Single droplet detection of immune checkpoints on a multiplexed electrohydrodynamic biosensor

Area of Science:

• Biomedical Engineering
• Immunology
• Analytical Chemistry

Background:

• Monitoring soluble immune checkpoints in bodily fluids is crucial for minimally-invasive diagnostics and personalized therapy in precision medicine.
• Sensitive detection of multiple immune checkpoints from small liquid biopsy volumes remains a significant challenge.

Purpose of the Study:

• To develop a multiplexed immune checkpoint biosensor (MICB) for parallel detection of soluble immune checkpoints PD-1, PD-L1, and LAG-3.
• To evaluate the assay performance and clinical applicability of the MICB for immune checkpoint monitoring.

Main Methods:

• Development of a microfluidic sandwich immunoassay utilizing engineered single chain variable fragments.
• Integration of alternating current electrohydrodynamic in situ nanofluidic mixing to enhance biosensor-target interaction and reduce non-specific binding.
• Utilizing yeast cell-derived single chain variable fragments as a cost-effective alternative to monoclonal antibodies.

Main Results:

• The MICB achieved simultaneous analysis of up to 28 samples in under 2 hours, requiring only 20 μL of sample per target.
• Demonstrated clinically-relevant detection levels with dynamic ranges of 5-200 pg/mL for PD-1/PD-L1 and 50-1000 pg/mL for LAG-3.
• Achieved sensitive detection limits of 5 pg/mL for PD-1, 5 pg/mL for PD-L1, and 50 pg/mL for LAG-3, with a coefficient of variation below 13.8%.

Conclusions:

• The MICB is a sensitive, multiplexed bioanalytical tool capable of parallel detection of key soluble immune checkpoints.
• Its ability to analyze small sample volumes efficiently makes it suitable for immune checkpoint therapy monitoring.
• The biosensor offers a promising advancement for precision medicine and personalized therapeutic strategies.