Target-regulated in-situ polymer dot formation for pre-synthesis-free sensing of biothiols and glutathione reductase activity

Biothiol analysis is crucial for evaluating redox homeostasis and monitoring various physiological processes. However, conventional fluorescence-based strategies often rely on structurally complex probes and lack straightforward multi-target discriminative capabilities. Herein, we present a versatile, pre-synthesis-free fluorogenic platform driven by the target-responsive inhibition of in-situ polymer dot (PD) formation. We discovered that dopamine (DA) spontaneously condenses with N -[3-(trimethoxysilyl)propyl]diethylenetriamine (AEEA) to yield robustly fluorescent PDs. Biothiols, acting as highly reactive competitors, intercept the DA precursor via a preferential Michael addition, fundamentally suppressing PD formation and inducing a marked fluorescence decrease. This mechanism enables the rapid, highly sensitive quantification of glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), with low limits of detection (LODs) of 0.095, 0.115, and 0.083 μM, respectively. Significantly, the introduction of a structurally distinct DA-DAMO analog facilitated the discrimination of these biothiols. This reaction-interference strategy was then successfully coupled with enzymatic cascades to evaluate glutathione reductase (GR) activity within 20 min, achieving a LOD of 0.046 mU/mL. The programmable analyte-dependent fluorescence outputs were successfully employed as signal readouts to construct molecular logic gates. This work establishes target-regulated in-situ nanomaterial formation as a powerful paradigm for biothiol sensing, enzyme assays, and advanced logic operations.