Confinement and digital counting of enzymatic reactions in microparticle-templated drops

Digital enzyme linked immunosorbent assays (dELISAs) offer exquisite sensitivity down to single molecule detection, and hold promise for early disease detection and patient monitoring [1]. Currently, the dELISA landscape is dominated by microwell and droplet-based platforms which leverage microfluidics to compartmentalize reactions into sub-nanoliter volumes to quickly concentrate signals from singular reporter enzymes. [2] These platforms require specialized skill and equipment or significant up-front capital investments on the user side; which has impeded the large-scale adoption of dELISA technologies for clinical research. Our transformative approach addresses several challenges to build an accessible dELISA platform by leveraging hydrogel particles to (1) provide solid support for affinity capture of biomarkers and enable washing to reduce assay background and (2) generate highly monodisperse particles by simple pipetting. This workflow can seamlessly integrate with pre-existing laboratory equipment for scale-up and large-scale testing required for clinical adoption.

Here, we prepared spherical biotinylated  polyethylene  glycol  (PEG)  hydrogel  particles using droplet microfluidic technologies [3] showing excellent uniformity (CV~5%) over a 10 hour  production period [4]; these can be stored long-term enabling scale-up and easy deployment. By simple pipetting and agitation, the particles template the formation of highly monodisperse droplets (CV~4%), called particle-drops or dropicles, within 90 seconds (5k dropicles/s). This batch approach can be easily scaled to larger quantities of dropicles for a larger dynamic range and higher resolution. In this approach, every droplet is deterministically associated with a single particle improving on Poisson limited loading observed in traditional microfluidic platforms. As  a  proof-of-concept  assay,  varying  concentrations  of  beta-galactosidase  (b-gal) labeled streptavidin  were incubated with the biotinylated particles, excess washed, and emulsified with fluorescein-di-b-galactopyranoside as substrate. We achieved single molecule counting with a limit of detection of 4.5 fM and 3 orders of magnitude dynamic range. We are now expanding to an assay for N-terminal (NT)-pro hormone B-type natriuretic peptide (NT-proBNP) for improved monitoring of patients for heart failure.

Overall, we demonstrate singular counting based quantification of affinity captured enzyme labels in droplets formed around a solid hydrogel support. We envision that these particles can be prepared at a central facility and shipped to local laboratories. Upon receiving the particles, the entire assay workflow can integrate with pre-existing laboratory equipment and automation infrastructure (liquid handlers, plate washers, etc.) precluding purchase of specialized dELISA only platforms. Reducing the need for specialized instrumentation can help democratize ultra-sensitive immunoassays, advancing clinical research and biomarker discovery.

References:

[1] Schubert, Arendt, Lou; Sci Rep; 2015

[2] Basu; SLAS Technology; 2017

[3] de Rutte; Adv. Functional Materials; 2019

[4] Wang, Shah; Lab Chip; 2021