Teresa Basinska

Highly hydrophilic surfaces from polyglycidol grafts with dual antifouling and specific protein recognition properties.

Homopolymer grafts from α-tert-butoxy-ω-vinylbenzyl-polyglycidol (PGL) were prepared on gold and stainless steel (SS) substrates modified by 4-benzoyl-phenyl (BP) moieties derived from the electroreduction of the parent salt 4-benzoyl benzene diazonium tetrafluoroborate. The grafted BP aryl groups efficiently served to surface-initiate photopolymerization (SIPP) of PGL. In similar conditions, SIPP of hydroxyethyl methacrylate (HEMA) permitted the production of PHEMA grafts as model surfaces. Water contact angles were found to be 66°, 15°, and 0° for SS-BP, SS-PHEMA, and SS-PPGL, respectively. The spontaneous spreading of water drops on SS-PPGL was invariably observed with 1.5 μL water drops. PPGL thus appears as a superhydrophilic polymer. Resistance to nonspecific adsorption of proteins of PPGL and PHEMA grafts on gold was evaluated by surface plasmon resonance (SPR) using antibovine serum albumin (anti-BSA). The results conclusively show that PPGL-grafts exhibit enhanced resistance to anti-BSA adsorption compared to the well-known hydrophilic PHEMA. PPGL grafts were further modified with BSA through the carbonyldiimidazole activation of the OH groups providing immunosensing surfaces. The so-prepared PPGL-grafted BSA hybrids specifically interacted with anti-BSA in PBS as compared to antimyoglobin. It is clear that the superhydrophilic character of PPGL grafts opens new avenues for biomedical applications where surfaces with dual functionality, namely, specific protein grafting together with resistance to biofouling, are required.

The direct determination of protein concentration for proteins immobilized on polystyrene microspheres

In this paper we show how the Lowry method, designed for the determination of proteins in solution, can be used for the determination of proteins immobilized on the surface of microspheres (for protein concentrations higher than 10 micrograms/ml). Measurements were made for human serum albumin (HSA) and for immunoglobulins [rabbit immunoglobulins--antibodies against human fibrinogen (IgGF) and against fragment D of human fibrinogen (IgGFgD)] immobilized on the surface of polystyrene microspheres.

Attachment of horseradish peroxidase (HRP) onto the poly(styrene/acrolein) latexes and onto their derivatives with amino groups on the surface; activity of immobilized enzyme

The polystyrene (P(S)), poly(styrene/acrolein) (P(SA)), and polyacrolein (P(A)) latexes, with varied fraction of polyacrolein in the surface layer (fA=0, 0.50, 0.63, 0.84, 1.00), were used for the attachment of horseradish peroxidase. Surfaces of latexes were modified by reaction with ethylenediamine. In this step the aldehyde groups from polyacrolein were blocked and the primary amino groups were introduced. The carbohydrate portion of HRP was oxidized in the reaction leading to formation of aldehyde groups. The adsorption and covalent immobilization of HRP onto the P(S), P(SA), and P(A) latexes and of the oxidized HRP (HRP-OX) onto the modified latex particles, with amino groups on the surface (P(SA)-M and P(A)-M), were investigated. The activities of parent and oxidized HRP were compared with activities of the corresponding enzymes in solution. It has been found that whereas HRP is not suitable for the covalent immobilization on P(SA) latex and loses its activity after adsorption onto P(S) latex, HRP-OX can be adsorbed onto P(S) latex and is readily immobilized covalently onto the ethylenediamine modified P(SA) and P(A) latexes, retaining much of its former enzymatic reactivity.

Synthesis and Characterization of Polystyrene Core/Polyacrolein Shell Latexes

The poly(styrene/acrolein) latexes were synthesized in an emul sifier-free emulsion-precipitation polymerization. Monodisperse particles from 0.30 μm to 0.52 μm, depending on the acrolein monomer feed, were obtained. More acrolein in the monomer feed yielded latex particles with smaller di ameters. Analyses indicate that the particles have a core-shell morphology. The core is rich in the hydrophobic (polystyrene) component whereas the shell is composed mainly of hydrophilic polyacrolein. Significant changes in polyacro lein in the latexes (from 0.03 to 0.28) has less influence on the composition of the shell (from 0.5 to 0.84, respectively). The surface of the latex particles is smooth and can be penetrated by 2,4-dinitrophenyl hydrazine to the depth from 1.5 to 3.5 Å. These poly(styrene/acrolein) latexes are capable of binding ca. 3 mg of human globulins or ca. 1 mg of human serum albumin on 1 m2 of the latex surface.

Design of polyglycidol-containing microspheres for biomedical applications

The paper presents a short review on the synthesis, characterisation and selected medical applications of poly(styrene/α-tert-butoxy-ω-vinylbenzyl-polyglycidol) (P(S/PGL)) microspheres. The soap-free emulsion-polymerisation of styrene and α-tert-butoxy-ω-vinylbenzyl-polyglycidol macromonomer (PGL) in water yielded core-shell microspheres with a low particle-diameter dispersity (ratio of the weight average particle diameter and the number average particle diameter). The interfacial fraction of PGL units, estimated by XPS, was in the range of 0–42 mole % depending on the concentration of the macromonomer in the polymerisation feed. The studies of adsorption of model proteins showed that the surface fraction of adsorbed protein was significantly reduced when the PGL interfacial fraction was higher than 40 mole %. The P(S/PGL) particles with covalently immobilised proteins were used for the preparation of photonic crystal assemblies suitable for applications in optical biosensors and the medical diagnostic test for the detection of Helicobacter pylori antibodies in the blood serum.

Thermoresponsive Colloidal Crystals Built from Core−Shell Poly(styrene/α-tert-butoxy-ω-vinylbenzylpolyglycidol) Microspheres

Core-shell particles of poly(styrene/alpha-tert-butoxy-omega-vinylbenzylpolyglycidol) P(S/PGL) were used as new building blocks for the assembly of a colloidal crystal. The added-value properties of these particles for photonic crystal architectures are their high hydrophilicity together with their thermoresponsivity. Indeed, the poglycidol-rich shell undergoes a phase transition above 45 degrees C, which leads to its collapse at the particle surface accompanied by a decrease in the particle diameter. The three-dimensional crystalline arrays display Bragg diffraction properties, as judged by angle-resolved reflectance spectroscopy. The thermoresponsivity of the colloidal assemblies was observed through modifications of their optical properties with respect to the temperature used during the assembly process. The wetting properties of the crystalline material were also shown to reversibly switch from hydrophilic to hydrophobic as a function of the assembly temperature, thus evidencing the reorganization of the surface polyglycidol chains during the polymer phase transition. This work shows conclusively that P(S/PGL) particles are promising alternatives to poly(N-isopropylacrylamide) and poly(ethylene glycol) particles for the elaboration of thermoresponsive colloidal crystals, with a phase transition situated in between those of these two polymers.

Adsorption studies of human serum albumin, human γ-globulins, and human fibrinogen on the surface of P(S/PGL) microspheres

Adsorption of human serum albumin (HSA), human γ-globulins (γ G), and human fibrinogen (Fb) onto the surface of poly(styrene/α-t-butoxy-ω-vinylbenzyl-polyglycidol) microspheres (P(S/PGL)) with controlled fraction of polyglycidol in the interfacial layer was investigated. The microspheres were synthesized by the emulsifier-free radical copolymerization of styrene and α-tbutoxy-ω-vinylbenzyl-polyglycidol macromonomer (PGL). Macromonomers with number average molecular weights M n = 950 and 2700 were used in the syntheses. Fraction of polyglycidol in the microsphere surface layer was varied from 0.22 to 0.44, depending on the composition of the monomer feed. It was found that the maximal surface concentration of adsorbed proteins and the equilibrium constant of protein adsorption decreased with increased fraction of polyglycidol in the microsphere surface layer. For microspheres with the highest fraction of polyglycidol at the surface the maximal surface protein concentration was c. ten times lower and the adsorption equilibrium constant was c. one hundred times lower than for the reference polystyrene microspheres. The dependence of maximal surface concentration of adsorbed proteins on the fraction of polyglycidol in the particle interfacial layer indicated random distribution of polyglycidol chains without formation of polyglycidol and polystyrene patches at the microspheres surface.

Composite poly(methyl methacrylate-methacrylic acid-2-hydroxyethyl methacrylate) latex for immunoassay. The case of plasminogen

Poly(methyl methacrylate-methacrylic acid-2-hydroxyethyl methacrylate) latex (ACRYLAT) was synthesized by radical precipitation polymerization. The mass median diameter (MMD) and the geometrical standard deviation (GSD) of the ACRYLAT particles were 138 nm and 1.2, respectively. The concentration of the titrable carboxylic groups in the surface layer of latex particles was equal to 8.41 x 10(-6) mol m-2. Latex was able to bind up to 2.82 x 10(-7) mol of 1-aminopyrene per 1 m2 of the surface of the latex particles due to the ionic interactions between carboxylate anions and ammonium cations of protonated 1-aminopyrene. ACRYLAT was able to immobilize covalently human serum albumin in amounts up to 0.23 mg m-2. Aggregation of ACRYLAT with immobilized HSA, induced with specific antibodies (anti-HSA), was investigated turbidimetrically. The results indicated that in the model turbidimetric immunoassay, ACRYLAT coated with HSA can be used for the detection of anti-HSA in the goat anti-HSA serum diluted from 50 to 7000-fold. Immobilization of rabbit antibodies to plasminogen (anti-Plg) to ACRYLAT via the epsilon-aminocaproic acid linkers provided particles which were used for the development of the turbidimetric immunoassay for plasminogen. In this assay plasminogen could be detected in concentration ranging from 0.75 to 75 micrograms ml-1 in the blood plasma.

Polyglycidol, Its Derivatives, and Polyglycidol-Containing Copolymers—Synthesis and Medical Applications

Polyglycidol (or polyglycerol) is a biocompatible polymer with a main chain structure similar to that of poly(ethylene oxide) but with a –CH2OH reactive side group in every structural unit. The hydroxyl groups in polyglycidol not only increase the hydrophilicity of this polymer but also allow for its modification, leading to polymers with carboxyl, amine, and vinyl groups, as well as to polymers with bonded aliphatic chains, sugar moieties, and covalently immobilized bioactive compounds in particular proteins. The paper describes the current state of knowledge on the synthesis of polyglycidols with various topology (linear, branched, and star-like) and with various molar masses. We provide information on polyglycidol-rich surfaces with protein-repelling properties. We also describe methods for the synthesis of polyglycidol-containing copolymers and the preparation of nano- and microparticles that could be derived from these copolymers. The paper summarizes recent advances in the application of polyglycidol and polyglycidol-containing polymers as drug carriers, reagents for diagnostic systems, and elements of biosensors.

Polystyrene and Poly(Styrene/Acrolein) Latexes with Immobilized Proteins as a Basis of the Diagnostic Agglutination Test

Properties of the poly(styrenefacrolein) latexes (PSA), with controlled surface concentration of aldehyde groups, as carriers of proteins (human serum albumin (HSA) and gamma globulins (yG)) are described. It has been found that the maximal surface concentration of attached proteins decreases with increased fraction of polyacrolein in the surface layer. This suggests that initially proteins are attached flatly to the latex particles. At the later stages the already adsorbed protein macromolecules change their orientation, become packed protruding from the surface, and make possible accommodation of new protein macromolecules coming from the solution. Such rearrangement is suppressed in the case of proteins attached covalently. Determination of the surface concentrations of proteins, adsorbed and covalently immobilized onto the PSA latexes, indicate that for the latexes with higher content of polyacrolein the surface concentration of adsorbed protein is lower. From the adsorption and immobilization isotherms for HSA it follows that the physical adsorption is preferred. Apparently, the Hydrophobic patches, suitable for adsorption, are covered first and only thereafter the covalent immobilization begins to play an important role. For γ G the different picture has been observed. Namely, the areas suitable for the covalent immobilization are covered first. Presumably, denaturation of the “soft” HSA macromolecules, at the latex-solvent interface, facilitates the Hydrophobie protein-latex interactions. In the case of yG macromolecules, which are less prone to denaturation, the covalent immobilization competes successfully with adsorption. The described latexes were used in the model test for detection of anti-HSA. Some were used for manufacturing of the diagnostic test for determination of fibrinogen degradation products in the blood serum and in urine.