Multiplex Digital Immunoassays with Reduced Pre-partition Reaction via Massively Parallel Reagent Delivery on a Bead-Based SlipChip

The emergence of digital immunoassays has advanced thesensitivityof protein analysis to ultrahigh sensitivity at the attomolar level.However, the background signal generated by the premixing of immunocomplexesand fluorogenic substrates can limit the precise quantification, especiallyin multiplexed assays. Herein, a bead-based SlipChip (bb-SlipChip)microfluidic device capable of massively parallel two-step sampleloading is presented. The background signal can be suppressed througha two-step loading mechanism. Specifically, encapsulate the beadsinto the microwells first and then, through a slipping process, deliverthe fluorogenic substrate in parallel into 281,200 microwells of 68fL to perform the digital immunoassay. The quantification capabilityis demonstrated with a duplex assay of IL-6 and IL-10, achieving alimit of detection of 5.2 and 15.3 fg/mL, which is approximately 2-3times improved compared to a commercial Simoa system. The bb-SlipChipprovides a robust and universal method for digital immunoassay andcan be extended to higher multiplexed detection as well as other biomedicalapplications involving microbeads.

Automated summary

The advent of digital immunoassays has greatly enhanced the sensitivity of protein analysis to ultra-low levels, but the background signal from premixing can hinder precise quantification, especially in multiplexed assays. This study introduces a bead-based SlipChip microfluidic device that allows massively parallel two-step sample loading, effectively suppressing the background signal. The device achieves improved detection limits for IL-6 and IL-10 compared to a commercial system. The bb-SlipChip offers a robust and universal approach for digital immunoassays and has potential applications in multidimensional detection and other biomedical studies involving microbeads.