Beata Miksa

Polypyrrole core/polyacrolein shell latex for protein immobilization

The redox polymerization of pyrrole, with ferric chloride as oxidant, carried out in the presence of polyvinylpyrrolidone (PVP), yielded polypyrrole latex particles. The polypyrrole latex was used, as seed, for the radical polymerization of acrolein. The resulting polypyrrole core/polyacrolein shell latex (poly(P/A)) was suitable for immobilization of up to 11 mg of human serum albumin (HSA) and/or 33 mg of human gamma globulin (γG) per 1 g of latex particles.

Inorganic–organic systems with tailored properties controlled on molecular, macromolecular and microscopic level

Abstract Methods suitable for modification of quartz and glass (plates and beads) on molecular, macromolecular and microscopic levels are described in this paper. The modifications led to composite materials with the core containing quartz (fused silica) or glass and outer shells composed of layers of γ-aminopropyltriethoxysilane (small molecules), dendrimers with aldehyde groups (G5) and/or Starburst PAMAM dendrimers with amino groups (macromolecules), and with one particle thick assemblies of poly(styrene–divinylbenzene–acrolein) microspheres with aldehyde groups at their surface (microscopic objects). Monitoring by AFM quartz plates with immobilized dendrimers revealed that their surfaces are relatively smooth with roughness parameter R g close to 2 nm and with diameters and maximal height of objects covering surface in form of bumps close to 4 nm, i.e. to diameters of single macromolecules of dendrimers. XPS studies of surfaces of modified quartz plates and glass beads unequivocally confirmed attachment of γ-aminopropyltriethoxysilane, dendrimers, and microspheres. Quantitative analysis revealed that dendrimer layers are 3.7 and 3.2 nm thick, for G5 and PAMAM dendrimers, respectively, indicating that these macromolecules form monolayer structures. XPS and SEM studies of quartz surface covered with layers of γ-aminopropyltriethoxysilane, G5 and PAMAM denrimers and subsequently with poly(styrene–divinylbenzene–acrolein) microspheres indicated that saturation of the surface coverage with these particles is achieved at the degree of coverage close to 0.60, i.e. to the maximal value attainable in the case of rapid covalent immobilization of spherical particles. Surfaces of quartz plates and glass beads, with outer layers containing G5 dendrimers and/or poly(styrene–divinylbenzene–acrolein) microspheres and thus, equipped with aldehyde groups, were used for covalent immobilization of human serum albumin (HSA). AFM studies revealed that macromolecules of HSA from dilute protein solution are attached onto surface of G5 dendrimers in such a way that they form clusters composed of not less than 50 protein macromolecules each. X-ray photoelectron spectroscopy used for monitoring of surfaces with immobilized poly(styrene–divinylbenzene–acrolein) microspheres which were subsequently exposed to contact with solution of HSA revealed that macromolecules of HSA are readily attached onto immobilized microspheres.

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.

Application of ionic liquid matrices in spectral analysis of poly(lactide) - solid state NMR spectroscopy versus matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

The work presented here shows the complementarity of Solid State NMR (SS NMR) spectroscopy and Matrix-Assisted Laser Desorption/Ionization-time-of-flight (MALDI-TOF) mass spectrometry in spectral analysis of poly(L-lactide) (PLLA) using second-generation ionic liquid matrices (ILM II) prepared from N,N-diisopropylethylamine (DEA) and DHB (2,5-dihydroxybenzoic acid), IAA (3-indoleacrylic acid), and HABA (2-(4-hydroxyphenylazo) benzoic acid). The 13C cross-polarization (CP) magic angle spinning (CP/MAS) SS NMR technique was used to study the structure of ionic liquid matrices, their thermal stability, and the influence of ILM on the morphology of polymer. A comparison of MALDI-TOF spectra for samples prepared employing the dried droplet (DD) and the solvent free (SF) mode showed that the former approach gave better results (signal to noise ratio) very likely due to intimate contact between analyte and matrix domains. This hypothesis was verified by analysis of CP build-up curves for DHB–DEA–PLLA samples prepared employing both methods. We also showed that an alternative method of sample preparation based on the melting of ILM II together with a suspended polymer in the liquid matrix is unsatisfactory, particularly for those matrices which can undergo isomerization at higher temperatures (e.g., HABA–DEA and IAA–DEA).

Unique activity of ureases immobilized on poly(styrene-co-acrolein) microspheres

Poly(styrene-co-acrolein) (P(S/A)) microspheres were synthesized by emulsifier-free emulsion-dispersion radical copolymerization of styrene and acrolein. Particles with a number average diameter (øD n) of 373 nm, a narrow diameter distribution (øD w/øD n) of 1.008 and a surface concentration of aldehyde groups from polyacrolein units of 2.51 · 10−6 mol/m2 were obtained. Ureases from jack beans (Urs-JB) and from pigeonpea (Urs-PP) were immobilized onto these microspheres. Activity of free and immobilized urease was determined using a standard phenol method. Surface concentration of attached enzymes was varied in the range from 0.06 to 1.6 mg/m2. Specific activity of Urs-JB in solution was 1.9 times higher than that of Urs-PP, however, upon immobilization of enzymes onto P(S/A) microspheres the reverse was true. Due to very high denaturation activity of immobilized Urs-JB was 10 times lower than in solution. In the case of Urs-PP, denaturation of the enzyme upon immobilization was much lower and possibly due to a cooperative effect resulting from a local high concentration of immobilized enzyme, its specific activity was from 3 to 9 times higher than that of immobilized Urs-JB.

Photosensitive nanocapsules for use in imaging from poly(styrene-co-divinylbenzene) cross-linked with coumarin derivatives.

The study objective was to generate biocompatible probes and develop a stable macromolecule imaging system that are based on nanolipopolymersomes and can be used in living cells. We synthesized nanolipopolymersomes with a fluorescent polymer wall surrounded by an outer phospholipid shell that exhibits potential for the controlled delivery of diagnostic agents to cells. We describe a new type of probe suitable for dual detection methods (spectrophotometric and fluorescence). This aspect makes it unique among currently available probes because allows it to be detected with greater accuracy. We developed a highly fluorescent coumarinated polymer to overcome the limited brightness of conventional dyes with insufficient for long-term photostablility. Hydrophilic dyes (Lucifer yellow, Procion red, Procion blue) are entrapped in the aqueous core of stable polymeric nanocapsules with coumarin 6 embedded in a nanometre-thick poly(styrene-co-divinylbenzene) wall. Target compounds can be incorporated into nanocapsules in a single step. The hydrophilic phospholipids outer shell ensures biocompatibility and facilitates cell penetration. In this way, the novel fluorescent hybrid materials can help of nanotechnology.

Adsorption and covalent immobilization of human serum albumin (HSA) and gamma globulins (γG) onto poly(styrene/acrolein) latexes with pyrene, dansyl, and 2,4-dinitrophenyl labels

The poly(styrene/acrolein) latexes (P(SA)1 and P(SA)2), differing in poly(acrolein) content, were synthesized by the emulsifier-less emulsion-precipitation polymerization of styrene and acrolein. The fraction of poly(acrolein) in the surface layer was 0.35 and 0.50, for the P(SA)1 and P(SA)2 latex, respectively. Latexes were labelled with 2,4-dinitrophenylhydrazine (DNPH), dansylhdrazine (DAH), and 1-aminopyrene (APY). Surface concentration of labels varied from 4.20.10(-7) mol m-2 (for APY label on P(SA)1 latex) to 1.54.10(-6) mol m-2 (for DNPH label on P(SA)2 latex) reflecting the fraction of polyacrolein in the surface layer and bulkiness of the label. The differences between adsorption and covalent immobilization of human serum albumin and gamma globulins onto the P(SA)2 latex and onto its derivatives labelled with the 2,4-dinitrophenyl (DNP), dansyl (DA), and pyrene (PY) groups were small. The observation conforms to the hypothesis that polyacrolein forms domains on the surface of the P(SA) latexes and that after labelling some aldehyde groups are still available for the covalent immobilization of proteins. Labelled and parent latexes were used in the model slide and turbidimetric aggregation tests for the goat anti-HSA. The fluorescent latexes, labelled with APY and DAH, and latexes labelled and with DNPH were found to be suitable for the model tests, similarly as the nonlabelled ones, however, some differences in the sensitivity, depending on the presence and the nature of labels, were noticed. The standard goat anti-HSA serum (Sigma) was detected at maximum dilution equal to 2000 in the slide test, and in the dilution region from 1.8.10(3) to 4.7.10(6) times in the turbidimetric test.

Covalent immobilization of urease on polypyrrole microspheres for application as a urea biosensor

Abstract Urease has been covalently immobilized on polypyrrole microspheres chemically linked to conducting polypyrrole-polyvinyl sulfonate (PPY-PVS) films. These films were electrochemically prepared during 5 - 7 min at a constant current of 2 mA using indium - tin oxide (ITO) glass plates as the working electrode, and a standard calomel electrode as the reference electrode. Urease covalently linked to polypyrrole microspheres (by reaction of protein amino groups with aldehyde groups on the surface of the microspheres) was entrapped/adsorbed onto electrochemically prepared conducting PPY-PVS films deposited on ITO. Potentiometric measurements undertaken on these conducting polymer electrodes using an ammonium ion analyzer reveal that they can be used for estimating the urea concentration in solutions from 5·10-3 mol/l to 6·10-2 mol/l.

Application of parylene for surface (polymer) enhanced laser desorption/ionization of synthetic polymers.

RATIONALE Synthetic polymers of molecular masses up to a few kDa can be analyzed without the use of any matrix by direct laser desorption/ionization mass spectrometry (LDI-MS). In this technique, the surface of the sample plate plays a crucial role, and many attempts have been made to understand the influence of the surface on the ease of desorption. Since this technique requires no tedious sample pretreatment, it is a promising method for the rapid characterization of various synthetic polymers. METHODS Parylene (poly(p-xylylenes), PPX) was tested as a surface support for studying the molecular masses of biocompatible polymers: poly(ethylene glycol) (PEG), poly(L-lactide) (PLLA), and poly(methyl methacrylate) (PMMA). The average molecular masses of the polymers were: PEG (600.0 Da and 3.5 kDa), PMMA (2.0 kDa), and PLLA (2.8 kDa). RESULTS LDI mass spectra of polymers deposited on parylene were enhanced by a factor of two over those obtained directly from the gold target plate. CONCLUSIONS Modification of the surface of the target plate by the addition of a PPX layer extended the functionality of LDI-TOF MS, especially for the analysis of low-mass compounds. The LDI analysis using the PPX-coated target plate provided details of polymers including: end-group, composition, monomer unit, and molecular mass distribution. The average molecular weights of four tested polymers on the gold target plate and the PPX support were unchanged, indicating that sample degradation was not occurring despite the high energy of the laser beam. The LDI investigations showed that the PPX support boosted ion yields by a factor of two compared with the gold target plate.

Chlorambucil labelled with the phenosafranin scaffold as a new chemotherapeutic for imaging and cancer treatment

Here we report the first of the phenosafranin-chlorambucil conjugate as a new type of a chemotherapeutic agent suitable for dual detection methods (spectrophotometric and fluorescence) in imaging systems and cancer treatment. The synthetic cationic dye (3,7-diamino-5-phenylphenazinium chloride) is used as a fluorescent light-triggered scaffold that acts as a carrier for an anti-cancer drug. The chlorambucil was attached covalently via amide bonds to the bifunctional fluorophore, which facilitates tracking with visible light. Our studies revealed that the new photosensitive compound exhibits improved intrinsic activity in vitro in HeLa cells culture experiments; thus it could be a potential anti-cancer candidate in theranostic drug-delivery systems. In light of the urgent need for in vivo monitoring of the biodistribution of anti-cancer drugs, this strategy for the synthesis of innovative conjugates based on the phenosafranin backbone offers a promising possibility for drug control in anti-cancer therapy and diagnosis. This aspect makes the phenosafranin-chlorambucil conjugate unique among currently available biomarkers.