Oligo cyc‐DEP: On‐chip cyclic immunofluorescence profiling of cell‐derived nanoparticles

Abstract

AbstractWe present a follow‐on technique for the cyclic‐immunofluorescence profiling of suspension particles isolated using dielectrophoresis. The original lab‐on‐chip technique (“cyc‐DEP” [cyclic immunofluorescent imaging on dielectrophoretic chip]) was designed for the multiplex surveillance of circulating biomarkers. Nanoparticles were collected from low‐volume liquid biopsies using microfluidic dielectrophoretic chip technology. Subsequent rounds of cyclic immunofluorescent labeling and quenching were imaged and quantified with a custom algorithm to detect multiple proteins. While cyc‐DEP improved assay multiplicity, long runtimes threatened its clinical adoption. Here, we modify the original cyc‐DEP platform to reduce assay runtimes. Nanoparticles were formulated from human prostate adenocarcinoma cells and collected using dielectrophoresis. Three proteins were labeled on‐chip with a mixture of short oligonucleotide‐conjugated antibodies. The sample was then incubated with complementary fluorophore‐conjugated oligonucleotides, which were dehybridized using an ethylene carbonate buffer after each round of imaging. Oligonucleotide removal exhibited an average quenching efficiency of 98 ± 3% (n = 12 quenching events), matching the original cyc‐DEP platform. The presented “oligo cyc‐DEP” platform achieved clinically relevant sample‐to‐answer times, reducing the duration for three rounds of cyclic immunolabeling from approximately 20 to 6.5 h—a 67% decrease attributed to rapid fluorophore removal and the consolidated co‐incubation of antibodies.