Evaluation of the Spectral Response of Functionalized Silk Inverse Opals as Colorimetric Immunosensors

Regenerated silk fibroin is a high molecular weight protein obtained by purifying the cocoons of the Expose to domesticated silkworm, Bombyx mori. This report exploits the analyte aqueous processing and tunable beta sheet secondary structure of regenerated silk to produce nanostructures (i.e., inverse opals) that can be used as colorimetric immunosensors. Such sensors would enable direct detection of antigens by changes in reflectance spectra induced by binding events within the nanostructure. Silk inverse opals were prepared by solution casting and annealing in a humidified atmosphere to render the silk insoluble. Next, antigen sensing capabilities were imparted to silk through a three step synthesis: coupling of avidin to silk surfaces, coupling of biotin to antibodies, and lastly antibody attachment to silk through avidin biotin interactions. Varying the antibody enables detection of different antigens, as demonstrated using different protein antigens: antibodies, red fluorescent protein, and the beta subunit of cholera toxin. Antigen binding to sensors induces a red shift in the opal reflectance spectra, while sensors not exposed to antigen showed either no shift or a slight blue shift. This work constitutes a first step for the design of biopolymer-based optical systems that could directly detect antigens using commercially available reagents and environmentally friendly chemistries.