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

Abstract

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.

X-ray Spectromicroscopy Study of Protein Adsorption to a Polystyrene−Polylactide Blend

“…Recently, fluorescence microscopy treated by integral geometry analysis was used to quantify the adsorption of labeled lentil lectin (LcH) or concavalin A (ConA) to several polymeric surfaces including PEO crosslinked with PETA [31]. While this technique can spatially resolve and quantify lectin adsorption to the surface, it provides no information on the polymeric substrate.…”

Temperature and pH dual-responsive coatings of oligoperoxide-graft-poly(N-isopropylacrylamide): Wettability, morphology, and protein adsorption

“…Direct examination reveals significant change of proteins adsorption from not visible at 20°C to moderate at 26°C and finally strong at 29 and 34°C. A semiquantitative analysis was performed by the integral geometry approach, as described in [51,52]. It provides average values of fluorescence intensity hIi corresponding to each sample but normalized for all micrographs of the series.…”

“…The adsorbed amount of proteins, if assumed to be proportional to fluorescent intensity, is clearly reduced for the coatings in their hydrated state that can be obtained by lowering the temperature at alkaline conditions (pH 9) or by increase in pH at room temperature (22°C). However, due to the complex nature of comparative fluorescence microscopy (e.g., adjusting the recording conditions to the samples with maximal intensity [51]), it is hardly possible to confirm the antifouling character of the coatings at low temperature and high pH. To distinguish between no measurable protein adsorption and merely not detected adsorption, the TOF-SIMS technique is usually applied due to its superior chemical specificity [30,31,[53][54][55].…”

An X‐ray Spectromicroscopy Study of Albumin Adsorption to Crosslinked Polyethylene Oxide Films

“…[42] While this technique can spatially resolve and quantify lectin adsorption to the surface, it provides no information on the polymeric substrate. This fluorescence study suggested that lectin adsorption may be influenced to some extent by the presence of PETA; however, since the spun-cast PEO film studied was prepared using 15% PETA, the crosslinker is undoubtedly the dominant component of the film surface.…”