Microencapsulated Immunoassays for Detection of Cytokines in Human Blood

Cytokines are produced by leukocytes in blood and may be used as indicators of malignancies or infections. The objective of this study was to develop a strategy for immunosensing cytokines in whole, unprocessed human blood. Microfluidic droplet generation was employed to fabricate ∼400 μm diameter microcapsules with a hydrogel shell and an aqueous core containing sensing microbeads. The hydrogel shell was composed of poly(ethylene glycol) forming a thin (∼10 μm) immunoisolation layer protecting antibody-modified microbeads inside the capsule from immune cells on the outside. The microbeads were functionalized with antibodies against cytokines of interest: interferon (IFN)-γ and tumor necrosis factor (TNF)-α. While nonfouling, a hydrogel shell was permeable to cytokine molecules; these molecules were captured on microbeads and were detected with fluorescently labeled secondary antibodies. Calibration of encapsulated immunoassays with known concentrations of cytokines revealed a limit of detection of 14.8 and 14.4 pM for IFN-γ and TNF-α, respectively. We also demonstrated the concept of multi-cytokine detection by fabricating distinct populations of capsules carrying either anti-IFN-γ or anti-TNF-α microbeads and dispensing these capsules into a solution containing both cytokine types. Importantly, when placed into whole blood for 16 h, microcapsules were free of leukocytes, effectively protecting sensing beads from the blood components. To further demonstrate utility of this strategy, encapsulated microbeads were used for detection of IFN-γ in blood of patients with latent tuberculosis infection (LTBI) and unexposed healthy controls. When compared to gold standard technology (interferon gamma release assay or IGRA), our encapsulated immunoassay accurately predicted LTBI diagnosis in 11 out of 14 patients. Overall, encapsulation of immunoassays represents a promising strategy for keeping sensing elements operational in a highly fouling complex environment such as blood.