Immunoassays show great potential for the detection of
low levels
of cytokines, due to their high sensitivity and excellent specificity.
There is a particular demand for biosensors that enable both high-throughput
screening and continuous monitoring of clinically relevant cytokines
such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel
bioluminescent immunoassay based on the ratiometric plug-and-play
immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background
and an >80-fold increase in the luminescent signal. The new dRAPPID
assay, comprising a dimeric protein G adapter connected via a semiflexible
linker, was applied to detect the secretion of IL-6 by breast carcinoma
cells upon TNFα stimulation and the production of low concentrations
of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle
tissue model. Moreover, we integrated the dRAPPID assay in a newly
developed microfluidic device for the simultaneous and continuous
monitoring of changes in IL-6 and TNFα in the low-nanomolar
range. The luminescence-based read-out and the homogeneous nature
of the dRAPPID platform allowed for detection with a simple measurement
setup, consisting of a digital camera and a light-sealed box. This
permits the usage of the continuous dRAPPID monitoring chip at the
point of need, without the requirement for complex or expensive detection
techniques.