Article pubs.acs.org/Langmuir
Development of Novel Fluorescent Probes for the Analysis of Protein Interactions under Physiological Conditions with Medical Devices Marc-Krystelle Mafina,† Karin A. Hing,†,* and Alice C. Sullivan‡ †
School of Engineering and Materials Science, Queen Mary University of London, London E1 4NS, UK. School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK.
‡
ABSTRACT: In this article, a method to analyze protein adsorption on porous, clinically relevant samples under physiologically relevant conditions is described. The use of fluorescent probes was identified as a methodology that would facilitate analysis under a range of conditions including fully competitive conditions where a protein of interest may be labeled in isolation and then allowed to compete with unlabeled proteins on samples that require no specialized surface pretreatment. As a first step, this article describes the covalent labeling of isolated bovine serum albumin (BSA) with fluorescent fluoresceinthioureidoaminocaproic acid, FTCA, giving FTCA-BSA. The fluorescence intensity of FTCA-BSA was then used to monitor the adsorption and desorption of the protein under noncompetitive conditions with two forms of hydroxyapatite discs (silicate-substituted, SA and stoichiometric, HA) in phosphate-buffered saline (PBS) and minimum essential Eagles’ medium (MEM). Noncompetitive conditions were used to facilitate the validation of the technique in which data obtained from these experiments were corroborated against data obtained using an established total protein assay method (Quant-IT kit, Invitrogen). These experiments demonstrated that the FTCABSA probe had several advantages including a greater sensitivity at lower concentrations and a considerably longer lifetime. The results also demonstrated that the interaction of BSA with SA and HA was also highly temperature- and media-dependent. Under the most physiologically relevant conditions of MEM at 37 °C, BSA was more readily adsorbed to SA with significant differences between biomaterials, but no differences were observed during the desorption process. The use of this method to analyze adsorption under competitive conditions will be the subject of further investigations.
1. INTRODUCTION Protein adsorption to a biomaterial surface is one of the earliest events that occurs after implantation,1−3 occurring within seconds of introduction into the body. The adsorbed protein layer subsequently influences the host response by acting as an intermediary between the implant surface and the surrounding tissue through its ability to facilitate cell motility, attachment, and development. In the case of bone graft substitute materials, modulation of the species and conformation of proteins that adsorb to the surface have been demonstrated to be critical in promoting osteogenic cell attachment,4−7 spreading,8,9 proliferation,10,11 morphology,12−14 and development.15−17 In the quest to develop bone graft substitutes with enhanced bioactivity or osteoinductivity, monitoring protein adsorption and desorption behavior on surfaces has increasingly been recognized as vital to understanding the mechanisms and processes that occur between a material surface and the host environment.18−23 In many studies, protein adsorption or depletion is quantified indirectly using enzyme-linked immunosorbent assays (ELISAs) or total-protein-type assays, where radioactive, colorimetric, or fluorimetric probes are employed. Total protein assays quantify all proteins present irrespective of species or conformation. However, ELISAs are limited to the measurement of a specific protein, and as a result of the reliance on antibodies, they can also be conformation-dependent. Therefore, to carry out a competitive © 2012 American Chemical Society
study multiple analyses must be performed. To date, truly competitive analyses are restricted to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) methodologies, ellipsometry for the analysis of real-time adsorption kinetics,24 or matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) adapted for surface analysis determination.25 All of these methodologies require a high degree of operator expertise and skill to obtain truly quantitative data in addition to specialized equipment and sample presentation that are not always convenient. The aim of this study was to develop a method to facilitate the competitive evaluation of protein adsorption on dense apatite discs via labeling of the species of interest with a fluorescent probe so that it may be monitored independently of other protein species in the local environment. We report here on the development and validation of a quantification technique employing the direct fluorescent labeling of an isolated protein, BSA, that has the potential to be used under competitive conditions with a cocktail of additional proteins that are either in their native unlabeled states or have been covalently labeled with a different probe. The method was tested against a total protein assay procedure Received: October 26, 2012 Revised: December 18, 2012 Published: December 21, 2012 1420
dx.doi.org/10.1021/la304244s | Langmuir 2013, 29, 1420−1426
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appropriate. The FTCA-BSA fluorescence intensity of the calibration solutions was then analyzed using a FluoStar Galaxy microplate reader (BMG Laboratories, U.K.) set at 485 and 520 nm, excitation and emission, respectively, with 10 s of orbital shaking before each reading. Calibration curves were then generated by plotting the known protein concentration against the fluorescence intensity and then evaluating the shape and correlation coefficient of the resulting plots. These calibration curves were then used to analyze unknown protein concentrations in samples obtained from adsorption and desorption experiments with FTCA-BSA, HA, and SA discs through an analysis of the fluorescence intensity of the unknowns. The calibration curves were validated with labeled protein solutions of known concentrations. 2.5. Total Protein Analysis Using Quant-IT. Protein concentrations in adsorption and desorption studies were independently analyzed using the Quant-IT assay kit (Invitrogen, U.K.). A stock solution of unlabeled BSA (50 mg mL−1) in either PBS or MEM was prepared to make a test solution at a concentration of 4 mg mL−1. Manufacturers’ instructions were modified slightly to obtain calibration curves with high- and low-concentration detection limits to facilitate the analysis of unknown protein samples obtained from both adsorption and desorption experiments with unlabeled BSA, HA, and SA discs. The reagent fluorescence intensity was analyzed using a FluoStar Galaxy Microplate Reader (BMG Laboratories, U.K.) set at 485 and 520 nm, excitation and emission, respectively, with 10 s of orbital shaking before each reading. 2.6. Protein Adsorption/Desorption Methodology. A 1.5 mL quantity of either BSA or FTCA-BSA protein solution in PBS or MEM at a concentration of 4 mg mL−1 was placed in triplicate in clean glass vials, and 1 dense disc per vial was added. To analyze BSA adsorption, 100 μL aliquots of the BSA solution were removed at time intervals of 1, 5, 10, and 15 min. After 15 min, the dense discs were removed, and each one was placed in a second clean glass vial containing 1.5 mL of fresh PBS or MEM and gently agitated for 30 s to remove any loosely bound BSA. Discs were then removed and placed in a third clean glass vial containing 1.5 mL of fresh PBS or MEM to analyze desorption, where aliquots of 100 μL were removed at time intervals of 5, 60, 240, and 1440 min. 2.7. Statistical Analysis. The Krusal−Wallis (KW) test was used for statistical analysis between treatment groups (media type or methods), and to compare within the same treatment group at different time points, the Wilcoxon−Mann−Whitney (WMW) test was used, where for both P values
