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

Area of Science:

• Biotechnology
• Analytical Chemistry
• Microfluidics

Background:

• Digital enzyme-linked immunoassays (dELISA) offer ultrasensitive analyte quantification in complex samples.
• Current dELISA methods face challenges in achieving high-throughput multiplexing for billions of compartments.
• Ultrasensitive detection is crucial for clinical diagnostics and research.

Purpose of the Study:

• To develop a high-throughput optofluidic platform for multiplexed digital assays.
• To overcome limitations in generating and detecting multiplexed dELISA compartments.
• To enable simultaneous quantification of multiple analytes with high sensitivity and throughput.

Main Methods:

• Developed a novel optofluidic platform for quantitative fluorescence measurements.
• Utilized microbeads encoded with distinct ratios of two fluorescent dyes for multiplexing.
• Implemented parallelized droplet generation and detection with time-domain encoding.
• Employed high dynamic range (HDR) photography principles for enhanced imaging.

Main Results:

• Achieved high-throughput droplet generation at 6 × 10^6 droplets/min.
• Demonstrated accurate classification of five populations of dual-encoded microbeads (>99% accuracy).
• Successfully detected bead-bound streptavidin-horseradish peroxidase in a third fluorescence channel.

Conclusions:

• Established a technological foundation for high-multiplexing, high-throughput droplet digital assays.
• The platform enables advanced multiplexed ultrasensitive quantification.
• This work paves the way for broader clinical applications of dELISA.