A supramolecular bioactive surface for specific binding of protein
Graphical abstract
Introduction
Surfaces with immobilized bioactive molecules aimed specifically at promoting or supporting particular interactions are referred to as “bioactive surfaces” [1], [2], [3]. Such surfaces are of great importance for various biomedical and biomaterials applications ranging from tissue engineering and implant materials to biosensors and diagnostics [4], [5], [6], [7], [8], [9]. In particular, bioactive surfaces used as biosensor platforms are of interest due to their application in areas such as medical diagnostics, environmental monitoring, and bioprocess engineering [6], [10]. An ideal bioactive surface must provide high and well-controlled binding capacity for biomolecules, prevent non-specific biointeractions to increase the signal-to-noise ratio [11], [12], and must be convenient to prepare [13]. When employed as a sensor the surface should also be “reversible” to allow repeated re-use [14]. Considerable efforts have been made to fabricate bioactive surfaces that meet these requirements. The most widely used strategy is first to modify the substrate with a thin polymer layer or film as a matrix, and then incorporate a bioactive molecule having a specific functionality [15]. Most commonly the biofunctional molecule is immobilized by covalent chemical bonding [16]. This strategy, however, suffers from several drawbacks: the modification process is complex, usually involving multiple steps; organic solvents with possible toxic effects are usually required; and precise control of the density of immobilized molecules is difficult. In particular, once the biomolecule is covalently anchored on the surface, it cannot be easily removed or replaced, leading to difficulties in regeneration of the surface, as may be required for sensor chips [17], [18], [19].
To overcome these problems, we recently proposed a new strategy for the development of bioactive surfaces using non-covalent attachment methods [20]. A polyelectrolyte multilayered film containing “guest” groups is first deposited on a substrate using layer-by-layer (LbL) assembly technique. This film serves as a matrix, providing binding sites for the incorporation of “host” biomolecules via host-guest interactions. LbL deposition is a simple and cost-efficient technique for the fabrication of polymer thin films with predetermined, tunable composition and functionality [21]. Compared with other coating techniques, it has the important advantage that it is broadly applicable and can be used with a wide variety of substrates of almost any shape and size [22]. Also, LbL deposition can be performed at room temperature and under mild conditions in aqueous solution, which is favorable for subsequent incorporation of biomolecules [23], [24]. The resulting polyelectrolyte multilayered film provides three dimensional (3D) structure with many accessible binding sites for biomolecules; the density of the sites can readily be adjusted by changing the film thickness. In addition, the polyelectrolyte multilayers are inherently hydrophilic and upon exposure to salt-containing solutions (e.g. buffered protein solutions or cell culture media); salt ions and water are taken up and the multilayers become swollen to varying degrees. The associated water can inhibit protein adsorption and cell attachment, providing a passive “background” to reduce non-specific biointeractions [25], [26]. Moreover, host-guest interactions, based on noncovalent molecular recognition, are highly selective and dynamic, and bind host and guest molecules together in a specific, reversible and efficient manner [27], [28], [29], [30], [31], [32], [33].
In this work, taking the advantageous features of LbL technique and host-guest interactions, we prepared a surface with high capacity for incorporation of bioactive molecules, high specificity for detecting target molecules in the presence of interfering proteins, and good ability to regenerate for repeated re-use. Herein, silicon substrates were alternately deposited with the polyanion adamantane (Ada)-modified poly(acrylic acid) (P(AA-co-Ada)) and the polycation poly(allylamine hydrochloride) (PAH) to achieve a multilayered polyelectrolyte film containing “guest” Ada groups. The Ada groups provide binding sites for multivalent β-cyclodextrin (β-CD)-based ligands. In addition, non-specific protein adsorption is inhibited due to the inherent hydrophilicity of the surface, thereby decreasing interference from “bystander” proteins. The biotin-avidin system is widely used as a model system in binding studies due to the high affinity of biotin-avidin interactions (Kd = 10−15 M) [34], [35]. In addition, for present purposes, because a biotin “label” on CD will be stable and small, it is not expected to affect the ability of CD to form CD/Ada inclusion complexes [36], [37]. Thus the biotin-avidin system was used to evaluate the binding capacity and specificity of our materials. A β-CD derivative bearing seven biotin units (CD-B) was incorporated into the films via host-guest interactions. The resulting surfaces exhibited high binding capacity for avidin due to the high density and specificity of biotin residues. Moreover, since host-guest interactions are inherently reversible, the avidin-CD-biotin complex is easily released by treatment with the proper reagent (e.g. sodium dodecyl sulfate, SDS), and the “regenerated” surfaces, after re-introducing CD-B, can be used repeatedly for avidin binding [38].
Section snippets
Materials
P(AA-co-Ada) and CD-B were synthesized as reported previously [20], [39] and PAH (MW: 120,000 to 200,000 g/mol) was purchased from Alfa Aesar Chemicals Co., Ltd (China); their chemical structures are shown in Scheme 1. 3-Aminopropyltriethoxysilane (APTES, 99%, Sigma-Aldrich) were used as received. All organic solvents were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China) and purified according to standard methods before use. Silicon wafers [p-doped, (100)-oriented, 0.45 mm thick]
Surface preparation and characterization
The surface preparation procedure (Scheme 2) involved three steps including (i) surface activation to provide positive charge, (ii) LbL assembly to deposit multilayered films containing guest moieties, and (iii) host-guest inclusion to incorporate biofunctional host molecules. Silicon (Si) was adopted as a model substrate due to its intrinsic chemical stability and good amenability for various surface characterization techniques. In addition, silicon-based biochips are of interest on account of
Conclusion
In this work, a biosensor concept based on a multilayered polyelectrolyte film formed by a combination of LbL assembly and host-guest interactions was developed. The concept was tested using a biotin-containing surface for the sensing of avidin. The multilayered films consisted of alternating layers of Ada-containing P(AA-co-Ada) as polyanion and PAH as polycation. After incorporation of β-CD-biotin by guest-host interactions, the resulting LbL/CD-B surface exhibited a high capacity and good
Acknowledgements
This work was supported by the National Natural Science Foundation of China (21404076, 21334004, 21474071, 21674074 and 21504060), the Natural Science Foundation of Jiangsu Province (BK20140316), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Jiangsu Clinical Research Center for Cardiovascular Surgery. The authors thank Prof. John Brash for helpful discussion.
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