Tomáš Riedel

Interaction of blood plasma with antifouling surfaces.

Nonspecific adsorption of proteins is a crucial problem in the detection of analytes in complex biological media by affinity sensors operating with label-free detection. We modified the gold surface of surface plasmon resonance (SPR) sensors with three types of promising antifouling coatings: self-assembled monolayers (SAM)s of alkanethiolates terminated with diethylene glycol and carboxylic groups, poly(ethylene glycol) (PEG) grafted onto the SAMs, and zwitterionic polymer brushes of poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), and poly(phosphorylcholine methacrylate). Using SPR, we compared the efficacy of the coatings to reduce nonspecific adsorption from human blood plasma and from single-protein solutions of human serum albumin, immunoglobulin G, fibrinogen, and lysozyme. There was no direct relationship between values of water contact angles and plasma deposition on the coated surfaces. A rather high plasma deposition on SAMs was decreased by grafting PEG chains. Fouling on PEG was observed only from plasma fractions containing proteins with molecular mass higher than 350 000 Da. The adsorption kinetics from plasma collected from different healthy donors differed. Poly(carboxybetaine methacrylate) completely prevented the deposition from plasma, but the other more hydrophilic zwitterionic polymers prevented single-protein adsorption but did not prevent plasma deposition. The results suggest that neither wettability nor adsorption of the main plasma proteins was the main indicator of deposition from blood plasma.

Polymer Brushes Showing Non-Fouling in Blood Plasma Challenge the Currently Accepted Design of Protein Resistant Surfaces

Ultra-low-fouling poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)) brushes have been synthesized for the first time. Similar to the so far only ultra-low-fouling surface, poly(carboxybetaine acrylamide), the level of blood plasma fouling was below the detection limit of surface plasmon resonance (SPR, 0.03 ng·cm(-2)) despite being a hydrogen bond donor and displaying a moderate wettability, thus challenging the currently accepted views for the design of antifouling properties. The antifouling properties were preserved even after two years of storage. To demonstrate the potential of poly(HPMA) brushes for the preparation of bioactive ultra-low fouling surfaces a label-free SPR immunosensor for detection of G Streptococcus was prepared.

Complete identification of proteins responsible for human blood plasma fouling on poly(ethylene glycol)-based surfaces.

The resistance of poly(ethylene glycol) (PEG) against protein adsorption is crucial and has been widely utilized in various biomedical applications. In this work, the complete protein composition of biofilms deposited on PEG-based surfaces from human blood plasma (BP) was identified for the first time using nanoLC-MS/MS, a powerful tool in protein analysis. The mass of deposited BP and the number of different proteins contained in the deposits on individual surfaces decreased in the order of self-assembling monolayers of oligo(ethylene glycol) alkanethiolates (SAM) > poly(ethylene glycol) end-grafted onto a SAM > poly(oligo(ethylene glycol) methacrylate) brushes prepared by surface initiated polymerization (poly(OEGMA)). The BP deposit on the poly(OEGMA) surface was composed only of apolipoprotein A-I, apolipoprotein B-100, complement C3, complement C4-A, complement C4-B, histidine-rich glycoprotein, Ig mu chain C region, fibrinogen (Fbg), and serum albumin (HSA). The total resistance of the surface to the Fbg and HSA adsorption from single protein solutions suggested that their deposition from BP was mediated by some of the other proteins. Current theories of protein resistance are not sufficient to explain the observed plasma fouling. The research focused on the identified proteins, and the experimental approach used in this work can provide the basis for the understanding and rational design of plasma-resistant surfaces.

Functionalized ultra-low fouling carboxy- and hydroxy-functional surface platforms: functionalization capacity, biorecognition capability and resistance to fouling from undiluted biological media.

The non-specific binding of non-target species to functionalized surfaces of biosensors continues to be challenge for biosensing in real-world media. Three different low-fouling and functionalizable surface platforms were employed to study the effect of functionalization on fouling resistance from several types of undiluted media including blood plasma and food media. The surface platforms investigated in this work included two polymer brushes: hydroxy-functional poly(2-hydroxyethyl methacrylate) (pHEMA) and carboxy-functional poly(carboxybetaine acrylamide) (pCBAA), and a standard OEG-based carboxy-functional alkanethiolate self-assembled monolayer (AT-SAM). The wet and dry polymer brushes were analyzed by AFM, ellipsometry, FT-IRRAS, and surface plasmon resonance (SPR). The surfaces were functionalized by the covalent attachment of antibodies, streptavidin, and oligonucleotides and the binding and biorecognition characteristics of the coatings were compared. We found that functionalization did not substantially affect the ultra-low fouling properties of pCBAA (plasma fouling of ~20 ng/cm(2)), a finding in contrast with pHEMA that completely lost its resistance to fouling after the activation of hydroxyl groups. Blocking a functionalized AT-SAM covalently with BSA decreased fouling down to the level comparable to unblocked pCBAA. However, the biorecognition capability of blocked functionalized AT-SAM was poor in comparison with functionalized pCBAA. Limits of detection of Escherichia coli O157:H7 in undiluted milk were determined to be 6×10(4), 8×10(5), and 6×10(5) cells/ml for pCBAA, pHEMA, and AT-SAM-blocked, respectively. Effect of analyte size on biorecognition activity of functionalized coatings was investigated and it was shown that the best performance in terms of overall fouling resistance and biorecognition capability is provided by pCBAA.

Use of pooled blood plasmas in the assessment of fouling resistance

The ability of a surface to resist fouling from blood plasma cannot be realistically estimated without measuring adsorption from real samples directly. Due to the variability of biological samples, pooled blood plasma is normally used. We show that even when using pooled plasma, a comparison of antifouling surfaces strongly depends on its source.

Controlled preparation of thin fibrin films immobilized at solid surfaces

A technique for coating surfaces with attached fibrin structures without the formation of fibrin gel in bulk solution was developed. It is based on the catalytic effect of the surface-bound thrombin on fibrinogen stabilized with inhibitor which inhibits thrombin in solution but not the thrombin on the surface. Such an inhibitor is antithrombin, the effect of which may be enhanced with heparin. Fibrinogen is first adsorbed on the substrate surface and then incubated with thrombin. The unbound thrombin is washed out and the surface is incubated with fibrinogen solution containing antithrombin III and heparin. A fibrin gel forms at the surface by the action of surface-bound thrombin on ambient fibrinogen solution; however, the gel formation in bulk solution catalyzed by thrombin partially released from the surface is suppressed. By utilizing antithrombin-independent inhibitors or repeating thrombin binding and incubation with fibrinogen solution, the amount of surface-attached fibrin gel can be controlled. The formation of immobilized fibrin networks was observed using surface plasmon resonance and turbidity measurements and morphology was observed by TEM, SEM, and AFM. Using this technique, a porous scaffold made of polylactide fibers was coated with fibrin without filling the space between fibers with a bulk fibrin gel. The technique makes it possible to coat the inner surface of porous scaffolds with surface-attached fibrin gel while preserving free volume for cell migration into the pores.

Vascular endothelial cells on two-and three-dimensional fibrin assemblies for biomaterial coatings

Various techniques for coating synthetic surfaces with fibrin structures were tested for seeding bovine pulmonary artery endothelial cells (EC). Two-dimensional fibrin (Fb) structures (2D Fb) were obtained by successively repeating adsorption of fibrinogen (Fbg), incubating the surface with thrombin (Thr) solution, and inhibiting surface-attached Thr. Three-dimensional fibrin networks immobilized at the surface (3D Fb) were formed by catalytic action of surface-attached thrombin on an ambient Fbg solution. Ultra-thin 3D Fbs were obtained if thrombin inhibitors antithrombin III and heparin were added into an Fbg solution. The formation of surface fibrin structures was observed in situ using surface plasmon resonance. The morphology of the structures was studied by transmission and scanning electron microscopy. A polylactide fibrous scaffold was modified with a surface fibrin film without filling the inner pores with a bulk gel. The growth of EC seeded on a polystyrene surface coated with the Fb films was evaluated by the number and morphology of the adhering ECs and the concentration of beta-actin, vinculin, alpha(v)-intergrin, and von Willebrand factor (vWF). The best initial cell spreading after 1 day was observed on 2D Fb and ultra-thin 3D Fb. The highest concentration of vWF, a marker of EC differentiation, was observed after 3 days on thick 3D Fbs. The highest EC population densities after 7 days were observed on 2D Fb and thick 3D Fb.

Polymer Brushes Interfacing Blood as a Route Toward High Performance Blood Contacting Devices

In the current study, well-defined polymer brushes are shown as an effective surface modification to resist the adhesion of whole blood and its components. Poly[oligo(ethylene glycol)methylether methacrylate] (poly(MeOEGMA)), poly(hydroxyethyl methacrylate) (poly(HEMA)), poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)), and poly(carboxybetaine acrylamide) (poly(CBAA)) brushes were grown by surface initiated atom transfer radical polymerization (SI-ATRP) and subsequently characterized by Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), dynamic contact angle measurements, atomic force microscopy (AFM), and surface plasmon resonance (SPR) spectroscopy. All brushes decreased the fouling from blood plasma over 95% and prevented the adhesion of platelets, erythrocytes, and leukocytes as evidenced by SPR and SEM measurements.

Hepatitis B plasmonic biosensor for the analysis of clinical serum samples

A plasmonic biosensor for rapid detection of protein biomarkers in complex media is reported. Clinical serum samples were analyzed by using a novel biointerface architecture based on poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] brushes functionalized with bioreceptors. This biointerface provided an excellent resistance to fouling even after the functionalization and allowed for the first time the direct detection of antibodies against hepatitis B surface antigen (anti-HBs) in clinical serum samples using surface plasmon resonance (SPR). The fabricated SPR biosensor allowed discrimination of anti-HBs positive and negative clinical samples in 10min. Results are validated by enzyme-linked immunoassays of the sera in a certified laboratory. The sensor could be regenerated by simple treatment with glycine buffer.

Improved adhesion and differentiation of endothelial cells on surface-attached fibrin structures containing extracellular matrix proteins.

Currently used vascular prostheses are hydrophobic and do not allow endothelial cell (EC) adhesion and growth. The aim of this study was to prepare fibrin (Fb)-based two-dimensional (2D) and three-dimensional (3D) assemblies coated with extracellular matrix (ECM) proteins and to evaluate the EC adhesion, proliferation and differentiation on these assemblies in vitro. Coating of Fb with collagen, laminin (LM), and fibronectin (FN) was proved using the surface plasmon resonance technique. On all Fb assemblies, ECs reached higher cell densities than on polystyrene after 3 and 7 days of culture. Immunoflurescence staining showed better assembly of talin and vinculin into focal adhesion plaques, and also more apparent staining of vascular endothelial cadherin on surface-attached 3D Fb and protein-coated Fb assemblies. On these samples, ECs also contained a lower concentration of intercellular adhesion molecule-1, measured by enzyme-linked immunosorbent assay. Higher concentrations of CD31 (platelet-endothelial cell adhesion molecule-1) were found on 3D Fb coated with LM, and higher concentrations of von Willebrand factor were found on 3D Fb coated with type I collagen or LM in comparison to 2D Fb layers. The results indicate that ECM protein-coated 2D and 3D Fb assemblies can be used for versatile applications in various tissue replacements where endothelialization is desirable, for example, vascular prostheses and heart valves.