Yes/No Quantitative Analysis of Single-Stranded Oligonucleotides with Lateral Flow Assays

Abstract

Accessible molecular diagnostics is fundamental to effective healthcare. While most current point-of-care devices detect only the presence of a molecular biomarker(s), biomarker quantification can be equally important for decision-making on disease treatment and containment. Here, we present a diagnostic platform that enables the equipment-free quantification of molecular biomarkers with the simplicity of a binary (yes/no) readout. This capability is achieved by integrating a stoichiometric quantitative approach with widely available and easy-to-use lateral flow dipsticks. To implement the approach, we engineer negative cooperativity into target–probe binding interactions for oligonucleotide targets as a model system. The resulting threshold-based semi-quantitative assay with lateral flow dipsticks quantifies targets in the low-nanomolar range and operates reliably in complex biological backgrounds. A key advantage of this platform is its potential adaptability to new and emerging targets: repurposing will require only reagent redesign, without the need for additional fabrication.