Whole Blood-Based Point-of-Care Assay for Single-Tier Lyme Disease Testing

Abstract

Rapid point-of-care diagnostics that operate directly on whole blood can accelerate clinical decision-making by bypassing complex sample preprocessing and centralized laboratory infrastructure. However, most serologic assays still require serum or plasma, limiting near-patient deployment. This challenge is particularly evident for Lyme disease, whose diagnosis relies on centralized two-tier serology. Here, we present a whole-blood multiplexed vertical flow assay (WB-xVFA) that performs single-tier Lyme serology directly from 50 microliter of whole blood. The assay combines multilayer blood filtration with a peptide-patterned nitrocellulose membrane to separate cellular components and detect Lyme-associated IgM/IgG responses on a cartridge. Coupled with smartphone-based imaging and machine-learning analysis, the WB-xVFA provides results in 20 min. The assay remained robust across hematocrits up to 60% and sample volumes ranging from 25 to 100 microliter. In blinded testing of 62 whole-blood measurements, it achieved ~97% sensitivity and specificity, demonstrating the feasibility of rapid, near-patient whole-blood Lyme serology.