Protein engineering is a technique that has allowed researchers to overcome billions of years of evolution to create unnatural proteins that have been re-programmed to function and interact in ways unfounded in nature. It has been used to engineer proteins for a breadth of new purposes. This technique allows researchers to change the function of the protein by modifying its structure. Though a theoretical understanding of the relationship between the protein structure and function is helpful, unfortunately, the structure-function relation in the vast majority of proteins remains unknown.  Directed evolution is a powerful protein engineering method that circumvents the need to define the structure-function relation of a protein. Briefly, after introducing random mutations to the protein, the collection of mutated proteins is screened to identify proteins with the new, desired function. The proteins with beneficial mutations are then isolated and mutated once more, and the process is repeated until the new protein function has been optimized. Scientists use this strategy to modify and/or improve protein properties, including stability, fluorescence, activity, and affinity to a specific target. One primary challenge in this method is developing a high-throughput screen for evaluating protein function. Researchers have established several high-throughput techniques to screen protein activities effectively using, for example, fluorescence-activated cell sorting or in vitro compartmentalization. However, these methods are largely limited to specific proteins and cannot be readily generalized.

In our work, we use single-walled carbon nanotubes (SWCNTs) in a novel platform for investigating the functionality of a protein. SWCNTs emit fluorescence in the near-infrared spectral region between 900 nm and 1500 nm, a region that lies outside the optical window of most proteins and biomaterials in general. Further, SWCNTs demonstrate single-molecule sensitivities, with fluorescence emissions changing in response to perturbations in the charge, dielectric constant, and pH of the environment. We have exploited this sensitivity by interfacing SWCNTs with proteins that induce local environmental changes when active, thus allowing us to monitor protein activity by measuring the resulting changes to the SWCNT fluorescence. Combined with a moderate to high-throughput approach for acquiring SWCNT fluorescence, this platform provides biologists with a new tool for expediting the protein discovery process and engineering previously intractable proteins.