A 2D Covalently Bound Continuous Protein Gradient Assay
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A 2D Covalently Bound Continuous Protein Gradient Assay Benjamin Mintz1 and James A. Cooper, Jr.1 1
Musculoskeletal &Translational Tissue Engineering Research (MATTER) Lab, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 8th St, Troy, NY 12180, U.S.A. ABSTRACT Traditional methods of quantifying cell movement in response to a chemotactic factors provide either a binary count of cell migration in response to a known concentration of the factor of interest in solution, as in Boyden chamber assays, or a method of tracking cells to determine velocities across a solubilized protein gradient where exact concentrations vary over time and are difficult to define, as in the Ibidi chemotaxis gradient assay. Using a silane self-assembling monolayer (SAM)-based procedure pioneered by V Hlady and associates, we have developed an assay capable of covalently binding a wide variety of proteins to an optically transparent surface in a 2D pattern via amine linkages. The pattern was then verified by contact angle and Raman and X-ray photoelectron spectroscopy. This new assay provides greater control of protein concentration and gradient intensity than when using only solubilized proteins.
INTRODUCTION Biochemical gradients play an important role in many biological processes from embryonic development to wound healing. To better understand the effect of chemical concentration on cells, classical in vitro experiments involve incorporation of the factor in culture media and assays at several discrete concentrations for subsequent analyses. While essential in determining effective dose size for the chemical of interest, these experiments do not indicate the ability of the protein to affect cells in a directional manner such as motility or polarity. Diffusion-based gradients formed by microbead encapsulation and layer by layer entrapment has been incorporated in many biomaterials-based drug delivery approaches.[1-4]. Although capable of forming gradients, diffusion-based methods introduce variability in changes of both local concentration over time, (initial burst release for example) and microbead size/ encapsulated concentration make these methods less ideal for quantification of specific activity of specific protein concentrations present in a static pattern. A commercially available laboratory scale microfluidics-based gradient assay is currently available from Ibidi (Chemotaxis Slide Assay, Ibidi, Germany) however many of the same limitations exist including a limited range of effective concentrations for stable gradient formation and changes in gradient intensity over time. In this study, patterning of the self-assembling monolayer (SAM) is achieved using a selfassembling monolayer derived from 3-mercaptopropyltrimethoxysilane (MTS) on fused quartz
modified by UV irradiation. The selective irradiation of UV light oxidizes the MTS surface thiols (-SH) into negatively-charged sulfonate (-SO3) groups providing two chemically distinct moieties for further modification. [5-9] The conventional method of patte
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