Analysis of inflammatory biomarkers by Arrayed Imaging Reflectometry.

Levels of serum cytokines are important markers for a broad range of human health conditions, ranging from infectious disease and cancer, to pollutant exposure and stress. In the interest of developing new rapid label-free methods for profiling serum cytokines, we have examined the utility of Arrayed Imaging Reflectometry (AIR) microarrays for this application. We find that AIR is readily able to profile 14 cytokines and other inflammatory biomarker proteins in a background of buffered bovine serum albumin or 1% bovine serum with performance metrics comparable to singleplex ELISA, but in a multiplex, chip-based, reagentless format. Further experiments with interferon-gamma (IFN-γ) demonstrated that the concentration of bovine serum could be increased to at least 20% without changing the overall analytical profile or limit of detection (<10 pg/mL).

Single-atom imino substitutions at A9 and A10 reveal distinct effects on the fold and function of the hairpin ribozyme catalytic core.

The hairpin ribozyme cleaves a phosphodiester bond within a cognate substrate. Structural and biochemical data indicate the conserved A9 and A10 bases reside close to the scissile bond but make distinct contributions to catalysis. To investigate these residues, we replaced the imino moiety of each base with N1-deazaadenosine. This single-atom change resulted in an 8-fold loss in k(obs) for A9 and displacement of the base from the active site; no effects were observed for A10. We propose that the imino moiety of A9 promotes a key water-mediated contact that favors transition-state formation, which suggests an enhanced chemical repertoire for RNA.