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

Autor: Gustafson KT; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.; Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon, USA., Sayar Z; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.; Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon, USA., Modestino A; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.; Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon, USA., Le HH; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.; Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon, USA., Gower A; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA., Civitci F; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA., Esener SC; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.; Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon, USA., Heller MJ; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA., Eksi SE; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.; Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon, USA.; Division of Oncological Sciences, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.
Jazyk: angličtina
Zdroj: Electrophoresis [Electrophoresis] 2024 Oct; Vol. 45 (19-20), pp. 1715-1720. Date of Electronic Publication: 2024 Jul 25.
DOI: 10.1002/elps.202400088
Abstrakt: We 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.
(© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)
Databáze: MEDLINE
Externí odkaz: