Milan Mrksich

Early on as an independent investigator, Mrksich developed and executed the concept of dynamic substrates for cell culture. Here, self-assembled monolayers (SAMs) present cell adhesive ligands with perfect control over density and orientation against a non-adhesive, inert background, such as ethylene glycol. These monolayers can be further modified with electroactive groups that selectively release immobilized ligand when stimulated with an electric potential. Several strategies using this approach were studied in the context of cell signaling, migration, and co-culture.[9][10][11] Subsequent cell-based work focused on developing methods to pattern cells on the aforementioned SAMs. The work has mostly utilized microcontact printing to confine adherent cells into defined positions, shapes, and sizes. Ultimately, his group's work has revealed examples of how cellular mechanics and cytoskeletal structure influence phenotype. A primary example of this involved investigating how cell shape exerts control over the differentiation of mesenchymal stem cells.[12] Further work utilized these patterned monolayers to investigate the relationship between various cytoskeletal elements and to observe complex phenotypic differences in patient-derived neuroprogenitor cells.[13][14] Recent work in the group investigating cell patterning has utilized photoactive adhesive peptides, allowing for local, spatiotemporal control of cell adhesion to study gap junction formation.[15]

While performing much of the early dynamic substrate and cell patterning work, Mrksich also pioneered an assay platform that utilizes SAMs of alkanethiolates on gold.[16][17] The monolayers contain capture ligands (e.g. biotin or maleimide) that can selectively immobilize a peptide of interest. Subsequently, the monolayer can treated with a specific enzyme or a complex mixture, such as cell lysate, that can modify the peptide through various biological processes (e.g. phosphorylation). For quality control, the monolayers present these peptides against a background of tri(ethylene glycol) groups to prevent the nonspecific adsorption of protein to the surface that could obfuscate the reaction signal and, therefore, enable quantitative and reproducible assays. Most significantly, the monolayers can be characterized with MALDI mass spectrometry in a technique known as SAMDI-MS, which provides the masses of the substituted alkanethiolates and, therefore, the mass change of the immobilized peptide that results from enzyme activity. The method is compatible with standard array formats and liquid handling robotics, allowing a throughput in the tens of thousands of reactions per day. Importantly, the matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) analysis provides a fast and quantitative mass shift readout without the need for labels.

Most recently, Mrksich's group has focused on developing a technique for assembling large molecular structures with perfectly defined structures and orientations, known as Megamolecules. This is primarily done through use of fusion proteins and irreversible inhibitor linkers that assemble stable intermediates.[18] Structure-function relationships, including synthesis of cyclic and antibody-mimic structures have been investigated for potential therapeutic application.[19][20]