Article pubs.acs.org/Biomac
CMC-Modified Cellulose Biointerface for Antibody Conjugation Hannes Orelma,*,† Tuija Teerinen,‡ Leena-Sisko Johansson,† Susanna Holappa,† and Janne Laine† †
Aalto University, School of Chemical Technology, Department of Forest Products Technology, Espoo, Finland VTT Technical Research Centre of Finland, 02044 VTT, Espoo, Finland
‡
ABSTRACT: In this Article, we present a new strategy for preparing an antihemoglobin biointerface on cellulose. The preparation method is based on functionalization of the cellulose surface by the irreversible adsorption of CMC, followed by covalent linking of antibodies to CMC. This would provide the means for affordable and stable cellulosebased biointerfaces for immunoassays. The preparation and characterization of the biointerface were studied on Langmuir−Schaefer cellulose model surfaces in real time using the quartz crystal microbalance with dissipation and surface plasmon resonance techniques. The stable attachment of antihemoglobin to adsorbed CMC was achieved, and a linear calibration of hemoglobin was obtained. CMC modification was also observed to prevent nonspecific protein adsorption. The antihemoglobin-CMC surface regenerated well, enabling repeated immunodetection cycles of hemoglobin on the same surface.
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INTRODUCTION There is a growing interest in developing new biomedical diagnostic platforms using renewable support matrices for biomolecules such as antibodies, enzymes, and aptamers. Cellulose, the most abundant biopolymer on earth, has desirable inherent characteristics such as low toxicity, hydrophilicity, affordability, biocompatibility, porous structure, disposability, and flexibility. These properties make it preferable to conventional bioassay supports, such as plastics, especially in rapid diagnostics outside clinical settings. Bioassays rely on the specific binding activity between anchored sensing (probe) molecules and soluble target molecules.1 Therefore, for a successful diagnostic assay system, the key requirement is that the sensing molecule selectively binds the desired target, even in a complex biological milieu, for example, serum. Nonspecific adsorption causes problems either by disturbing the binding of the target via blocking binding sites or by giving false positive responses. Furthermore, for high-quality assay performance, the stability of the captured molecule in its native conformation can be a critical issue.2 In diagnostic applications, the sensitivity and specificity of paper-based assays must at least achieve present standards to provide a competitive alternative. Sensing molecules are immobilized onto solid supports via three main mechanisms: physical adsorption, direct conjugation, and assisted conjugation.3 At present, research in bioactive paper has mainly been focused on immobilization of sensing molecules on conventional paper surfaces.4 Furthermore, the adsorption of sensing elements on surfaces of paper-based assays has been largely based on physical adsorption. In this system, the main driving forces are hydrophobic, van der Waals, and ionic interactions. However, direct adsorption of the capturing substances, especially proteins and aptamers, on cellulose has led to low surface densities,5 probably because the cellulose surface is highly hydrated and has a slightly negative © 2012 American Chemical Society
charge. In addition, the degree of charged groups in woodderived fibers may vary according to the content of other accompanying compounds in cellulose, such as hemicelluloses or the chemicals used during paper manufacturing.6 Because of this, a specific covalent immobilization method is much preferred. Frequently, the hydroxyl groups of cellulose have been employed as sites for direct covalent conjugation of proteins to cellulose or its derivatives. Activating chemicals such as carbonyldiimidazole (CDI),7 sodium periodate,8−10 fluoro-2nitro-4-azidobenzene,11 or cyanurchloride5 have been utilized. The problem with covalent coupling methods through a linker is that the stability of the conjugated proteins is sometimes affected, as indicated by up to 50% loss of the enzyme activity after chemical conjugation.12 Moreover, covalent modification of cellulose alters the structural integrity of paper in the case of sodium periodate.13 Quite recently, a third strategy to immobilize biomolecules on paper support has been introduced, called assisted conjugation. In this method, biomolecules are linked or loaded in a colloidal gel or polymer such as polyacrylamide microgels14 or dendronized cellulose,15 captured inside polyethyleneiminederived microcapsules,16 or entrapped in a sol−gel,17 followed by deposition of the modified polymers or gels onto the cellulose surface. Carboxymethyl cellulose (CMC) has the potential to act as a linker molecule when adsorbed on cellulose. CMC was chosen because of its chemical and physical characteristics; that is, it possesses carboxyl groups (suitable for EDC/NHS chemistry) and it adsorbs irreversibly on cellulose fibers under suitable Received: December 13, 2011 Revised: January 31, 2012 Published: February 23, 2012 1051
dx.doi.org/10.1021/bm201771m | Biomacromolecules 2012, 13, 1051−1058
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Scheme 1. Preparation of Immunochemical Hemoglobin Assay on the Cellulose Surfacea
a
(a) Carboxymethyl cellulose (CMC) is irreversibly adsorbed on cellulose and then antibodies are covalently conjugated on CMC-modified cellulose and (b) antibodies are pre-conjugated with CMC and then the pre-conjugated conjugate is irreversibly adsorbed on cellulose.
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conditions.18−20 Irreversible adsorption is caused not only by reduced charge repulsion but also by hydrogen bonding between unsubstituted glucopyranosides of CMC and the glucopyranosides of cellulose. Apart from charge, CMC addition also changes other physical properties of the cellulose fibers, such as surface swelling, and probably also hydration. Enhanced swelling and hydrogel-like structure are expected to promote functionality and stability of immobilized biomolecules that favor the hydrophilic milieu. In this study, CMC was deposited on a model cellulose surface (Scheme 1) prepared by the Langmuir−Schaefer (LS) method.21 The carboxyl groups of CMC, activated with succinimide ester (EDC/NHS chemistry), were utilized for the conjugation of antihemoglobin. Finally, the prepared biointerface was used for detection of hemoglobin. This would be a relative robust method to prepare an immunochemical hemoglobin assay for use in, for example, fecal occult blood testing (FOBT), which is one of the most widely used routine methods for early screening of colorectal cancer.22 The formation, specificity, and sensitivity of the biointerface was monitored by the quartz crystal microbalance (QCM-D)23 technique and by the surface plasmon resonance (SPR)24 technique. Both methods allow real-time monitoring of the changes in surface coverage. The use of QCM-D in a real-time immunoassay has been previously reported by Carrigan et al.20,25,26 We demonstrate that adsorbed CMC on cellulose can be used to introduce new reactive sites for antibody conjugation. This technique allows covalent linkage of antibodies and proteins to cellulose without desorption. To our knowledge, the use of carboxymethyl cellulose (CMC) as a conjugation anchor on cellulose has not been previously reported.
MATERIALS AND METHODS
Materials. CMC (DP 300, DS 0.6), provided by Noviant, Finland, was dissolved in deionized water (Milli-Q system, Millipore) and dialyzed to salt-free form with a 10−12 kDa mesh dialyzation membrane tube (SpectraPor, Spectrumlabs) and freeze-dried to dry form. The purified CMC was dissolved in the solutions of calcium chloride (CaCl2) in water for grafting on cellulose surfaces. The conductivities of 0, 10, and 100 mM CaCl2 in water, measured at 20 °C, were 0, 2.2, and 17.9 mS/cm, respectively. Mouse monoclonal antihemoglobin IgG 7202 (antihemoglobin) and 7204 (secondary antihemoglobin) were obtained from Medix Biochemica, Finland. Hemoglobin, bovine serum albumin (BSA), and HEPES ((4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) were purchased from Sigma Aldrich. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine hydrochloride, and the blocking agent, Superblock, were purchased from Pierce Biotechnology (Rockford, IL). Glycine-hydrochloride was obtained from Merck. Centrifugal filter columns (Amicon Ultra-15 filter) from Millipore were used for purification of the preconjugated antihemoglobin-CMC conjugate. Trimethylsilyl cellulose (TMSC) was prepared by silylation of microcrystalline cellulose powder from spruce (Fluka) with hexamethyl disilazane (Fluka), as described in ref 27. The polystyrene-coated sensor crystals for QCM-D were 5-MHz AT-cut quartz crystals (QSX 301) from Q-Sense (Sweden). Gold substrates (SIA Au kit) and the surfactant, p20, used in SPR measurements were purchased from Biacore (GE Healthcare, Sweden). The polystyrene (MW 280 000 g/mol) used with SPR substrates was obtained from Sigma Aldrich. The water used in all studies was deionized and further purified using a Millipore Synergy UV unit. All laboratory chemicals used were analytical grade. Quartz Crystal Microbalance with Dissipation Monitoring. The adsorption experiments were performed using a Q-Sense E4 system (Q-Sense, Sweden). The resonance frequency and dissipation shifts of the oscillating crystals were simultaneously monitored at the fundamental frequency (5 MHz) and its six overtones (15, 25, 35, 45, 55, and 65 MHz). The working principle for QCM-D has been described elsewhere by Rodahl et al.28 Increased mass on the oscillating surface decreases the oscillation frequency of the QCM-D crystal, whereas the shift in dissipation is proportional to the 1052
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viscoelasticity of the adsorbed layer. If the adsorbed mass on the crystal is evenly distributed, rigid, and sufficiently small compared with the mass of crystal, then the surface coverage can be calculated by Sauerbrey’s equation29 Δm = −
C·Δf n
preconjugated antihemoglobin-CMC conjugate was allowed to react with 1 M ethanolamine at pH 8.5 and purified by Amicon Ultra-15 columns. Preconjugated antihemoglobin-CMC conjugate was finally diluted to antihemoglobin concentration of 0.2 mg/mL with PBS and injected on the cellulose surface in QCM-D. The adsorption of hemoglobin (Hb) on the prepared antihemoglobin biointerface was studied in QCM-D by injecting hemoglobin concentrations varying from 0.1 to 1 μg/mL in phosphate buffered saline (PBS) at pH 7.4 until saturation of hemoglobin binding was reached. After each injection step, the surface was then regenerated with 0.1 M glycine-HCl at pH 2.7 to remove bound hemoglobin; the surface was stabilized with the PBS buffer. In the SPR experiments, hemoglobin concentrations varying from 0.75 to 15 μg/mL in 10 mM HEPES at pH 7.4 with 150 mM NaCl and 0.005% p20 (surfactant) were injected using 35 μL injection volumes. After each injection step, the antihemoglobin-CMC surface was regenerated with 0.1 M glycineHCl at pH 2.7. Nonspecific protein adsorption on the antihemoglobin-CMC surface was studied in QCM-D by adsorbing 200 μg/mL BSA in PBS at pH 7.4. The adsorption of secondary antihemoglobin (sandwich assay) was studied using 200 μg/mL of secondary antihemoglobin with and without linker hemoglobin. Atomic Force Microscope (AFM). Topographical changes on the prepared antihemoglobin-CMC surface were imaged ex situ in air using a Nanoscope IIIa multimode scanning probe microscope from Digital Instruments (Santa Barbara, CA). Prior to the measurements, the samples were allowed to dry at room temperature at least overnight. The images were scanned using tapping mode with silicon cantilevers. No image processing except flattening was done, and at least three areas on the each sample were measured. The image analysis was performed using Nanoscope 6.0 software. The rms surface roughnesses were measured from 5 × 5 μm2 images. X-ray Photoelectron Spectroscopy (XPS). The surface chemistry changes on the antihemoglobin-CMC surface were characterized using a Kratos Analytical AXIS 165 electron spectrometer with a monochromatic Al Kα X-ray source. The XPS experiments were performed on dry samples, together with an in situ reference sample.37 Spectra were collected normal to the surface. The sampling area was
