We describe a high-throughput screening (HTS) assay for transglutaminase (TG) enzyme activity using plasmonic fluorescent nanocomposites. We used TG to covalently crosslink 500 μM solution of 5′-biotinamidopentylamine (BP) to N,N′-dimethylcasein (DMC) which was adsorbed onto 384-well microplates. We then bound 0.2 – 2.0 × 1011/mL of 10 nm gold nanoparticles-streptavidin conjugate (10 nm AuNPs-SA) to BP via biotin-streptavidin interactions. Finally, J-aggregation of cyanine 1 (25 μM) or 2 (10 μM) upon the 10 nm AuNPs elicited absorption and fluorescence signaling of TG catalysis. The cyanines could be added sequentially to elicit green (590 nm) and red (700 nm) spectral responses from the same set of reactions. Catalysis was linear (r2 > 0.98) up to 10 min within a linear dynamic range (LDR) of 0.1 – 5 μg/mL enzyme. The multi-wavelength interrogation offered fast results (< 5 min), sensitivity (limit of detection, LOD of 5 ng or 64 fmol TG) and intermediate precision (relative standard deviation, RSD of < 20% over 42 days). Plasmonic fluorescent nanocomposites offer new ways of interrogating biomolecules in HTS format.
2012 by The Japan Society for Analytical Chemistry