A Three-Dimensional Electrochemiluminescence Sensor Integrated with Peptide Hydrogel for Detection of H₂O₂ Released from Different Subtypes of Breast Cancer Cells

Breast cancer is a malignant tumor, with various subtypes showing different behaviors. Endogenous H₂O₂ is an important marker of tumor progression, which makes it important to study the relationship between breast cancer subtypes and H₂O₂ for pathogenesis and treatment strategies, but this has rarely been reported so far. In this work, we constructed a three-dimensional (3D) electrochemiluminescence (ECL) sensing platform for the detection of H₂O₂ released from two typical subtypes of breast cancer cells (MCF-7 cells for luminal A-type and MDA-MB-231 cells for three negative breast cancers, TNBCs). To adequately replicate the tumor microenvironment, the peptide hydrogel was introduced as a scaffold for 3D cell culture. The titanium foam (TF) was used as a 3D electrode to better match the 3D culture substrate. <i>N</i>-(4-Aminobutyl)-<i>N</i>-ethylisoluminol (ABEI) was selected as the ECL emitter and assembled into the peptide hydrogel by hydrogen bonding and π-stacking, which resulted in a stable and homogeneous distribution of ABEI along the hydrogel fibers. Furthermore, basic amino acids were introduced to provide alkaline microenvironment for ABEI. Therefore, ABEI exhibited high ECL efficiency, resulting in a high sensitivity with an ultralow detection limit of 0.023 nM (S/N = 3) for H₂O₂ of the proposed ECL biosensor. MCF-7 and MDA-MB-231 cells were cultured in a 3D peptide hydrogel/ABEI/TF electrode, respectively, and endogenous H₂O₂ was successfully monitored. A notably significant difference of H₂O₂ released between MDA-MB-231 cells and MCF-7 cells without stimulation but similar extra release with stimulation were observed. These findings may help understand the physiological mechanisms behind the various subtypes and reactive oxygen species (ROS)-related treatment for breast cancer.