A protein nanofiber hydrogel for sensitive immunoassays

Abstract

Amyloid-like protein nanofibers were assembled in vitro using a recombinant protein fusion, with yeast Sup35 and human SSB/La proteins as assembly units, where the length of the nanofibers was mostly between 100 and 400 nm. The protein nanofibers, hereafter referred to as Sup35-based protein nanofiber probes (SuPNPs), were used to sensitively detect anti-SSB/La antibodies [Sjogren's syndrome (SS)-specific marker]. After, the SuPNPs were vinylated and subsequently linked to acrylamide. The polymerization reaction with acrylamide formed a SuPNP-hydrogel with uniform porosity, where the SuPNPs were directly cross-linked to polyacrylamide. Alternatively, biotinylated SuPNPs (bt-SuPNP) were attached to a streptavidin-hydrogel, resulting in the formation of a bt-SuPNP-hydrogel. When both the SuPNP-hydrogel and bt-SuPNP-hydrogel were used as 3D assay platforms for the detection of anti-SSB/La antibodies in a buffer solution, the LODs (limit of detection) were found to be 10 pM for both, showing 100-fold enhancement in sensitivity compared to conventional 2D polystyrene (PS) plate-based assays. It seems that the exposed surface and uniform distribution of the SuPNPs within the 3D space of the porous hydrogel matrix interacted more effectively with the anti-SSB/La antibodies, leading to more sensitive detection. The equal sensitivity demonstrated by the SuPNP- and bt-SuPNP-hydrogels above indicates that the target binding activity of the SuPNPs remains unchanged when either directly crosslinked to the hydrogel or indirectly immobilized to the hydrogel via streptavidin. When used to detect anti-SSB/La antibodies in human serum, the SuPNP-hydrogel is 1000 times more sensitive than a 2D PS plate. It seems that non-specific adsorption of the serum proteins occurs heavily on the 2D PS plate. While diagnostic assays for Sjogren's syndrome were demonstrated as proof-of-concept in this study, the SuPNP-hydrogel can be generally applied for the sensitive and specific detection of many other disease markers.