In contrast to the classical design of a probe with one binding site to target one specific analyte, probes with multiple interaction sites or, alternatively, with single sites promoting tandem reactions to target one or multiple analytes, have been developed. They have been used in addressing the inherent challenges of selective targeting in the presence of structurally similar compounds and in complex matrices, as well as the visualization of the in vivo interaction or crosstalk between the analytes. Examples of analytes include reactive sulfur species, reactive oxygen species, nucleotides and enzymes. This review focuses on recent innovations in probe design, detection mechanisms and the investigation of biological processes. The vision is to promote the ongoing development of fluorescent probes to enable deeper insight into the physiology of bioactive analytes.

Intracellular pH values are some of the most important factors that govern biological processes and the acid–base homeostasis in cells, body fluids and organs sustains the normal operations of the body.