Sugar additives improve signal fidelity for implementing two-phase resorufin-based enzyme immunoassays

Enzymatic signal amplification based on fluorogenic substrates is commonly used for immunoassays; however, when transitioning these assays to a digital format in water-in-mineral oil emulsions, such amplification methods have been limited by the leakage of small reporting fluorescent probes. In the present study, we used a microfluidic system to study leakage from aqueous droplets in a controlled manner and confirmed that the leakage of fluorescent resorufin derivatives is mostly due to the presence of the lipophilic surfactant Span80, which is commonly used to preserve emulsion stability. This leakage can be overcome by the addition of specific sugars that most strongly interfered with the surfactants ability to form micelles in water. The application of the microfluidic system to the quantitative analysis of droplets and the implementation of the described sugar additives would allow for alternatives to fluorinated surfactant-based platforms and improve the signal fidelity in enzyme immunoassays implemented through multiphase microfluidics.