Droplet-Based Digital Ratiometric Fluorescence Coding for Multiplex Nucleic Acid Amplification Testing

Multiplexed NAAT is essential for molecular diagnosis of various diseases. Currently, encoding each nucleic acid target with a specific fluorescently-labeled probe has been the most commonly employed approach for multiplexed detection. Unfortunately, this "one-color-one-target" approach is limited by spectral overlap between fluorophores and cannot achieve highly multiplexed detection. This paper proposes a novel method termed Digital Ratiometric Fluorescence Coding to expand multiplexing capacity of NAAT. We encode each nucleic acid target with a specific ratio between two standard fluorophores in single droplet via a microfluidic system, allowing multiplexed detection of targets while obviating spectral overlap restriction. For demonstration, we detected DNA targets from six sexually transmitted diseases and demonstrated the potential for further expanding the multiplexing capability of our method.