A New Ratiometric Fluorescent Probe for Specific Monitoring of hROS under Physiological Conditions Using Boric Acid-Protected l-DOPA Gold Nanoclusters

The specific monitoring of physiological highly reactive oxygen species (hROS) using fluorescent gold nanoclusters (AuNCs) remains a challenge for scientists. Herein, SLB-AuNC was first synthesized via an ecofriendly one-pot method using starch as a template, l-3,4-dihydroxyphenylalanine (l-DOPA) as a reducing and a capping agent, and boric acid as a protecting agent for the catechol moiety of l-DOPA. The ingenious introduction of starch and boric acid enhanced the dispersibility, quantum yield, and photostability of fluorescent SLB-AuNCs. The obtained SLB-AuNCs possessed good monodispersity with an average diameter of 2.9 ± 0.8 nm and exhibited highly stable fluorescence with maximum emission at 480 nm under physiological conditions. A ratiometric fluorescent probe for hROS was developed through an oxidization-regulated Förster-resonance-energy-transfer process between SLB-AuNCs and 2,3-diaminophenazine (the oxidative product of hROS and o-phenylenediamine, with maximum fluorescence emission at 560 nm). With increasing amount of hROS, the outstanding fluorescence variation of the probe (I₅₆₀ ₙₘ/I₄₈₀ ₙₘ) enhanced about 300-fold, accompanied with a distinguishable color change from cyan to yellow. The detection limits of •OH, ClO–, and ONOO– were calculated as 0.11, 0.50, and 0.69 μM, respectively. High selectivity was achieved using o-phenylenediamine as a specific signal response for hROS to enable no interference reaction of other ROS toward SLB-AuNCs. The practicability of the proposed probe with super biocompatibility was evaluated by measuring exogenous and endogenous hROS levels in HeLa cells through fluorescence imaging. This work provides a novel strategy to design fluorescent AuNC probes for physiological hROS with great potential for the application of bioassay and bioimaging.