NIR-II-Absorbing TMB Derivative for 1064 nm-Excited Photothermal Immunoassay

Materials exhibiting strong absorption in the NIR-II region are appealing for photothermal conversion-based imaging, diagnosis, and therapy, due to better thermal effect and decreased absorption of water in such a region. 3,3',5,5'-Tetramethylbenzidine (TMB), the typical substrate in ELISA, has been explored in photothermal immunoassay, since its oxidation product (oxTMB) is photothermally active in the NIR region. However, its absorption at 1064 nm (the most often used laser wavelength in photothermal studies) is not appreciable, thus limiting the assay sensitivity. Here, we proposed a derivative of TMB (3,3'-dimethoxy-5,5'-dimethylbenzidine, 2-OCH₃) bearing higher NIR-II absorption for 1064 nm-excited photothermal immunoassay. Since electron-donating groups can help decrease the energy gap of molecules (here -CH3 → -OCH₃), the oxidation product of 2-OCH₃ exhibited substantially red-shifted absorption as compared with oxTMB, leading to a more than twofold higher absorption coefficient at 1064 nm. As a result, 2-OCH₃ showed enhanced sensitivity over TMB in a photothermal immunoassay (PTIA), yielding a limit of detection (LOD) of 0.1 ng/mL for prostate-specific antigen (PSA). The feasibility of 2-OCH₃-based PTIA for diagnosis was further validated by analyzing PSA in 61 serum samples. Considering its superior photothermal performance, 2-OCH₃ can be explored for a broad range of photothermal applications.