A Microwell–Printing Fabrication Strategy for the On-Chip Templated Biosynthesis of Protein Microarrays for Surface Plasmon Resonance Imaging

A two-step templated, ribosomal biosynthesis–printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a 16-component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein. In the second step, the protein microwell array is used to contact print one or more protein microarrays onto nitrilotriacetic acid (NTA)-functionalized gold thin-film SPRI chips for real-time SPRI surface bioaffinity adsorption measurements. Even though each microwell array element contains only approximately 40 picomoles of protein, the concentration is sufficiently high for the efficient bioaffinity adsorption and capture of the approximately 100 femtomoles of hexahistidine-tagged protein required to create each SPRI microarray element. As a first example, the protein biosynthesis process is verified with fluorescence imaging measurements of a microwell array containing His-tagged green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry (RFP), and then the fidelity of SPRI chips printed from this protein microwell array is ascertained by measuring the real-time adsorption of various antibodies specific to these three structurally related proteins. This greatly simplified two-step synthesis–printing fabrication methodology eliminates most of the handling, purification, and processing steps normally required in the synthesis of multiple protein probes and enables the rapid fabrication of SPRI protein microarrays from DNA templates for the study of protein–protein bioaffinity interactions.