title: 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 [Sjögren'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 cross-linked 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 Sjögren'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.
authors.affiliation: Department of Chemical and Biological Engineering, College of Engineering, Korea University, Anam-dong 5-1, Seoul 136-713, Republic of Korea.
authors.forename: Dae-Sung
authors.initials: DS
authors.lastname: Lee
authors.forename: Jin-Seung
authors.initials: JS
authors.lastname: Park
authors.forename: Eun Jung
authors.initials: EJ
authors.lastname: Lee
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Kim
authors.forename: Jeewon
authors.initials: J
authors.lastname: Lee
mesh_terms: D001323:Autoantibodies / Q000032:analysis / Q000097:blood / Q000276:immunology; D015415:Biomarkers / Q000032:analysis / Q000097:blood; D001710:Biotin / Q000378:metabolism; D002021:Buffers; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D008958:Models, Molecular; D008968:Molecular Conformation; D057139:Nanofibers / Q000737:chemistry*; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011137:Polystyrenes / Q000737:chemistry; D016062:Porosity; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D012859:Sjogren's Syndrome / Q000276:immunology; D019809:Streptavidin / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D015415:Biomarkers; D002021:Buffers; D020100:Hydrogels; D055767:Immobilized Proteins; D010454:Peptide Termination Factors; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D001710:Biotin; D019809:Streptavidin