Generic Method for Attaching Biomolecules via Avidin−Biotin

Jul 25, 2012 - modification of affordable, recyclable cellulose with CMC under mild conditions seems to offer a viable alternative for a generic bioin...
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Generic Method for Attaching Biomolecules via Avidin−Biotin Complexes Immobilized on Films of Regenerated and Nanofibrillar Cellulose Hannes Orelma,† Leena-sisko Johansson,† Ilari Filpponen,*,† Orlando J. Rojas,†,‡ and Janne Laine† †

Department of Forest Products Technology, School of Chemical Technology, Aalto University, Espoo, Finland Department of Forest Biomaterials, North Carolina State University, Raleigh, North Carolina 27695, United States



S Supporting Information *

ABSTRACT: We investigated the adsorption and chemical conjugation of avidin and its deglycosylated form, neutravidin, on films of regenerated and nanofibrillar cellulose. The dynamics and extent of biomolecular attachment were monitored in situ by quartz crystal microbalance microgravimetry and ex situ via surface analyses with atomic force microscopy and X-ray photoelectron spectroscopy. The installation of carboxyl groups on cellulose after modification with carboxymethylated cellulose (CMC) or TEMPO-oxidation significantly increases physisorption of avidins, which can be then covalently conjugated by using 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDS/NHS) coupling chemistries. The developed cellulose−avidin biointerfaces are able to scavenge biotinylated molecules from solution as demonstrated by successful surface complexation of biotinylated bovine serum albumin (Biotin-BSA) and antihuman immunoglobulin G (Biotin-anti-hIgG). Finally, we show that cellulose substrates carrying immobilized anti-hIgG are effective in detecting human immunoglobulin G (hIgG) from fluid matrices.



INTRODUCTION Development of functional materials from renewable, nontoxic plant-derived biopolymers for diagnostic and medical applications has sparked a renewed interest in academia and industry. Cellulose is the principal structural component of plants and the most abundant natural biopolymer in the biosphere.1 It is a high molecular weight linear homopolymer of D-anhydroglucopyranose units connected through β-(1−4) linkages. Moreover, cellulose is a protein-inert material,2 which expands its suitability for biomedical applications and to develop robust bioassay platforms. The interaction between avidin and biotin (vitamin H) characterized by a dissociation constant of KD ∼ 10−15 M is the strongest noncovalent interaction known.3 Avidin−biotin complexes have been widely employed in immunoassays,4 drug carriers5−7 and systems for linking antibodies, enzymes, and other molecules. Avidin is a glycoprotein (pI 10.5, ∼66 kDa, 5.6 nm × 5 nm × 4 nm) that contains four identical subunits for biotin binding.8,9 However, avidin’s tendency to bind nonspecifically to molecules other than biotin, via its oligosaccharide regions,10 combined with its high pI are limiting factors for developing avidin-based immune-assays. Neutravidin (pI 6.3, ∼60 kDa), a deglycosylated version of avidin,11 displays biotin binding properties similar to those of avidin, but the absence of oligosaccharide regions significantly increases its binding specificity.12 The lowest nonspecific binding tendency of avidins can be obtained from bacterial © 2012 American Chemical Society

avidin (streptavidin), which is structurally quite similar to deglycosylated egg white avidin.13 Recently, several modifications aiming to alter avidin−biotin binding properties have been introduced.14 For example, reversible systems with monomeric and whole avidins have been developed to allow for regeneration of the respective biointerface.15−17 Bifunctional avidins with two different biotin binding pockets18,19 and that bind covalently to biotin have been reported,20 and the thermal stability (denaturation temperature over 90°) of avidin has been improved via chemical modification.21 The utilization of avidin−biotin complexes for biomolecular binding onto cellulose has been reported only in few publications, and the role of electrostatic interactions between avidin and carboxymethylated cellulose (CMC) has been presented.3 For example, chemically functionalized filter paper has been used as a template for covalent conjugation of avidin.22,23 In addition, the immobilization of streptavidin on a paper matrix through the carboxylic poly(N-isopropylacrylamide) microgel has been demonstrated.24 In contrast, the adsorption of avidin and neutravidin on substrates other than cellulose is well documented. For example, investigations with avidin and neutravidin using quartz crystal microgravity have revealed a strong adsorption tendency onto gold,25 silica,25 and Received: May 19, 2012 Revised: July 19, 2012 Published: July 25, 2012 2802

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Scheme 1. Schematic Representation of the Sequential Preparation of Cellulose-Based Biointerfaces for Capturing Biotinylated Molecules

polypyrrole26 surfaces. In addition, the adsorption of avidin on highly hydrophobic polystyrene has been studied by atomic force microscopy (AFM).27 The conjugation of avidin on carbon nanotubes28 and gold29 have also been demonstrated. Finally, the covalent attachment of avidin on sol−gel-modified films of polymethylmethacrylate has been established.30 It has been previously demonstrated that hydrosoluble CMC adsorbs irreversibly on cellulose substrates, increasing their surface charge and carboxyl group content.31 Importantly, installed carboxyl groups allow the use of conjugation techniques, such as 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/ NHS) activation, for irreversible linking proteins onto cellulose.32 Another method to introduce carboxyl groups on cellulose is TEMPO-mediated oxidation: here the primary OHgroups in the D-glucopyranose repeating units are selectively converted to carboxyl anion forms.33 This method accompanied by conjugation chemistry has been employed for introducing chemical functionalities on nanofibrillar cellulose (NFC) and cellulose nanocrystals (CNCs).34−36 Such nanosized cellulose substrates are renewable, light-weight, and highstrength materials of low toxicity and therefore can be considered as promising candidates for the development of platforms in diagnostics.1,37 The kinetics of bimolecular binding and its effects on topographical and chemical characteristics of the substrate can be investigated by using many surface-sensitive techniques. Native cellulose (such as in plant fibers) is a chemically and topographically complex substrate that limits adsorption studies. Thus, ultrathin films of pure cellulose are better suited for such investigations.38 In this paper, we report on the irreversible attachment of avidin and neutravidin on ultrathin films of cellulose and NFC by using in situ quartz crystal microbalance with dissipation monitoring (QCM-D), AFM, and X-ray photoelectron spectroscopy (XPS). The adsorption of avidin and neutravidin on cellulose and carboxylated cellulose was investigated (Scheme 1). The binding of avidins on CMC-modified cellulose was performed by using two different strategies, i.e., physical

adsorption and covalent conjugation. It was found that avidins adsorb irreversibly on cellulose. Furthermore, adsorption was significantly increased by using carboxylated cellulose (via CMC adsorption or TEMPO-oxidation). The binding of biotinylated bovine serum albumin (Biotin-BSA) and antihuman immunoglobulin G (Biotin-anti-hIgG) on the cellulosebased biointerfaces was validated. It was also demonstrated that the biointerface containing antihuman immunoglobulin is capable of detecting human immunoglobulin G (hIgG) from a solution. It is expected that the universal and robust nature of the proposed methods, coupled with the mild reaction conditions used, will open new venues for immobilizing antibodies and other functionalities on cellulosic materials.



MATERIALS AND METHODS

Materials. Avidin (#21121), neutravidin (#31000), Biotin-BSA (#29130), NHS (#24500) and EDC (#22980) were purchased from Pierce (Rockford, IL, USA). Glycine hydrochloride (Glycine-HCl, #G2879), ethanolamine (EA, #398136), CMC (Mw 250 000, degree of substitution (DS) = 0.7 , #419311), anti-hIgG (#I9764), and Biotinanti-hIgG (#I9010) were obtained from Sigma-Aldrich (Helsinki, Finland). Water used in all solutions was deionized and further purified with a Millipore Synergy UV unit. QCM-D sensors were ATcut quartz crystals supplied by Q-Sense AB (Västra Frölunda, Sweden). Methods. Adsorption and Conjugation of Avidins on Cellulose Films. Cellulose films were modified by irreversibly adsorbing 0.5 mg/ mL CMC solution (25 mM CaCl2) at pH 6, thus obtaining “CMCmodified cellulose”. Adsorption of avidin and neutravidin (100 μg/ mL) on cellulose or CMC-modified cellulose was studied from aqueous NaOAc (10 mM, pH 5, 3 mS/cm conductivity) or phosphate (10 mM, pH 7.4, 3 mS/cm conductivity) buffer solutions. Activation of the CMC-modified cellulose was performed by applying a NaOAc buffer solution (10 mM, pH 5, 3mS/cm) containing EDC (0.1 M) and NHS (0.4 M). Avidin or neutravidin (100 μg/mL) was conjugated on the activated CMC-modified cellulose from aqueous NaOAc buffer solution (10 mM, pH 5, 3 mS/cm). The excess of NHS-esters formed during the conjugation reaction were removed by washing the system with 0.1 M ethanol amine at pH 8.5. Nonspecific binding was prevented by using a superblock treatment, that is, a commercial protein solution for filling the surface, 100 mM glycine-HCl solution in Milli-Q-water (pH adjusted to 2.5 with HCl and NaOH) was used to 2803

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remove the electrostatically bound avidins. Biotin-BSA (10 μg/mL) was dissolved in phosphate buffer (10 mM, pH 7.4, 3 mS/cm) and then adsorbed on avidin-decorated cellulose substrates. Conjugation of Avidins on TEMPO-Oxidized Nanofibrillated Cellulose. TEMPO-solution (0.13 mmol TEMPO, 4.7 mmol NaBr, and 5.65 mmol NaClO in Milli-Q-water, pH of 8.5) was allowed to flow over films of NFC for 2−70 min. The oxidation was then stopped by ethanol addition and rinsing with aqueous buffer solution. The obtained carboxylated NFC films were rinsed with Milli-Q-water to remove excess oxidation reagents and then stabilized with 10 mM NaOAc buffer solution at pH 5. Activation of the NFC model surface was performed by using NaOAc buffer solution (10 mM, pH 5, 3mS/ cm) containing EDC (0.1 M) and NHS (0.4 M). Next, avidin or neutravidin (100 μg/mL) was conjugated on the activated TEMPOoxidized NFC cellulose in NaOAc buffer (10 mM, pH 5, 3 mS/cm). The excess of NHS-esters formed during the conjugation reaction were then removed by washing the surface with 0.1 M ethanol amine at pH 8.5. The nonspecific binding was prevented by using a superblock treatment. 100 mM Glycine-HCl solution in Milli-Q-water (pH adjusted to 2.5 with HCl and NaOH) was used to remove the electrostatically bound avidins. Biotin-anti-hIgG (100 μg/mL) was dissolved in phosphate buffer (10 mM, pH 7.4, 3 mS/cm) and then adsorbed on the avidin-decorated NFC substrates. The antigen binding test was employed with hIgG (10 μg/mL) in phosphate buffer (10 mM, pH 7.4, 3 mS/cm). Preparation of Cellulose Films. Two types of cellulose substrate were used in this work: ultrathin films of regenerated cellulose and NFC. In order to prepare the films of regenerated cellulose, a precursor solution of trimethylsilylcellulose (TMSC) was diluted in toluene and then spin coated (3000 rpm) on silica-coated QCM-D quartz crystals (Q-Sense).39 The deposited TMSC films were converted to cellulose by desilylation with hydrochloric acid vapor according to reported procedures.40 The thin films of NFC were prepared as described by Ahola et al.41 Briefly, NFC gels were obtained from the microfluidization (M110P Microfluics Corp., Newton, U.S.) of bleached sulphite hardwood pulp (birch) after 20 passes. The individual cellulose nanofibrils were then dispersed by sonication with an ultrasonic microtip (10 min at 25% amplitude) and centrifuged (10400 rpm for 45 min) in order to collect (manual pipetting) the transparent upper phase dispersion containing the finest nanofibrils. The NFC dispersion (0.148 w% NFC in Milli-Q-water) was then spin coated (WS-650SX-6NPP spin coater, Laurell Technologies, North Wales, PA, USA) at 3000 rpm for 1.5 min on silica crystals carrying a preadsorbed polyethyleneimine (PEI) layer. The NFC-coated QCM-D crystals were stored in a desiccator until use. Prior to QCM-D measurements, the NFC-surfaces were allowed to stabilize overnight in Milli-Q-water. Quartz Crystal Microbalance with Dissipation Monitoring. The adsorption of avidin and neutravidin on the unmodified or modified cellulose supports was investigated with a QCM-D apparatus (model E4, Q-Sense, Västra Frölunda, Sweden). The basic principles of the QCM-D technique have been described by Rodahl et al.42 and Höök et al.43 The QCM-D measurements were conducted at the fundamental frequency of 5 MHz and 15, 25, 35, 45, 55, and 75 MHz overtones at 25 °C under constant fluid flow (0.1 mL/min). The cellulose surfaces were allowed to swell overnight in the respective buffer solution prior to QCM-D measurements. All the experiments were repeated at least two times. Only the changes in the normalized frequencies and dissipations of the fifth overtone are reported. The sensitivity constant (C) of 17.7 ngHz−1cm−2 (provided by the manufacturer) was used. The QCM-D data was fitted with the Johanssmanńs model44 in order to calculate the attached protein masses. Atomic Force Microscopy. Cellulose films with and without adsorbed layers were characterized with an AFM (Nanoscope IIIa Multimode scanning probe microscope from Digital Instruments Inc., Santa Barbara, CA, USA). The AFM scans (5 × 5 and 1 × 1 μm2) were collected by using tapping mode in air with silicon cantilevers. At least three different areas on each sample were analyzed, and no image processing was used except flattening.

X-ray Photoelectron Spectroscopy. The surface chemical composition of the dried samples was determined via XPS. The measurements were performed using a Kratos Analytical AXIS 165 electron spectrometer and monochromatic Al Kα X-ray irradiation at 100W (ca. 1 mm2 and