polystyrene (PS), poly(methyl methacrylate) (PMMA), poly( n-butyl methacrylate) (PnBMA), poly( tert-butyl methacrylate) (PtBMA), and PS(PETA) and cross-linked poly(ethylene oxide) (PEO*(PETA)), admixed with pentaerythritol triacrylate (PETA). The polymeric surfaces were incubated for 15 min in phosphate-buffered saline (pH 7.4) containing 125 mug/mL fluorescently labeled lectins, either lentil lectin (LcH) or concanavalin A (ConA). Fluorescence images were recorded at identical conditions (physiological buffer, same exposure time, magnification, gain). For each image, taken a few times for each polymer, the distribution and average value of the normalized intensity were determined. The results show that the binding of LcH to PS(PETA), PtBMA, PS, PnBMA, PMMA, and PEO*(PETA) can be expressed by the ratio of the following values (mean +/- 95% confidence interval): 0.356 +/- 0.022, 0.298 +/- 0.030, 0.241 +/- 0.014, 0.083 +/- 0.008, 0.039 +/- 0.008, and 0.010 +/- 0.006, respectively. In turn, the relative adsorption of ConA is described by the values 0.252 +/- 0.016, 0.217 +/- 0.014, 0.222 +/- 0.016, 0.046 +/- 0.006, 0.116 +/- 0.008, and 0.006 +/- 0.002, respectively. Low dispersions of fluorescence intensity around average values indicate homogeneous distribution of adsorbed proteins. The introduced approach enables a fast and easy way not only to quantify the relative amount of bound proteins but also to characterize quantitatively the organization of their surface distribution, as demonstrated for patchlike protein adsorption onto the polymer blend surface.

Synthesis and Postpolymerization Modification of Thermoresponsive Coatings Based on Pentaerythritol Monomethacrylate: Surface Analysis, Wettability, and Protein Adsorption

Properties of novel temperature-responsive hydroxyl-containing poly(pentaerythritol monomethacrylate) (PPM) coatings, polymerized from oligoperoxide grafted to glass surface premodified with (3-aminopropyl)triethoxysilane, are presented. Molecular composition, chemical state, thickness, and wettability are examined with time of flight-secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), ellipsometry, and contact angle measurements, respectively. Temperature-induced changes in hydrophobicity of grafted PPM brushes are revealed by water contact angle and ellipsometric measurements. Partial postpolymerization modification of hydroxyl groups (maximum a few percent), performed with acetyl chloride or pyromellitic acid chloride, is demonstrated to preserve thermal response of coatings. Adsorption of bovine serum albumin to PPM brushes, observed with fluorescence microscopy, is higher than on glass in contrast to similar hydroxyl-containing layers reported as nonfouling. Enhanced and temperature-controlled protein adsorption is obtained after postpolymerization modification with pyromellitic acid chloride.

Temperature-Controlled Three-Stage Switching of Wetting, Morphology, and Protein Adsorption

The novel polymeric coatings of oligoperoxide-graft-poly(4-vinylpyridine-co-oligo(ethylene glycol)ethyl ether methacrylate246) [oligoperoxide-graft-P(4VP-co-OEGMA246)] attached to glass were successfully fabricated. The composition, thickness, morphology, and wettability of resulting coatings were analyzed using X-ray photoelectron spectroscopy, ellipsometry, atomic force microscopy, and contact angle measurements, respectively. In addition, adsorption of the bovine serum albumin was examined with fluorescence microscopy. The thermal response of wettability and morphology of the coatings followed by that of protein adsorption revealed two distinct transitions at 10 and 23 °C. For the first time, three stage switching was observed not only for surface wetting but also for morphology and protein adsorption. Moreover, the influence of the pH on thermo-sensitivity of modified surfaces was shown.

Temperature-responsive properties of poly(4-vinylpyridine) coatings: influence of temperature on the wettability, morphology, and protein adsorption

Although the pH-response of poly(vinylpyridine)-based systems is well-known and indeed used in several biomedical applications, the impact of temperature on the properties of this polymer has not been investigated in detail so far. Herein, we demonstrate the temperature-responsiveness and switchable wettability of two poly(4-vinylpyridine) coatings, mimicking the behavior of materials with lower critical solution temperature. The thermal response of P4VP spin-coated films, solvent cast on a glass, is weaker than that observed for P4VP-grafted brushes, fabricated via polymerization from an oligoperoxide grafted on an amino-silanized glass. Both the P4VP coatings exhibit a temperature dependence of the water contact angle with a well-defined transition at 13–14 °C. This transition is absent at acid pH levels wherein almost all pyridyl groups are protonated. The P4VP-grafted brushes were used to examine the impact of temperature on the surface morphology and protein adsorption. The coating surface, recorded with atomic force microscopy, evolved noticeably at alkaline pH, from being relatively smooth at 10 °C to structured and rough at 20 °C. In turn, at acid pH levels, flat surfaces with rare elevations were observed at both temperatures. The adsorption of bovine serum albumin and human fibrinogen was observed with fluorescence microscopy to be significantly more efficient for temperatures above the transition, indicating that P4VP coatings can act as a noteworthy switching material.

Binding activity of patterned concanavalin A studied by atomic force microscopy

The mode of protein immobilization plays a crucial role in the preparation of protein microarrays used for a wide spectrum of applications in analytical biochemistry. The microcontact printing technique was used to form a protein pattern using concanavalin A (Con A) since Con A belongs to a group of proteins widely used in analytical assays due to their selectivity as regards different kinds of carbohydrates. Atomic force microscopy was used to image surface topography, delivering information about the quality of the protein pattern. The force spectroscopy mode was used to verify the functional activity of deposited proteins via determination of the forces of interaction between Con A and carboxypeptidase Y bearing carbohydrate structure recognized by Con A. The calculated binding force between Con A and CaY was 105 ± 2 pN and it was compared with that measured for Con A deposited directly from the protein solution. The similarity of the value obtained for the interaction force was independent of the mode of protein deposition, thereby verifying that the microcontact printing technique did not influence the carbohydrate binding activity of Con A. The correlation between the surface topography of patterned samples and adhesion maps obtained showed the possible use of AFM for studying the chemical properties of different regions of the micropatterns produced.