Ratio type nanoprobe with boric acid as recognition unit for imaging intracellular H₂O₂ with SERS

Fluctuations of intracellular H₂O₂ level are intricately linked to diverse biological processes, e.g., cellular proliferation, biosynthesis, and metabolic activities. The dynamics of intracellular H₂O₂ levels holds immense significance as it represents a pivotal metabolite within the realm of free radicals. This study describes a novel internal standard assisted surface enhanced Raman scattering (SERS) nanoprobe based on H₂O₂ boronic acid oxide group for precise detection of H₂O₂ levels in vitro and within living cells. The nanoprobe consists of a core molecule shell Au nanostar (Au@MPBN@Au NPs) embedded with internal marker (4-mercaptobenzonitrile, 4-MPBN) as a high plasma active SERS substrate and a 4-mercaptophenylboronic acid (4-MPBA) molecule fixed on its surface as H₂O₂ recognition unit. The presence of H₂O₂ leads to a decrease of the band intensity for B-O group at 998 cm^-1, while the absorption for the internal standard molecule 4-MPBN at 2223 cm^-1 remains unaffected by the variation of external environment. H₂O₂ quantification may be achieved by analyzing the ratio of band intensities at I_{2223 cm}^-1/I_{998 cm}^-1, with a linear relationship between I_2223cm^-1/I_998cm^-1 and H₂O₂ concentration within the range of 5-100 μM, along with a limit of detection (LOD) of 1.97 μM. Meanwhile, due to the presence of internal marker molecules, measurement errors caused by environmental or instrumental fluctuations can be calibrated in real-time. The AuNPs/4-MPBN/4-MPBA nanoprobe provides an accurate tool for dynamic monitoring and quantitative characterization of intracellular H₂O₂ content during cell apoptosis or other cell growth processes.