Label-free evaluation of protein aggregation by a point-of-care fluorometric device

Protein aggregation is a hallmark of numerous neurological disorders, necessitating rapid, sensitive, and label-free platforms to monitor protein structural alterations. This study reports the development of a cost-effective, portable, light-emitting diode-based point-of-care fluorometric device operating at 285 nm for intrinsic protein fluorescence detection. The device employs an orthogonal optical configuration combined with externally metal-coated cuvettes to enhance fluorescence collection while minimizing background scattering. Among the evaluated cuvettes, aluminum-coated cuvettes provided the highest signal enhancement with minimal spectral distortion, enabling reliable nanomolar-level detection. Thermally induced destabilization of bovine serum albumin and human serum albumin was used as a well-established model to induce protein unfolding and aggregation. The device sensitively detected concentration-dependent and thermally induced changes in intrinsic fluorescence arising from tryptophan and tyrosine residues, primarily reflected as intensity variations and progressive red shifts rather than discrete spectral transitions. Independent validation using ultraviolet-visible spectroscopy, fluorescence dye assays, scanning electron microscopy, fluorescence microscopy, X-ray powder diffraction, circular dichroism spectroscopy, dynamic light scattering, and Fourier transform infrared spectroscopy confirmed that thermal treatment leads to partial unfolding and aggregation. Importantly, these complementary techniques consistently revealed heterogeneous and mixed aggregate populations containing amorphous and beta-sheet-associated structural features, rather than uniformly fibrillar or crystalline amyloid assemblies. The strong agreement between intrinsic fluorescence measurements and established biophysical methods demonstrates that the developed device reliably tracks protein structural destabilization and aggregation independent of aggregate morphology. Owing to its simplicity, portability, and label-free operation, this platform offers a practical alternative to conventional laboratory-based instrumentation for monitoring protein stability.