posted on 2024-02-21, 12:36authored byEva A. van Aalen, Joep J. J. Lurvink, Leandra Vermeulen, Benice van Gerven, Yan Ni, Remco Arts, Maarten Merkx
Here we present LUCOS (Luminescent Competition Sensor),
a modular
and broadly applicable bioluminescent diagnostic platform enabling
the detection of both small molecules and protein biomarkers. The
construction of LUCOS sensors entails the covalent and site-specific
coupling of a bioluminescent sensor component to an analyte-specific
antibody via protein G-mediated photoconjugation. Target detection
is accomplished through intramolecular competition with a tethered
analyte competitor for antibody binding. We established two variants
of LUCOS: an inherent ratiometric LUCOSR variant and an intensiometric
LUCOSI version, which can be used for ratiometric detection upon the
addition of a split calibrator luciferase. To demonstrate the versatility
of the LUCOS platform, sensors were developed for the detection of
the small molecule cortisol and the protein biomarker NT-proBNP. Sensors
for both targets displayed analyte-dependent changes in the emission
ratio and enabled detection in the micromolar concentration range
(KD,app = 16–92 μM). Furthermore,
we showed that the response range of the LUCOS sensor can be adjusted
by attenuating the affinity of the tethered NT-proBNP competitor,
which enabled detection in the nanomolar concentration range (KD,app = 317 ± 26 nM). Overall, the LUCOS
platform offers a highly versatile and easy method to convert commercially
available monoclonal antibodies into bioluminescent biosensors that
provide a homogeneous alternative for the competitive immunoassay.