Mechanically tunable multiphoton fabricated protein hydrogels investigated using atomic force microscopy

Recent work has demonstrated the feasibility of employing three dimensional (3D) protein hydrogels, fabricated using multiphoton-induced photochemistry, as chemically responsive microactuators in “lab on a chip” devices. In addition, these materials show great promise as cell capture/incubation devices, allowing single bacterial cells to reproduce into multicellular constructs with “user-defined” 3D geometries. However, to date, the mechanical properties of these materials, critical for these applications, have not been quantitatively characterized. In this work, we develop and apply a method to measure the elastic modulus of microfabricated protein hydrogels in situ under dynamic physical and chemical environments. We fabricated protein microcantilevers using a wide range of protein building blocks (albumin, lysozyme, avidin) and probed their mechanical properties using atomic force microscopy (AFM). The length dependence of the spring constant displayed by protein cantilevers followed the predicted cantilever model, yielding the elastic modulus of the material. By varying laser dwell time, the modulus of protein cantilevers could be tuned over 2 orders of magnitude (from 0.03 to 3 MPa for albumin), a range that encompasses modulus values for a number of biological tissues (e.g., cartilage, basement membrane). Further, the modulus was shown to vary strongly over a range of pH values (pH 2–12). Distinct profiles of pH vs. modulus for albumin, lysozyme and avidin cantilevers were observed, which correlate to structural transitions of the incorporated protein. Modification of protein cantilevers via ligand binding (biotin to avidin), increased cantilever stiffness. Finally, using the modulus of a hydrogel microchamber calculated in situ, we determined the pressure generated by a replicating bacterial colony entrapped in the microchamber to be 2.7 ± 1.3 kPa. This work demonstrates an ability to quantify mechanical properties under both chemically and biologically dynamic microenvironments and will enable the development of a robust platform to investigate cell/microenvironmental interactions with high spatial resolution, in three dimensions, using mechanically tunable biological materials.