Counting of enzymatically amplified affinity reactions in hydrogel particle-templated drops

Counting of numerous compartmentalized enzymatic reactions underlies quantitative and high sensitivity immunodiagnostic assays. However, digital enzyme-linked immunosorbent assays (ELISA) require specialized instruments which have slowed adoption in research and clinical labs. We present a lab-on-a-particle solution to digital counting of thousands of single enzymatic reactions. Hydrogel particles are used to bind enzymes and template the formation of droplets that compartmentalize reactions with simple pipetting steps. These hydrogel particles can be made at a high throughput, stored, and used during the assay to create ∼500 000 compartments within 2 minutes. These particles can also be dried and rehydrated with sample, amplifying the sensitivity of the assay by driving affinity interactions on the hydrogel surface. We demonstrate digital counting of β-galactosidase enzyme at a femtomolar detection limit with a dynamic range of 3 orders of magnitude using standard benchtop equipment and experiment techniques. This approach can faciliate the development of digital ELISAs with reduced need for specialized microfluidic devices, instruments, or imaging systems.