Selective, Rapid, and Ratiometric Fluorescence Sensing of Homocysteine in Live Neurons via a Reaction-Kinetics/Sequence-Differentiation Strategy Based on a Small Molecular Probe

Small molecular biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play essential roles in maintaining the redox homeostasis of biological systems, the disorders of which are closely associated with neuropathology. To date, many probes have been developed to identify Cys and GSH; however, due to the relatively low content and the high structural homology with Cys, there is still a lack of effective strategies to design probes enabling Hcy detection in physiological environments with high selectivity, high sensitivity, and rapid response. Herein, we developed a reaction-kinetics/sequence-differentiation strategy based on a dual-binding-site boron-dipyrrin (BODIPY) fluorophore, which was able to selectively distinguish Hcy from Cys and GSH within 50 s though a ratiometric fluorescence response mode. Benefiting from these features, the probe is capable of real-time imaging and quantitative analysis of intracellular Hcy in living neurons. Moreover, results of the disease-model experiments at the cellular level indicated a gradual increase of the Hcy level in neurons during the processes of aggregated amyloid-β (Aβ) peptide or ischemia treatment, which would further promote the neuron apoptosis. These findings provide the first direct experimental evidence for the impact of Alzheimer's disease and ischemic stroke on the Hcy metabolism of brain neurons and the associated neuron injury.