Development of a portable low-cost customizable immunoassay robot for multiplexed diagnostics using enzymatic silver metallization

Automation can improve the efficiency, reproducibility, and affordability of clinical immunodiagnostics, yet most ELISA automation platforms are bulky and expensive and remain poorly suited to decentralized, resource-poor settings. Enzymatic silver metallization immunoassays on low-cost glass or plastic slides enable multiplexed microscale measurements from a single drop of sample with a dry readout compatible with cellphone imaging, but fluid handling is still largely manual. We present AutoENZ, a low-cost, 3D-printed, modular immunoassay robot that executes customizable enzymatic metallization protocols with minimal user intervention. The user inserts a prepared diagnostic chip, loads the required reagents, and provides a protocol file, after which AutoENZ automates reagent delivery, washing, timed waiting, and drying steps to produce a metallized readout that can be quantified using a cellphone camera. AutoENZ is controlled by a microcontroller and uses integrated pumping, using inexpensive off-the-shelf components instead of custom microfluidics, for both continuous flow and calibrated microliter-scale dispensing. Built from accessible components, AutoENZ reduces hands-on time while maintaining quantitative performance. We validate AutoENZ using a model binding assay and multiplexed detection of antigen-specific antibodies in clinical tuberculosis samples, demonstrating its utility for affordable diagnostics in decentralized settings.