Figure 2.

Optimization of NAD‐display bead‐surface sensing and flow cytometry‐based detection of dehydrogenase activity. A) Schematic depiction of the commercially available analogue of NAD^′ in which biotin and a linker are attached to the adenine end of NAD⁺ (chemical structure shown in Figure S4). B) The in vitro functioning of three different fluorescent protein‐based sensors of NAD(H) redox state was tested with biotin‐17‐NAD⁺ and its reduced form, both in the presence and absence of Tamavidin‐2‐HOT. C) Reduction and re‐oxidation of biotin‐11‐NAD⁺ immobilised on bead. Immobilised co‐factor reduction was brought about by treating beads with LDH and sodium lactate, while the reverse was achieved with LDH and sodium pyruvate. D) Monitoring of bead redox state using SoNar‐SpyCatcher and flow cytometry.