Red-Emissive Copper-Gold Bimetallic Metal-Organic Gel Nanozyme as a Highly Sensitive and Selective Ratiometric Photoluminescent Probe for Cellular Thiamine Detection.

Accurate detection of cellular thiamine is crucial for biological research and disease diagnosis but is often compromised by complex environments and interfering structural analogs, such as thiamine monophosphate and thiamine pyrophosphate. To address this challenge, we designed copper-gold bimetallic metal-organic gels (Cu-Au MOGs) that functioned as an all-in-one ratiometric photoluminescent biosensing platform. The Cu-Au MOGs synergistically integrated a red-emissive luminophore and a peroxidase-like nanozyme into a unified material, enabling rapid, selective, and sensitive detection of cellular thiamine. Synthesized via a facile one-pot in situ assembly of 6-mercaptopurine coordinated to Cu/Au centers (assisted by ascorbic acid), the Cu-Au MOGs exhibited red emission at 656 nm with a large Stokes shift of 286 nm, providing an ideal internal reference signal. Leveraging the peroxidase-like activity of the Cu sites, the Cu-Au MOGs catalyzed the hydrogen peroxide-mediated oxidation of nonemissive thiamine to photoluminescent thiochrome (emission: 464 nm) via hydroxyl radical formation and electron transfer. The process provided a label-free, turn-on ratiometric readout based on the intensity ratio (I464/I656). The biosensor demonstrated two linear ranges (0.005-0.5 μM and 0.5-40 μM), a detection limit of 1.58 nM, high selectivity over phosphorylated thiamine derivatives, and high stability. Its practical utility was validated by accurately quantifying thiamine levels in different cell lines (RSC96, HeLa, and MCF-7), showing good agreement with a reference high-performance liquid chromatography method. This work not only establishes a reliable tool for cellular thiamine bioanalysis, but also showcases the potential of heterometallic MOGs as versatile multifunctional biosensing platforms.