Evaporation-driven digital ELISA with micro-droplet arrays for ultrafast detection of low-abundance proteins

Early detection of ultra-low-abundance protein biomarkers is critical for improving clinical outcomes. However, many conventional immunoassays and proteomic platforms exhibit high limits of detection (LOD) and long incubation times, complicating low-to sub-femtomolar measurements. In addition, state-of-the-art digital ELISAs often rely on magnetic beads, oil emulsions, and pump-valve benchtop analyzers-bulky and costly systems that hinder broad deployment. Here, we present a digital ELISA (dELISA) that lowers LOD and shortens time-to-result by harnessing rapid, repeated femtoliter-scale droplet evaporation on a bead-free, oil-free micro-droplet array. Cyclic ≈30-fL droplet generation/evaporation at the air-water interface enriches analytes at antibody-functionalized surfaces, while evaporation-induced convection and a shortened diffusion path increase antigen-antibody encounter frequency. For cardiac troponin I, the platform achieved an LOD of 64.23 fM with 300 evaporation cycles, a >280-fold improvement over 96-well ELISA. The same capture obtained after 120 min of static soaking occurred in 1.76 min with cyclic evaporation. Counts matched a Poisson capture model, with binding increasing linearly with cycle number. In diluted human plasma, the assay maintained >86 % recovery with minimal non-specific adsorption. Simple fabrication, the absence of beads, oils, and complex fluidic hardware, and high-throughput compatibility indicate strong potential for point-of-care use and integration into diverse digital immunoassay applications.