Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1 H NM
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Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1H NMR Jonathan M. Zatorski 1 & Alyssa N. Montalbine 1 & Jennifer E. Ortiz-Cárdenas 1 & Rebecca R. Pompano 1 Received: 20 May 2020 / Revised: 19 June 2020 / Accepted: 25 June 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 3D cell culture in protein-based hydrogels often begins with chemical functionalization of proteins with cross-linking agents such as methacryloyl or norbornene. An important and variable characteristic of these materials is the degree of functionalization (DoF), which controls the reactivity of the protein for cross-linking and therefore impacts the mechanical properties and stability of the hydrogel. Although 1H NMR has emerged as the most accurate technique for quantifying absolute DoF of chemically modified proteins, colorimetric techniques still dominate in actual use and may be more useful for quantifying fractional or percent DoF. In this work, we sought to develop an optimized colorimetric assay for DoF of common gelatin-based biomaterials and validate it versus NMR; along the way, we developed a set of best practices for both methods and considerations for their most appropriate use. First, the amine-reactive ninhydrin assay was optimized in terms of solvent properties, temperature, ninhydrin concentration, and range of gelatin standards. The optimized assay produced a linear response to protein concentration in a convenient, 96-well plate format and yielded a fractional DoF similar to NMR in most cases. In comparing with NMR, we identified that DoF can be expressed as fractional or absolute, and that fractional DoF can be inaccurate if the amino acid content of the parent protein is not properly accounted for. In summary, the fractional DoF of methacryloyl- and norbornenefunctionalized gelatins was quantified by an optimized colorimetric ninhydrin assay and orthogonally by 1H NMR. These methods will be valuable for quality control analysis of protein-based hydrogels and 3D cell culture biomaterials. Keywords Gelatin methacryloyl . GelMA . Gelatin norbornene . GelNB . Assay development . Protein NMR . Biomaterials
Introduction Hydrogels composed of functionalized, protein-based polymers have been used increasingly in regenerative medicine and tissue engineering [1–3]. Applications such as controlled release and tunable 3D cell culture require precisely controlled cross-linking density, which mediates stiffness, extrudability, and shear-thinning properties. Naturally derived hydrogel materials such as gelatin are a mainstay of the field due to their Published in the topical collection featuring Female Role Models in Analytical Chemistry. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02792-5) contains supplementary material, which is available to authorized users. * Rebecca R. Pompano [email protected] 1
Department of Chemistry, University of Virginia, Charlottesvill
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