Next, we investigated the reaction cascade using 1 H NMR to examine all species, including the amplification of the SH-trigger analyte 4. First, we explored the background reaction (no thiol 4) using 13.5 mM of 1 (1.0 eq.) and 1.5 eq. of 3 in aqueous buffer. The experiment revealed no conversion of 1 without signal initiation after 24 hours (Supplementary Figure ), which is a highly desirable feature for an amplification reaction 3 .