A protein nanofiber hydrogel for sensitive immunoassays

Area of Science:

• Biomaterials Science
• Nanotechnology
• Immunodiagnostics

Background:

• Conventional 2D assays for disease markers like anti-SSB/La antibodies (Sjögren's syndrome) lack sensitivity.
• Amyloid-like protein nanofibers offer potential for novel diagnostic platforms.
• Integrating nanofibers into 3D hydrogels can enhance assay performance.

Purpose of the Study:

• To develop and evaluate Sup35-based protein nanofiber probes (SuPNPs) within 3D hydrogel matrices.
• To assess the sensitivity and specificity of SuPNP-hydrogels for detecting anti-SSB/La antibodies.
• To compare the diagnostic performance of 3D SuPNP-hydrogels with conventional 2D assays.

Main Methods:

• In vitro assembly of recombinant Sup35 and human SSB/La proteins into amyloid-like nanofibers (SuPNPs).
• Vinylating SuPNPs and cross-linking them into polyacrylamide hydrogels (SuPNP-hydrogels) or attaching biotinylated SuPNPs to streptavidin-hydrogels (bt-SuPNP-hydrogels).
• Testing hydrogel platforms for anti-SSB/La antibody detection in buffer and human serum using varying concentrations.

Main Results:

• SuPNP-hydrogels and bt-SuPNP-hydrogels achieved a limit of detection (LOD) of 10 pM for anti-SSB/La antibodies in buffer, a 100-fold improvement over 2D assays.
• In human serum, SuPNP-hydrogels demonstrated 1000-fold higher sensitivity compared to 2D polystyrene plates, likely due to reduced non-specific adsorption.
• The detection sensitivity remained consistent whether SuPNPs were directly cross-linked or indirectly immobilized in the hydrogel.

Conclusions:

• 3D SuPNP-hydrogel platforms offer significantly enhanced sensitivity and specificity for detecting disease biomarkers like anti-SSB/La antibodies.
• The 3D architecture and uniform nanofiber distribution in hydrogels improve antibody interaction and diagnostic performance.
• This technology holds promise for developing sensitive diagnostic assays for Sjögren's syndrome and other diseases.