Covalent immobilization of human serum albumin

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/248840492

Covalent immobilization of human serum albumin onto reactive polypyrrolecoated polystyrene latex particles Article  in  Journal of Materials Chemistry · August 2005 DOI: 10.1039/b500982k

CITATIONS

READS

27

47

5 authors, including: Yann Yip Cheung Sang

Mohamed Chehimi

Paris Diderot University

Institut de Chimie et des Matériaux Paris-Est

14 PUBLICATIONS   196 CITATIONS   

315 PUBLICATIONS   7,321 CITATIONS   

SEE PROFILE

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Coming soon: CHEMISTRY AFRICA (Springer) - A Journal of The Tunisian Chemical Society View project

Empreintes moléculaires View project

All content following this page was uploaded by Yann Yip Cheung Sang on 17 July 2015.

The user has requested enhancement of the downloaded file.

PAPER

www.rsc.org/materials | Journal of Materials Chemistry

Covalent immobilization of human serum albumin onto reactive polypyrrole-coated polystyrene latex particles Smain Bousalem, Sihem Benabderrahmane, Yann Yip Cheung Sang, Claire Mangeney* and Mohamed M. Chehimi Received 20th January 2005, Accepted 19th May 2005 First published as an Advance Article on the web 20th June 2005 DOI: 10.1039/b500982k Polypyrrole-coated polystyrene latex particles bearing reactive N-succinimidyl functional groups (PS-PPyNSE) were prepared by the in-situ copolymerization of pyrrole (Py) and the active ester-functionalized pyrrole (PyNSE) in the presence of 1 mm diameter-sized polystyrene (PS) latex particles. These particles were prepared by dispersion polymerization of styrene using poly(N-vinylpyrrolidone), PNVP, as a steric stabilizer. The initial comonomer fractions (in mol%) were 25/75, 50/50 and 75/25 for pyrrole and pyrrole-NSE, respectively. The functionalized polypyrrole-coated PS particles (PS-PPyNSE) were characterized in terms of their particle size and surface morphology using transmission electron microscopy (TEM). Infrared and X-ray photoelectron spectroscopy (XPS) detected pyrrole-NSE repeat units at the surface of the latex particles, indicating that this monomer had indeed copolymerized with pyrrole. Furthermore, the core–shell structure of the PS-PPyNSE particles was confirmed by etching the polystyrene core in THF, leading to the formation of hollow conducting polymer capsules. Reactivity of the PS-PPyNSE75 particles was investigated using 2-aminoethanol and 2-mercaptoethanol, two model molecules bearing functional groups borne by proteins. Incubation of the particles with these model molecules clearly showed that the particles are reactive towards amine and thiol groups leading to the formation of interfacial amide and thioester bonds, respectively. The PS-PPyNSE particles were further evaluated as bioadsorbents of human serum albumin (HSA) used as a test protein. It was shown that the HSA macromolecules were immobilized at the surface of PS-PPyNSE particles by forming interfacial amide groups. Incubation of the HSA-grafted latex particles with anti-HSA resulted in immediate flocculation. This indicates that, despite probable conformational changes resulting from contact with NSE-functionalized latexes, HSA proteins retain their biological activity when immobilized on the particle surface. These PS-PPyNSE particles are therefore alternative candidates for visual diagnostic assays.

1. Introduction The interaction between biomolecules and latexes has considerable importance in the development of particle-enhanced immunoassays. Several procedures for physical and covalent coupling of proteic ligands on latex particles have been previously described.1,2 Particularly, great attention has been paid to the synthesis of latexes with functional groups, which could be used for the immobilization of proteins.3,4 In this regard, colloidal dispersions of inherently conducting polymers (ICPs) such as polypyrrole (PPy) were found to be particularly suitable for the covalent attachment of proteins due to their high environmental stability and biocompatibility.5,6 These properties, together with its electronic conductivity and ion exchange capacity, have made PPy a popular constituent of planar electrochemical biosensors.7 For the synthesis of PPy particles, several strategies have been reported.8 The main methods of preparation of such Interfaces, Traitements, Organisation et Dynamique des Syste`mes (ITODYS), Universite´ Paris 7 Denis Diderot, CNRS (UMR 7086), 1 rue Guy de la Brosse, 75005 Paris, France. E-mail: [email protected]

This journal is ß The Royal Society of Chemistry 2005

dispersions are: (i) sterically-stabilized polypyrrole particles; (ii) polypyrrole–silica nanocomposites; (iii) polypyrrolecoated latex particles. The preparation of polypyrrole latex in aqueous media has been described by Armes et al.9,10 and Bjorklung and Liedberg.11 Subsequently, Tarcha et al.12 developed methods for the surface derivatization of poly(vinyl alcohol)-stabilized PPy particles in organic solvents such as N-methylpyrrolidone. Polypyrrole latex was acetylated in N-methylpyrrolidone by using bromoacetyl bromide. The bromoacetylated latex could be converted into latexes with carboxylic or amino groups by reaction with thioacetic acid and triethylene tetramine. Latexes obtained in this way were suitable for the covalent immobilization of proteins and the development of diagnostic assays for the human hormone chorionic gonadotropin (HCG), HIV antibody, and hepatitis B surface antigen. However, multistep procedures and several transfers between aqueous and non-aqueous solvents are the disadvantages of this method. Miksa and Slomkowski13 prepared polypyrrole/polyacrolein core–shell latex particles in order to introduce aldehyde groups capable of reacting with proteins via Schiff base formation. J. Mater. Chem., 2005, 15, 3109–3116 | 3109

In the early 1990s, nanocomposite particles of ultrafine silica sols and polypyrrole, in which the silica provides charge stabilization, were developed by Maeda and Armes.14 Furthermore, one-step syntheses were developed in order to obtain polypyrrole–silica surface carboxylation of these nanocomposites employing the comonomer 1-(2-carboxyethyl)pyrrole in the polymerization.15,16 Amine-functionalized polypyrrole–silica nanocomposites were also synthesized using either copolymerization of an N-substituted aminofunctional comonomer with pyrrole or the treatment of non-functionalized nanocomposites with 3-aminopropyltriethoxysilane (APTES). Immobilization of DNA was possible using surface functionalized PPy-silica-COOH or PPy-silica-NH2 particles.17 Pope et al.18 used N-carboxylic acid functionalized polypyrrole–silica nanocomposites as marker particles in a simple strip assay for the human pregnancy hormone hCG. However, covalent attachment of the monoclonal anti-hCG required two surface treatment steps. More recently, Azioune et al.19 copolymerized pyrrole and N-alkyl substituted pyrrole with N-succinimidyl esters (NSE) in the presence of silica sols for HSA immobilization. The high specific area of these raspberry-like particles allowed the immobilization of high amounts of HSA. However, the hydrophilic nature of the silica surface did not allow a homogeneous coating of the conducting polymer around the particles which results in a heterogeneous nature of the nanoparticle surfaces. It follows that the free silica surface sites may interact in a non-specific way with proteins. Furthermore, the porous nature of these particles may also lead to non-specific insertion of proteins inside the particles pores. With regard to conducting polymer deposition onto latex substrates, Yassar et al.20 have described the synthesis of polypyrrole-coated polystyrene latexes by the polymerization of pyrrole in the presence of 0.1 mm sized PS particles having sulfonic or carboxylic groups at the surface. These negatively charged groups counterbalanced the positive charges from the PPy backbone. Later, Lascelles and Armes21 described the preparation in aqueous media of polypyrrole-coated PS latex particles, in the 1.6–1.8 mm size range, having good colloidal stability. Based on the work of Azioune et al.19 for the copolymerization of pyrrole and N-alkyl substituted pyrrole with N-succinimidyl esters (NSE) in the presence of silica sols, we proposed to modify the surface of latex particles by active ester groups such as N-succinimidyl ester. Polystyrene was selected as a ‘model’ colloidal substrate since it has a relatively high Tg (i.e. the particles are rigid and non-deformable) and latexes can be readily synthesized with narrow size distributions over a wide particle size range (50 nm–10 mm). The rationale for choosing the N-succinimidyl ester (NSE) group is that it is well known to react readily with amines or thiols (e.g. from a protein residue) under very mild conditions to form the corresponding amides or thioesters in high yields.22 This approach has also been suggested for the functionalization of carbon nanotubes in order to attach proteins.23 Given the previously published work of Lascelles and Armes21 showing the formation of thick polypyrrole coatings on PS particles, on the one hand, and the effective copolymerisation of pyrrole and NSE-pyrrole in the presence of 3110 | J. Mater. Chem., 2005, 15, 3109–3116

silica sols,19 on the other hand, it is reasonable to expect that on PS it is possible to obtain reactive polypyrrole coatings. The reactive polypyrrole-coated PS latex particles, with reactive polypyrrole-rich surface, could then serve as novel, model conducting polymer particles for the covalent immobilization of proteins. In the present work, we describe the preparation, characterization and reactivity study of polypyrrole-coated polystyrene latex particles bearing surface ester reactive groups towards proteins (see Fig. 1). The polystyrene core, around 1 mm in diameter, was prepared by dispersion polymerization of styrene, using poly(N-vinylpyrrolidone) as a steric stabiliser. Large micrometer-sized PS latexes were chosen as they are expected to induce enhanced light scattering which can be useful for sensitive assays. In addition, as mentioned above, large micrometer-sized PS latex particles permit the formation of thick and continuous coatings of polypyrrole, which are essential to develop model particles of reactive polypyrrole for the study and understanding of the reactivity of these conducting polymers. The reactive coatings consist of copolymers of pyrrole and N-alkyl substituted pyrrole with N-succinimidyl esters (NSE) as easily replaceable leaving groups at the alkyl chain end. Various poly(pyrrole/pyrroleNSE)-coated PS latex particles were prepared with four different comonomer feed ratios (PS-PPyNSEx; x being the initial fraction of PyNSE). The core–shell particles were characterized by means of transmission electron microscopy (TEM), FTIR and X-ray photoelectron spectroscopy (XPS). They were then used as precursors for the formation of hollow capsules by selective extraction of the polystyrene core. Selected batches of particles were incubated with model molecules, 2-aminoethanol (R-NH2) and 2-mercaptoethanol (R-SH) in order to monitor the formation of interfacial bonds between the carrier (NSE functionalized polypyrrole particles) and the target molecules. These model molecules bear functional reactive groups (amine, thiol), which are contained in proteins. Furthermore, the N-ethyl succinimidyl ester-functionalized particles were evaluated as a support for the bioconjugate system HSA/anti-HSA. First HSA was immobilized on the functionalized polypyrrole particles, and then the HSA-decorated particles were incubated with anti-HSA, in order to evaluate the propensity of particles as carriers in diagnostic assays.

Fig. 1 Schematic representation of the reactivity of a polypyrrolecoated, PNVP-stabilized polystyrene latex particles bearing surface reactive N-ester succinimidyl (NSE) groups. Chemical reactivity towards proteins is shown.

This journal is ß The Royal Society of Chemistry 2005

2. Experimental 2.1 Materials Styrene (Aldrich) was purified by passing through a column of activated neutral alumina. Poly(N-vinylpyrrolidone), with a nominal molecular weight of 360 000, was purchased from Aldrich and used without further purification. Pyrrole (Fluka) was purified by passing through a column of activated basic alumina (Acros) prior to use. 1-(2-Cyanoethyl)pyrrole (Acros), FeCl3?6H2O (Aldrich) and a-azoisobutyronitrile (AIBN) (Fluka) were used without further purification. 2-Aminoethanol, 2-mercaptoethanol (Aldrich), HSA (Sigma, Cohn fractions V) and anti-HSA serum (Sigma) were used as received. All aqueous solutions were prepared with deionized water. 2.2 Synthesis of uncoated polystyrene latex particles The sterically stabilized polystyrene latex particles were prepared using the procedure described by Lascelles and Armes.21 Briefly, PNVP stabilizer (7.5 g) was dissolved in 400 ml of 2-propanol in a three-necked round-bottomed flask. The mixture was heated to 75 uC under a nitrogen purge for 24 h. A solution of styrene (83.3 ml) containing AIBN (0.75 g) was added dropwise to the vigorously stirred PNVP–2-propanol solution, which was maintained at 70 uC for 24 h. The polymerization was allowed to proceed for 24 h before cooling to room temperature. The resulting milky-white mixture was centrifuged, the supernatant was decanted and replaced with deionized water, and the white sediment was redispersed. This centrifugation–redispersion cycle was repeated several times in order to remove non-adsorbed PNVP stabilizer. 2.3 Synthesis of surface functionalized polypyrrole-coated polystyrene latex particles The coating procedure consists of the in-situ copolymerization of the monomers in the presence of polystyrene latex. Pyrrole and PyNSE (see reference 19 for its detailed synthesis and characterization) were premixed in 75 : 25 (1.05 6 1023 : 3.5 6 1024 mol), 50 : 50 (23 6 1023 : 23 6 1023 mol) and 25 : 75 (1.05 6 1023 : 3.5 6 1023 mol) molar ratios. This comonomer mixture was added to a vigorously stirred solution (13 mL) containing 1 g dry weight of PS latex and 1.8 g of FeCl3?6H2O. The solution was stirred at 75 uC for 24 h. The resulting colloidal particles were isolated by five centrifugation–redispersion cycles and redispersed in deionised water. The composite poly(pyrrole/pyrroleNSE)-coated PS particles are abbreviated as PS-PPyNSEx where x stands for the initial molar fraction of pyrroleNSE (x 5 0, 25, 50 or 75%). 2.4 Reactivity of PS-PPyNSE75 towards molecules and proteins All measurements were performed at room temperature and pressure. Phosphate-buffered saline (PBS) at pH 7.4 was prepared in distilled water using 10 mM phosphate, 138 mM NaCl and 2.7 mM KCl. The reactivity was investigated by the reaction of PSPPyNSE75 with 2-aminoethanol (R-NH2) and 2-mercaptoethanol This journal is ß The Royal Society of Chemistry 2005

(R-SH). In this reaction the nucleophilic amine (or thiol) undergoes acylation with the succinimidyl ester group to produce the amide (or thioester) product. Amine (or thiol) predissolved in PBS solution were added to the PS-PPyNSE75 latex particles (0.15 g dry weight, total volume 2 ml) and the mixture was left to react for 16 h. After incubation, the samples were centrifuged and washed thoroughly with distilled water to remove all the free and/or loosely bound amine (or thiol) molecules. The products were characterized by FTIR and XPS spectroscopy. 2.5 Immobilization of human serum albumin (HSA) on particles Immobilization of HSA was carried out by gentle mixing of protein and PS-PPyNSE75 latex particles in PBS (pH 7.4) for 20 h at room temperature. The product was isolated by several centrifugation–redispersion cycles to remove unreacted HSA, dried under vacuum and analysed by XPS and FTIR spectroscopy. Then 10 ml of diluted solutions of anti-HSA serum were added to 10 ml of the suspension of HSA-decorated PSPPyNSE75 particles on the test glass slide. In the control experiment 10 ml of latex were mixed with 10 ml of PBS. Aggregation was monitored visually, usually 10 min after mixing. 2.6 Analytical techniques Transmission Electron Microscopy (TEM) micrographs were obtained using a JEOL JEM 100CXII UHR operating at 100 kV. Solutions containing the latex particles were cast onto Formvar-coated copper grids and the solvent was allowed to evaporate. FT-IR spectra of PS particles and PS-PPyNSEx latexes particles (KBr disks) were recorded using a Nicolet Magna 550 Series II instrument. Spectra were typically averaged over 20 scans at 4 cm21 resolution. X-Ray photoelectron spectroscopy (XPS) measurements were performed using a Thermo VG ESCALAB 250 instrument equipped with a monochromatic Al Ka X-ray source (1486.6 eV). The X-ray spot size was 650 mm. The specimens were pressed against double sided adhesive tapes mounted on sample holders. The pass energy was set at 150 and 40 eV for the survey and the narrow scans, respectively. Additional high resolution C1s regions were recorded using a pass energy of 10 eV. Charge compensation was achieved with a combination of electron and argon ion flood guns. The energy and emission current of the electrons were 4 eV and 0.35 mA respectively. For the argon gun, the energy and the emission current were 0 eV and 0.1 mA, respectively. The partial pressure for the argon flood gun was 2 6 1028 mbar. These standard conditions of charge compensation resulted in a negative but perfectly uniform static charge. Data acquisition and processing were achieved with the Avantage software, version 1.85. Spectral calibration was determined by setting the main C1s component at 285 eV. The surface composition was determined using the manufacturer’s sensitivity factors. The fractional concentration of a particular element A (% A) was computed using: ðIA =sA Þ |100 % A~ P ðIn =sn Þ J. Mater. Chem., 2005, 15, 3109–3116 | 3111

where In and sn are the integrated peak areas and the sensitivity factors, respectively.

3. Results and discussion 3.1 Transmission electron microscopy (TEM) Fig. 2a–b displays TEM micrographs of PS and PS-PPyNSE75 particles. The number average diameter (Dn) and the polydispersity parameter (Dv/Dn) of particles were measured directly from the TEM images. Typically, the sizes of 50 particles were measured and the values were averaged. The values of Dn and Dv were calculated from the following equations: P P Ni D4i i Ni Di and Dv ~ Pi Dn ~ P 3 i Ni i Ni Di where Di means the diameters of individual particles and Ni refers to the number of particles corresponding to the diameters. It is worth noting that particles are spherical, have a relatively narrow size distribution and that the number average particle diameter increases from ca. 1.02 mm (Dv/Dn 5 1.03) for PS particles to ca. 1.12 mm (Dv/Dn 5 1.02) for PS-PPyNSE75 particles. The difference between these two diameters (100 nm) gives an estimation of the conductive overlayer thickness, i.e. around 50 nm. However, this is only a rough estimation of the overlayer thickness which is about 5% or less of the total diameter of particles. The comparison between PS and PS-PPyNSEx particles indicates significant modification of the surface morphology after covering with the conducting polymer overlayer. The uncoated PS particles (Fig. 2a) have a smooth, featureless surface morphology. In contrast, for PS-PPyNSEx particles, the conducting polymer overlayer induces roughening of the surface, which is exacerbated for higher initial PyNSE monomer concentration used for the synthesis of the conducting polymer particles. For example, PS-PPyNSE75 particles (Fig. 2b) exhibit small, raised granular polymer nodules. These modifications in the surface morphology and roughness of the PS-PPyNSEx latex particles are in line with previous results published by Lascelles and Armes,21 thus suggesting that homopolypyrrole and reactive copolymers of pyrrole grow in a similar manner at the surface of the polystyrene template. In order to check whether PS-PPyNSE particles are indeed of the core–shell type, hollow PPyNSE75 microcapsules were then prepared by extracting the polystyrene core in THF. TEM micrographs (Fig. 2c) of hollow PPyNSE75 microcapsules show that the sphericity of the templated polystyrene core is preserved. The noticeable difference in contrast of the spheres before and after infiltration of THF confirmed that hollow microcapsules were produced by the extraction of the

polystyrene core. The sphere shell thickness dTEM, determined from the TEM images, is approximately 40 nm. This value is of the same order of magnitude as that previously obtained by calculating the difference between PS-PPyNSE75 and PS particles diameters (i.e. 50 nm). It can also be compared to the values obtained by Lascelles and Armes21 who found evidence for weak flocculation of their PPy-coated PS latexes when the coating gets thicker than y20 nm in diameter. This relatively thick coating of the PPyNSE75 shell could hinder the latex stability but, nevertheless, presents the advantage of maintaining the capsule form of the conductive polymer shell even when the core has been removed. This contrasts with the case of homopolypyrrole-coated PS latex particles with thinner conducting polymer coating which results in broken shells after extraction of the core in THF.21 The granular nature of PPyNSE coatings on PS particles, observed in Fig. 2b, could explain that it is possible to leach out the PS core through micropores. This granular structure may indeed lead to mechanical and microporous properties that allow the selective extraction of the core. Still, it is important to know if PS is fully extracted through the PPyNSE75 unbroken shell. Work is in progress in this regard. From the sphere shell thickness (dTEM), TEM can be used as a semi-quantitative method for the determination of the mass fraction of the PPyNSE copolymers, as reported in ref. 21. Indeed, assuming a uniform coating of PPyNSE75 at the surface of monodisperse PS particles, one can relate the thickness x of conducting copolymer overlayer to the main fraction of the PS core and the conducting shell using: 8 9