Effective cell-based assays that enable cell culture preparation and protein analysis on the same chip would greatly facilitate quantification of molecular activities, significantly reduce the number of cells required for an assay, and enable the discovery of novel drug candidates. Despite its importance in cell development and disease, measuring enzymatic activities in a quick and accurate manner remains one of the most challenging problems. Current assays often rely on time-consuming preparation steps, which lead to loss of enzymatic activity, thereby limiting the validity of the readout. We have successfully developed an enzymatic assay based on polymer pen lithography (PPL) and self-assembled monolayer laser desorption/ionization mass spectrometry (SAMDI-MS) that overcomes the limitations of the methods available today. We used the nanopatterning PPL methodology to template discreet sites of extracellular matrix (ECM) attachment proteins over large area in an arrangement that is commensurate with the dimensions of a cell, while leaving &gt; 95% of the remaining surface available to display enzyme substrates that get covalently modified after lysis. In this manner, the nanoscale confinement of ECM proteins provides a surface suitable for cell adhesion without compromising the sensitivity of the assay, while the short peptides encode biochemical activities that can be quantitatively measured using SAMDI-MS, thus enabling cell culture and analysis on the same monolayer-coated surface. Our results demonstrate that cell attachment is directed toward the regions patterned with fibronectin. Following lysis, we quantified the enzymatic activity of cell lysate by measuring the conversion of the phosphatase reporter ligand displayed in the unpatterned area to the dephosphorylated product using SAMDI-MS. We have also expanded the capabilities of our assay design to introduce other extracellular matrix (ECM) attachment proteins, such as laminin and collagen, which would allow culture of a wider range of adherent cell types. Overall, given the high-throughput nature and powerful capabilities of PPL and SAMDI-MS, we envision the development of a broad range of cellular assays and biosensors based on our approach in drug discovery and research.

Nanopatterned matrices enable enzyme activity measurements using SAMDI-mass spectrometry

253rd National Meeting (2017)

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