Interaction of proteins with polymeric latexes

Syntheses, modifications and properties of latexes containing polyacrolein, suitable for the covalent immobilization of proteins, are described. Results of the studies of physical adsorption and covalent attachment of human serum albumin and gamma globulins are discussed. Procedures allowing immobilization of the enzyme, horseradish peroxidase, are described. Changes in protein molecules, owing to their contact with latex particles, are analyzed. Applicability of polyacrolein containing latexes for diagnostic purposes is discussed.     

High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates

The electrostatic adsorption onto charged surfaces of comb copolymers comprising a polyelectrolyte backbone and pendent PEG side chains, such as poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), has in previous studies provided protein-repellent thin coatings, particularly on metal oxide surfaces. A drawback of this approach is, however, the instability of such adsorbed layers under extreme pH values or high ionic strength. We have overcome this limitation in the present study by covalently immobilizing PLL-g-PEG copolymers onto aldehyde plasma-modified substrates. Silicon wafers, optical waveguide chips, and perfluorinated ethylene-co-propylene (FEP) polymer substrates were first coated with a thin plasma polymer layer using a propionaldehyde plasma, followed by covalent immobilization of PLL-g-PEG via reductive amination between amine groups of the PLL backbone with aldehyde groups on the plasma-deposited interlayer. The stability in high salt media and the protein resistance of different molecular architectures of immobilized PLL-g-PEG layers were quantitatively investigated by XPS, an optical waveguide technique (OWLS), and ToF-SIMS. The adsorption of bovine serum albumin was found to be below the detection limit (<2 ng/cm(2)), as for electrostatically adsorbed PLL-g-PEG layers. However, after 24 h of exposure of covalently immobilized layers of PLL-g-PEG to high ionic strength buffer (2400 mM NaCl), no significant change in the protein resistance was observed, whereas under the same conditions electrostatically adsorbed PLL-g-PEG coatings lost their protein resistance. Moreover, covalent immobilization via an aldehyde plasma interlayer enabled the application of PLL-g-PEG layers onto substrates such as FEP onto which electrostatic binding is not possible. These findings create a generic platform for the covalent immobilization of PLL-g-PEG onto a wide variety of substrates.     

Surface functionalization of polymer latex particles. III. A convenient method of producing ultrafine poly(methylstyrene) latexes with aldehyde groups on the surface

A convenient method of preparing ultrafine poly(methylstyrene) (PMS) latex particles with aldehyde groups on the surface is developed. PMS latexes in the size range 33–81 nm were prepared via microemulsion polymerization, using cetyltrimethylammonium bromide (CTAB) as surfactant. The surface of the PMS latexes was oxidized in the presence of tert-butyl hydroperoxide catalyzed by copper(II) chloride. As the degree of oxidation increased up to 6 h, the amounts of aldehyde group increased. Bigger particles were found to have a slightly higher rate of oxidation than small ones under the same oxidation conditions. The particle size underwent little change during oxidation; thus, the amounts of functional groups and the particle size could be controlled concurrently. Dialysis study of the oxidized PMS microlatexes indicated that the instability of highly oxidized PMS microlatexes was caused by the reduction of total surface charge density due to the presence of carboxylic acid groups. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 2103–2109, 1998     

Adsorption of Bovine Serum Albumin onto Polystyrene Latex Particles Bearing Saccharidic Moieties

The adsorption of bovine serum albumin (BSA) onto polystyrene latexes bearing various amounts of sugar moieties has been investigated as a function of pH and ionic strength and the results were compared to those for bare polystyrene latexes having negative surface charges. The functionalized latexes were produced by seeded copolymerization of (0.3 μm) liposaccharidic monomer onto polystyrene particles obtained by soap-free emulsion polymerization of styrene using potassium persulfate as initiator. At first, the electrophoretic mobility behavior of the various latexes was examined as a function of pH: a significant decrease was observed in the case of saccharide-containing latex particles compared to the bare particles. The adsorption of BSA onto these latexes exhibited a reduced amount of adsorbed BSA for those latex particles bearing saccharide groups. This adsorbed amount depends on the yield of saccharidic monomer incorporated onto the surfaces of the latex particles.     

Anchoring of a [Mn(salen)Cl] complex onto mesoporous carbon xerogels

Carbon xerogels were synthesized by the conventional sol-gel approach using formaldehyde and resorcinol. The wet gel was dried by two different procedures followed by carbonization, leading to mesoporous carbon xerogels with considerably different pore size distributions. The materials were subsequently oxidized with air, in order to introduce functional groups on the surface, in particular phenols, anhydrides and carbonyls. The capacity of the carbon xerogels for direct immobilization of metal complexes was tested with a manganese(III) salen complex which possesses an extended ligand pi system and two reactive hydroxyl groups on the aldehyde fragment. The manganese loadings of the various samples indicate that larger amounts of Mn(III) complex were immobilized in the oxidized carbon xerogels when compared with the parent unactivated materials, suggesting that complex immobilization took place preferably by covalent bond between the surface oxygen functional groups and the ligand reactive groups, rather than by pi-pi interactions. The size and shape of the carbon xerogel pores were also shown to play an important role in the final loading of the manganese(III) salen complex.     

Surface morphology of polypyrrole core/polyacrolein shell latex by atomic force microscopy (AFM)

 Polypyrrole latex (P(P)), synthesized in Redox polymerization of pyrrole, was used as seed for radical polymerization of acrolein initiated with K₂S₂O₈. In this process the polypyrrole core/polyacrolein shell latex (P(P–A)) was obtained. Morphology of the surface of P(P–A) particles was investigated by atomic force microscopy (AFM). It was found that macromolecules of polyacrolein are not randomly distributed on the surface of polypyrrole but form patches. Apparently, attraction between macromolecules of poly-acrolein in the surface layer is high and the enthalpy of formation of polyacrolein macromolecule clusters is sufficient to compensate, at least, the negative entropy change due to ordering of these macromolecules. Thickness of the polyacrolein layer on the surface of polypyrrole particles, which were covered only partially with polyacrolein, was equal to only 1.6 nm (standard deviation σ= 0.2 nm) and thus, it was reasonable to assume that it corresponded to the monolayer coverage. Received: 30 April 1997 Accepted: 25 August 1997     

Polymer mediated peptide immobilization onto amino-containing N-isopropylacrylamide-styrene core-shell particles

Monodisperse cationic core-shell latex particles have been prepared using a shot polymerization process, with N-(3-aminopropyl)-methacrylamide-hydrochloride (APMH) as the functional monomer. The final latexes were characterized with respect to final polymerization conversion, water soluble polymer formation, particle size and size distribution, surface charge density and electrokinetic properties. Then the covalent grafting of maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer onto aminated latex particles was investigated. The most efficient conditions to obtain derivatised particles with no alteration of the colloidal stability were to control both polymer amount/latex particles concentration ratio and the mixing method of the two species. The charge inversion of the hydrolysed MAMVE functionalized particles was demonstrated by measuring the electrophoretic mobility as a function of pH. Finally, the covalent binding approach was implemented with peptide-MAMVE conjugates, confirming the great potential of this promising methodology for the preparation of reactive latex particles bearing peptides.     

Hydrophilic magnetic polymer latexes. 1. Adsorption of magnetic iron oxide nanoparticles onto various cationic latexes

With a view to preparing monosized hydrophilic functional magnetic latex particles based on a two-step strategy using anionic iron oxide and cationic polymer latexes, the adsorption step was systematically investigated for a better control of the subsequent encapsulation step. The iron oxide nanoparticles were first obtained according to the classical precipitation method of ferric and ferrous chloride salt using a concentrated sodium hydroxide solution, whereas the polystyrene (PS), P(S/N-isopropylacrylamide (NIPAM)) core–shell and PNIPAM latexes were produced via emulsion and precipitation polymerizations, respectively. The polymer and inorganic colloids were then characterised. The adsorption of iron oxide nanoparticles onto the three types of polymer latexes via electrostatic interaction was studied as a function of iron oxide particle concentration, charge density and the cross-linking density of the hydrophilic layer. The maximum amounts of magnetic nanoparticles adsorbed onto the various latexes were found to increase in the following order: PS < P(S/NIPAM) < P(NIPAM). This significant difference is discussed by taking into account the charge distribution in the hydrogel layer and diffusion phenomena inside the cross-linked hydrophilic shell. Received: 28 December 1998 Accepted in revised form: 15 April 1999     

Grafting epoxy-modified hydrophilic polymers onto poly(dimethylsiloxane) microfluidic chip to resist nonspecific protein adsorption

In order to achieve a simple covalent hydrophilic polymer coating on poly(dimethylsiloxane) (PDMS) microfluidic chip, epoxy modified hydrophilic polymers were synthesized in aqueous solution with a persulfate radical initiation system, and crosslinked onto PDMS pretreated by oxygen plasma and silanized with 3-aminopropyl-triethoxysilanes (APTES). Glycidyl methacrylate (GMA) was copolymerized with acrylamide (poly(AAM-co-GMA)) or dimethylacrylamide (poly(DAM-co-GMA)), and graft polymerized with polyvinylpyrrolidone (PVP-g-GMA) or polyvinylalcohol (PVA-g-GMA). The epoxy groups in the polymers were determined by UV spectra after derivation with benzylamine. Reflection absorption infrared spectroscopy (RAIRS) confirmed covalent grafting of GMA-modified polymers onto PDMS surface. Electroosmotic flow (EOF) in the polymer grafted microchannel was strongly suppressed within the range pH 3-11. Surface adsorption of lysozyme and bovine serum albumin (BSA) was reduced to less than 10% relative to that on the native PDMS surface. On the GMA-modified polymer coated PDMS microchip, basic proteins, peptides, and sodium dodecyl sulfate (SDS) denatured proteins were separated successfully.     

Immobilization of Thermomyces lanuginosus Lipase by Different Techniques on Immobead 150 Support: Characterization and Applications

Thermomyces lanuginosus lipase (TLL) was immobilized on native and modified Immobead 150, with epoxy groups removed by hydrolysis and oxidized to add aldehyde on its surface. Immobilizations on both supports were performed by adsorption, adsorption and cross-linking, covalent attachment, multipoint covalent attachment, and, for the modified support, multipoint covalent attachment using ethylenediamine. Biocatalysts were evaluated for thermal and solvent stabilities, and the best biocatalyst was also tested after incubation in ionic liquids and used in the synthesis of butyl butyrate and isoamyl butyrate. Multipoint covalent immobilized TLL on the native Immobead 150 (Emulti) showed a half-life of 5.32 h at 70 °C, being approximately 30 times more stable than its soluble form; it showed high stability in acetone, hexane, and isooctane. Its enzymatic activity was up to 40 % when incubated in ionic liquids. Ester synthesis produced yields of esterification above 60 % in 24 h. Of all immobilization protocols, the Emulti performed best concerning the thermal, solvent, and ionic liquids stabilities. Emulti was successfully applied to the synthesis of butyl butyrate and isoamyl butyrate, which are very important products for the food and beverage industries.     

NHS-ester functionalized poly(PEGMA) brushes on silicon surface for covalent protein immobilization

Poly(PEGMA) homopolymer brushes were developed by atom transfer radical polymerization (ATRP) on the initiator-modified silicon surface (Si-initiator). Through covalent binding, protein immobilization on the poly(PEGMA) films was enabled by further NHS-ester functionalization of the poly(PEGMA) chain ends. The formation of polymer brushes was confirmed by assessing the surface composition (XPS) and morphology (atomic force microscopy (AFM), scanning electronic microscopy (SEM)) of the modified silicon wafer. The binding performance of the NHS-ester functionalized surfaces with two proteins horseradish peroxidase (HRP) and chicken immunoglobulin (IgG) was monitored by direct observation. These results suggest that this method which incorporates the properties of polymer brush onto the binding surfaces may be a good strategy suitable for covalent protein immobilization.     

Enzymatic Decolorization of Anthraquinone and Diazo Dyes Using Horseradish Peroxidase Enzyme Immobilized onto Various Polysulfone Supports

In this study, covalent immobilization of the horseradish peroxidase (HRP) onto various polysulfone supports was investigated. For this purpose, different polysulfones were methacrylated with methacryloyl chloride, and then, nonwoven fabric samples were coated by using solutions of these methacrylated polysulfones. Finally, support materials were immersed into aquatic solution of HRP enzyme for covalent immobilization. Structural analysis of enzyme immobilization onto various polysulfones was confirmed with Fourier transform infrared spectroscopy, atomic force microscopy, and proton nuclear magnetic resonance spectroscopy. Decolorization of textile diazo (Acid Black 1) and anthraquinone (Reactive Blue 19) dyes was investigated by UV–visible spectrophotometer. Covalently immobilized enzyme has been used seven times in freshly prepared dye solutions through 63 days. Dye decolorization performance of the immobilized systems was observed that still remained high (70 %) after reusing three times. Enzyme activities of immobilized systems were determined and compared to free enzyme activity at different conditions (pH, temperature, thermal stability, storage stability). Enzyme activities of immobilized systems and free enzyme were also investigated at the different temperatures and effects of temperature and thermal resistance for different incubation time at 50 °C. In addition, storage activity of free and immobilized enzymes was determined at 4 °C at different incubation days.     

The Modification of Screen‐Printed Carbon Electrodes with Amino Group and Its Application to Construct a H2O2 Biosensor

Electrooxidation of thionine on screen‐printed carbon electrode gives rise to the modification of the surface with amino groups for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The biosensor was constructed using multilayer enzymes which covalently immobilized onto the surface of amino groups modified screen‐printed carbon electrode using glutaraldehyde as a bifunctional reagent. The multilayer assemble of HRP has been characterized with the cyclic voltammetry and the faradaic impedance spectroscopy. The H₂O₂ biosensor exhibited a fast response (2 s) and low detection limit (0.5 μM).     

Synthesis and characterization of surface-cyanofunctionalized poly (N-isopropylacrylamide) latexes

 A series of P[N-isopropylacrylamide (NIPAM)] latexes with different contents of cyano groups were successfully prepared by either seeded or shot-growth polymerizations of an aqueous solution containing acrylonitrile (AN) onto a seed P[NIPAM] latex, respectively, and further characterized by FT-IR, ¹H-NMR, elemental analysis, as well as by quasielastic light scattering (QELS) and scanning electron microscopy (SEM). All prepared surface-cyanofunctionalized P[NIPAM] latexes exhibited the same range of lower critical solution temperature (LCST) as a pure P[NIPAM] latex. The shot polymerization process proved more efficient at yielding cyano derivatized latexes than the seeded polymerization technique. The amount of incorporated cyano groups onto the particles was determined with a good correlation both by ¹H-NMR and elemental analysis. The higher the amount of initially introduced AN monomer in the reaction mixture, the more cyano groups were incorporated onto the particles. The surface of the particles with high content of cyano groups appeared quite rough by SEM in comparison with that of the pure P[NIPAM] particles. Received: 25 February 1998 Accepted: 23 June 1998     

One-step immobilization of aminated and thiolated DNA onto poly(methylmethacrylate) (PMMA) substrates

Direct immobilisation of modified DNA oligonucleotides (aminated or thiolated) onto a plastic substrate, poly(methylmethacrylate), (PMMA) is described. Using the methyl esters present on non-modified PMMA, it was possible to establish a covalent bond between the electron donor of a DNA probe and the C terminal ester of the PMMA substrate. Since the procedure consists of a single brief wash in isopropanol or ethanol, the procedure is simple and environmentally friendly. The new immobilization strategy was characterized by analysing DNA microarray performance. The new procedure resulted in probe- and hybridization densities that were greater or equivalent to those obtained with commercially available surfaces and other procedures to immobilize DNA onto PMMA. The described chemistry selectively immobilized the DNA via terminal thiol or amine groups indicating that probe orientation could be controlled. Furthermore, the chemical bond between the immobilized DNA and the PMMA could endure repeated heat cycling with only 50% probe loss after 20 cycles, indicating that the chemistry could be used in integrated PCR/microarray devices.     

Potassium Acyltrifluoroborate (KAT) Ligations are Orthogonal to Thiol‐Michael and SPAAC Reactions: Covalent Dual Immobilization of Proteins onto Synthetic PEG Hydrogels

The covalent immobilization of peptides, proteins, and other biomolecules to hydrogels provides a biologically mimicking environment for cell and tissue growth. Bioorthogonal chemical reactions can serve as a tool for this, but the paucity of such reactions and mutual incompatibilities limits the number of distinct molecules that can be introduced. We now report that the potassium acyltrifluoroborate (KAT ) amide‐forming ligation is orthogonal to both thiol‐Michael and strain promoted azide alkyne cycloadditions (SPAAC ) and the requisite functional groups – KAT s and hydroxylamines – are stable and compatible to hydrogel formation, protein modification, and post‐assembly immobilization of biomolecules onto hydrogels. In combination these ligations enables stepwise covalent protein immobilization of multiple BSA ‐derivatives onto the hydrogel scaffold regardless of the order of addition.     

The surface characterization of styrene-acrylamide copolymer latex particles and their deposition onto fibers

The surface characteristics of styrene-acrylamide (St-AAm) copolymer latex particles were investigated and their deposition onto polyamide (Nylon 6), polyester (polyethylene terephthalate) and polyacrylonitrile fibers was studied. Conductometric titrations and viscosity measurements of latex dispersions revealed the presence of a water-soluble polymer layer on the particle surface and the thickness of its polymer layer increased with increasing acrylamide fraction in a latex particle. The deposition rates of St-AAm copolymer latices onto Nylon 6 and polyester fibers increased with increasing acrylamide fraction and decreasing pH at a constant ionic strength. These deposition phenomena onto Nylon 6 and polyester fibers agreed qualitatively with prediction based on the electrokinetic data of the latices and the fibers. However, a participation of attractive interaction due to an increase in acrylamide fraction was also suggested.The deposition rate onto polyacrylonitrile fiber decreased with increasing acrylamide fraction in spite of a decrease in electrostatic repulsive interaction, and it was found that a specific large repulsive interaction acts between polyacrylonitrile fiber and St-AAm copolymer latex particles.This paper is part VIII in a series on Interfacial electrical studies on the deposition of polymer latexes onto fabrics and the removal of these deposited latexes. Part VII: Tamai H, Kimura I,Suaza T Coll Polym Sci 261: 661 (1983)     

Surface modification. I. Polyacrolein microspheres covalently bonded onto polyethylene

The synthesis and characterization of novel surfaces composed of polyacrolein microspheres covalently bonded onto polyethylene films are described. These surfaces were prepared through a sequence of reactions carried out onto polyethylene films in order to form primary amine groups at the w position. Polyacrolein microspheres in water were then covalently bonded to these modified polyethylene surfaces. The binding between the microspheres and the modified surfaces is due to the interaction between the aldehyde groups of the microspheres and the amine groups of the modified surfaces to form the polyvalent Schiff base bonds. Fourier transform-infrared/attenuated total reflection, electron spectroscopy for chemical analysis, contact angle measurements, and scanning electron microscopy have been used for the characterization of the modified polyethylene surfaces.     

Determination of Fluoride,Cyanide, and Thiocyanate Using Horseradish Peroxidase Immobilized on Modified Silica Gel

Abstract

Silica gels modified with different functional groups (amino, epoxy, cycloepoxy, isocyanate, and thiocyanate) were used for the covalent immobilization of horseradish peroxidase (HRP). The catalytic activity and stability of the obtained enzyme preparations were studied using the reaction of o‐dianisidine oxidation with hydrogen peroxide as an indicator. The covalent immobilization of horseradish peroxidase using silica gel modified with thiocyanate groups provided not only the improvement of the enzyme stability, but also the development of the sensitive, rapid, and simple procedures for the determination of fluoride, cyanide, and thiocyanate. Enzymatic determination of inorganic anions is based on their inhibitory effect on the enzyme as the ligands capable to form stable complexes with Fe(III)‐HRP cofactor. The proposed procedures were applied for the determination of F^? in mineral and drinking waters; CN^? and SCN^?—in biological fluids (blood and saliva).     

The increased termal stability associated with humic acid anchored onto silica gel

Commercial humic acid (HA) was anchored onto silica gel (SiAPTS) previously modified with 3-aminopropyltrimethoxysilane (APTS). HA was anchored onto SiAPTS through two routes: adsorption and covalent chemical immobilization onto the surface. The adsorption occurred by adding SiAPTS to HA in an aqueous solution, producing SiHA1, while chemical immobilization was performed by reacting HA suspended in N,N-dimethylformamide with SiAPTS, to yield SiHA2. The infrared spectra confirm HA immobilization using both procedures and the termogravimetric results showed that the anchored compounds have significantly thermal stability increased. While natural HA presents a thermal stability up to 200°C, the anchored compound presents a thermal stability near to 750°C. This revised version was published online in July 2006 with corrections to the Cover Date.