A stepwise assembly strategy to enhance sensitivity in fluorogenic ALP assays

The creation of fluorescent probes with exceptional brightness holds the key to unlocking sensitive, reproducible, and reliable performance of fluorometry. Other than synthetizing novel fluorescent probes, enhancing the functionality of well-established, mass-produced existing probes is also very meaningful although thousands of organic small molecule fluorescent probes have been synthesized in recent years. Here, we demonstrate an unexpected and significant enhancement of brightness of Alizarin Red S (ARS)/PyB(OH)₂ (A/P) complexes, which we reported recently by coordination induced assembly with Al³⁺. A novel fluorescence turn-on method for Al³⁺ detection was developed based on the significant emission change. The introduction of Al³⁺ to the formed A/P/Al³⁺ ensembles make them highly suitable for constructing sensitive fluorescence methods because Al³⁺ provides recognition sites. The observed performance was rationalized through theoretical calculations, including HOMO-LUMO gaps and configuration changes. Thus, a highly sensitive sensing system for PPi and ALP was proposed. Unlike other Cu²⁺ or Cd²⁺ based ALP on-off assay, the proposed Al³⁺ based ALP assay allows off-on readout. In addition, the practicality of the proposed sensing system has been validated by detecting ALP in complex fetal bovine serum. The strategy can also be extended to enhance the brightness of other existing fluorescent probes, and related work is currently underway in our laboratory.