Combining time domain modulation optofluidics and high dynamic range imaging for multiplexed, high throughput digital droplet assays

Digital enzyme-linked immunoassays (dELISA) have been successfully applied to the ultrasensitive quantification of analytes, including nucleic acids, proteins, cells, and extracellular vesicles, achieving robust detection limits in complex clinical specimens such as blood, and demonstrating utility across a broad range of clinical applications. The ultrasensitivity of dELISA comes from partitioning single analytes, captured onto a microbead, into millions of compartments so that they can be counted individually. There is particular interest in using dELISA for multiplexed measurements, but generating and detecting the billions of compartments necessary to perform multiplexed ultrasensitive dELISA remains a challenge. To address this, we have developed a high-throughput, optofluidic platform that performs quantitative fluorescence measurements on five populations of microbeads, each encoded with distinct ratios of two fluorescent dyes, for digital assays. The key innovation of our work is the parallelization of droplet generation and detection, combined with time-domain encoding of the excitation sources into distinct patterns that barcode the emission signal of both dyes within each bead, achieving high throughput (6 × 10⁶ droplets/min) and accurate readout. Additionally, we modulate the exposure settings of the digital camera, capturing images of multiplexed beads and the droplet fluorescent substrate in consecutive frames, a method inspired by high dynamic range (HDR) photography. Our platform accurately classifies five populations of dual-encoded beads (accuracy > 99%) and detects bead-bound streptavidin-horseradish peroxidase molecules in a third fluorescence channel. This work establishes the technological foundation to combine high multiplexing and high throughput for droplet digital assays.