Analytical and Structural Evaluation of Recombinant Human Serum Albumin and Fragment F8 for Aptamer-Based Urinary Biomarker Detection

Abstract

Human serum albumin (HSA) is a key biomarker for early detection of kidney disorders such as microalbuminuria. However, its structural complexity and susceptibility to degradation in urine challenge current diagnostic assays, particularly immunoturbidimetric methods that depend on epitope stability. In this study, we expressed and characterized full-length recombinant HSA (rHSA) and its proteolytic fragment F8 (residues 496–585) in both Escherichia coli and rice-based systems to explore their suitability for biosensor validation and diagnostic use. Comparative analysis via SDS-PAGE, LC–MS/MS, and dynamic light scattering revealed differences in solubility and aggregation profiles between expression systems, with rice-derived rHSA showing greater resistance to proteolytic degradation. The F8 fragment retained structural integrity and remained detectable in both native and protease-treated samples. Using a previously developed Cy5-labeled ssDNA aptamer-based fluorescence aptasensor, we evaluated binding to rHSA and F8 across a clinically relevant concentration range (0.37–1500 μg/mL). Sigmoidal fluorescence recovery curves with R²> 0.99 were observed for all recombinant proteins. Notably, the aptasensor successfully detected both targets even under conditions mimicking nonfresh urine, highlighting its diagnostic robustness. These findings establish recombinant F8 as a stable, diagnostically relevant biomarker candidate and confirm the practical utility of aptamer-based platforms for albumin detection in diverse testing scenarios.