3D nanoporous hydrogel-integrated portable platform for pretreatment-free, rapid multiplexed detection of small-molecule biomarkers in whole blood.

Point-of-care testing (POCT) demands rapid, user-friendly solutions for instant medical diagnosis. Traditional colorimetric whole-blood analysis faces challenges from red blood cell and hemoglobin interference, often necessitating complex pretreatment. Herein, we introduce a three-dimensional (3D) nanoporous hydrogel-integrated portable platform designed for pretreatment-free, rapid multiplexed detection of small-molecule biomarkers directly from whole blood. The platform utilizes a polystyrene Eppendorf (EP)-tube as a miniaturized laboratory, featuring a TiO2-sodium alginate hydrogel-modified cap with a 3D nanoscale porous architecture. This innovative structure serves dual roles: embedding enzymatic systems for cascade reactions and excluding macromolecules (e.g., hemoglobin, erythrocytes) through size-selective exclusion. Small-molecule biomarkers (e.g., glucose, uric acid, lactate) diffuse into the hydrogel matrix, triggering concentration-proportional colorimetric signals via enzymatic cascade reactions. A 15-min "flip-and-scan" protocol-comprising tube inversion for biomarker diffusion and smartphone RGB analysis for quantitative readout-enables simultaneous quantification of three biomarkers with detection limits of 0.11 mM for glucose, 16 μM for uric acid, and 20 μM for lactate. The clinical validation with diverse patient samples (diabetic, gout, and composite metabolic cases) demonstrates that the tri-marker detection strategy enables precise differential diagnosis of overlapping metabolic syndromes by distinguishing biomarker patterns. Clinical validation with 15 diverse patient samples (5 diabetic patients, 5 gout patients, and 5 patients with composite metabolic abnormalities) demonstrated strong concordance with a commercial clinical biochemistry analyzer, with Pearson correlation coefficient > 0.95 and negligible bias in Bland-Altman analysis with all data points falling within the 95% limits of agreement. Compared with conventional microfluidic and paper-based sensors, this approach eliminates pretreatment steps, reduces assay time, ensures closed-loop biosafety, and prevents cross-contamination, offering a versatile and accessible analytical platform that represents a significant step toward practical multiplexed biomarker detection in POCT.