A High-Sensitive and Low-Probe-Dosage Nanoprobe for Quantifying Hydrogen Peroxide Fluctuations in Physiological Pathways

The latest research has revealed significant insights into the critical role of hydrogen peroxide (H₂O₂) signaling pathways. However, during the detection process, certain probes necessitate micromolar concentrations to achieve effective imaging, while others exhibit low sensitivity. Therefore, the development of methodologies capable of enabling high-sensitivity detection while minimizing the required dosage of the probe remains an urgent priority. As a proof-of-concept, the nanoprobe was designed by conjugating BHQ2, a quenching agent that effectively suppresses the fluorescence of wrappage, with benzylbenzeneboronic acid pinacol ester (Bpe), an H₂O₂ recognition site, to an alkynyl-functionalized poly(carbonate) backbone. Within this structure, SQ-dye, a squarylium dye capable of significantly enhancing fluorescence quantum yields through interactions with cytoplasmic proteins, was encapsulated to form a micelle-based nanoprobe, designated as PMPC-Bpe-BHQ2@SQ. The presence of H₂O₂ activates the micelles, thus releasing the SQ-dye and generating primary fluorescence enhancement. Moreover, the released SQ-dye can bind with cytoplasmic proteins, leading to secondary fluorescent enhancement. Owing to the secondary signal amplification effect of the probe, the required dosage of the probe in the imaging process is significantly reduced. Owing to the incorporation of the quencher BHQ2 and the signal amplification mechanism, the fluorescence intensity of the probe was enhanced by 265.83-fold, thereby enabling a detection limit as low as 1 nM. The proposed method mitigates the disruption of the dynamic balance of H₂O₂ in organisms caused by excessive probe usage, thereby enabling a more accurate elucidation of the growth factor-stimulated signaling pathways in cells and the in vivo mechanism of H₂O₂ production induced by testosterone.