Nanopatterned extracellular matrices enable cell-based assays with a mass spectrometric readout

A method of assaying activity of an intracellular enzyme, comprising:

• (a) printing a surface with an array of immobilized cell adhesion ligands and immobilized substrates for the intracellular enzyme by
• (i) coating a polymer pen lithography (PPL) tip array with a first monolayer reagent and printing the first monolayer reagent at selected positions on the surface to form an array of printed first monolayer reagent,
• (ii) incubating the array of printed first monolayer reagent with a second monolayer reagent such that the second monolayer reagent is adsorbed onto unprinted portions of the surface,
• wherein one of the first monolayer reagent and the second monolayer reagent comprises a monolayer reagent for adsorption of the cell adhesion ligand and the other comprises a monolayer reagent for chemical immobilization of the substrate for the intracellular enzyme,
• (iii) contacting the resulting array of step (ii) with the substrate for the intracellular enzyme under conditions to immobilize the substrate to the surface at the portion of the surface comprising the monolayer reagent for chemical immobilization,
• (iv) contacting the resulting array of step (ii) with the cell adhesion ligand under conditions to immobilize the cell adhesion ligand to the surface at the portion of the surface comprising the monolayer reagent for adsorption of the cell adhesion ligand;
• wherein steps (iii) and (iv) can be performed in either order;
• (b) contacting a cell and the surface of step (a), the contacting resulting in immobilization of the cell via interaction between the cell and the immobilized cell adhesion ligand;
• (c) contacting the immobilized cell with a lysing solution to form a cell lysate and release the enzyme, thereby allowing contact between the enzyme and the immobilized substrate to transform the immobilized substrate to a product, the product having a different mass than the substrate;
• and
• (d) measuring the amount of the product formed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to assay the activity of the enzyme.