Controlled microfluidic tank integrated AgNPs-coated paper-based impedimetric immunosensor for leukemia biomarker detection and complete validation through 3i-study in-silico, in-vitro, in-vivo.

Leukemia, a critical hematological malignancy, demands early and accurate diagnosis to improve outcomes. In present work a 3D-printed microfluidic platform enables sensitive, label-free PHB2 detection via an AgNP-based paper immunosensor. Silver nanoparticles enhance electron transfer kinetics and provide a high surface area for efficient antibody immobilization, improving sensor performance. Electrochemical impedance spectroscopy (EIS) measurements show a stepwise increase in charge-transfer resistance (Rct) upon antigen-antibody interaction, confirming the sensing mechanism. The immunosensor exhibits a linear detection range of 2.5-40 ng mL-1 with a limit of detection of 2.5 ng mL-1. The platform demonstrates high selectivity against interfering biomarkers, excellent reproducibility, satisfactory recovery in spiked samples, and stable performance for up to 15 days. Biological validation using serum samples (animal serum and synthetic serum), HL-60 cell lines, and a BALB/c mouse model confirms its applicability in complex matrices. In silico and in vitro studies further support PHB2 as a diagnostic target. The platform offers a sensitive, portable, and cost-effective approach for point-of-care leukemia screening.