Real Time and Spatiotemporal Quantification of pH and H 2 O 2 Imbalances with a Multiplex Surface-Enhanced Raman Spectroscopy Nanosensor.

Oxidative stress is involved in many aging-related pathological disorders and is the result of defective cellular management of redox reactions. Particularly, hydrogen peroxide (H 2 O 2 ), is a major byproduct and a common oxidative stress biomarker. Monitoring its dynamics and a direct correlation to diseases remains a challenge due to the complexity of redox reactions. Sensitivity and specificity are major drawbacks for H 2 O 2 sensors regardless of their readout. Luminiscent boronate-based probes such as 3-mercaptophenylboronic acid (3-MPBA) are emerging as the most effective quantitation tool due to their specificity and sensitivity. Problems associated with these probes are limited intracellular sensing, water solubility, selectivity, and quenching. We have synthesized a boronate-based nanosensor with a surface-enhanced Raman spectroscopy (SERS) readout to solve these challenges. Furthermore, we found out that environmental pH gradients, as found in biological samples, affect the sensitivity of boronate-based sensors. When the sensor is in an alkaline environment, the oxidation of 3-MPBA by H 2 O 2 is more favored than in an acidic environment. This leads to different H 2 O 2 measurements depending on pH. To solve this issue, we synthesized a multiplex nanosensor capable of concomitantly quantifying pH and H 2 O 2 . Our nanosensor first measures the local pH and based on this value, provides the amount of H 2 O 2 . It seems that this pH-dependent sensitivity effect applies to all boronic acid based probes. We tested the multiplexing ability by quantitatively measuring intra- and extracellular pH and H 2 O 2 dynamics under physiological and pathological conditions on healthy cells and cells in which H + and/or H 2 O 2 homeostasis has been altered.