Label-Free, Single Protein Detection on a Near-Infrared Fluorescent Single-Walled Carbon Nanotube/Protein Microarray Fabricated by Cell-Free Synthesis

Excessive sample volumes continue to be a major limitation in the analysis of protein–protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni²⁺ chelated by Nα,Nα-bis(carboxymethyl)-l-lysine grafted to chitosan surrounding the SWNT. The Ni²⁺ acts as a proximity quencher with the Ni²⁺/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein–protein interactions in the staurosporine-induced apoptosis of SH-SY5Y cells, confirming literature predictions.