Integrated Bioluminescent Immunoassays for High-Throughput Sampling and Continuous Monitoring of Cytokines

Abstract

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.

Development of Luminescent Biosensors for Calprotectin

“…NanoBiT-based technology has recently been developed with various synthetic scaffolds and applied for different tests, including but not limited to SARS-CoV-2 (COVID-19), , Clostridium difficile infections, , and botulinum neurotoxins . While efforts have been reported in combining NanoBiT with full-length antibodies, − the most common and versatile protein-based binder so far, such antibody engineering usually relies on chemical conjugation of antibody with LgBiT or SmBiT, which necessitates not only the costly production of antibodies, but also extensive purifications due to the heterogeneity of products. While primary efforts suggested Fab binder tagged with a short peptide (peptide β9 from NanoLuc) is compatible with the NanoBiT system, we reasoned that directly fusing NanoBiT with Fab could address the challenges associated with antibody conjugation, thereby fully leveraging Fab’s versatility.…”

Point-of-care therapeutic drug monitoring of tumour necrosis factor-α inhibitors using a single step immunoassay

“…We applied and adapted the recently established bioluminescent dRAPPID platform to realize a homogeneous sensor for infliximab and adalimumab TDM, with a simple read-out that allows applications in POC testing. 30 The dRAPPID sensors consist of two analyte-specific binders connected to either a Large Bit (LB) or a Small Bit (SB, K D = 2.5 μM) domain, the two fragments of split NLuc. 31 In the presence of the furimazine substrate of NLuc and upon analyte-induced reconstitution of split NLuc, blue light is emitted.…”

“…25–29 The recently developed dRAPPID (dimeric Ratiometric Plug-and-Play Immunodiagnostics) sensor format is based on analyte-induced split NanoLuc (NLuc) complementation. 30 Binding of the protein of interest by analyte-specific antibodies, covalently coupled to the split NLuc domains by photo-cross-linkable protein G adapters, engenders the formation of a luminescent ternary complex. 31,32 The mix-and-measure dRAPPID assay has a fast and simple sample-to-result workflow, requires low sample volumes (<10 μL) and, due to its stable ratiometric light output, allows for easy readout techniques and reliable analyte quantification.…”

Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays

“…Photoconjugation reactions were subsequently performed using a UV lamp (Thorlabs M365LP1 with a Thorlabs LEDD1B T-Cube LED Driver) for 15 min. 6 After conjugation, the LUCOS sensors were not further purified and stored at 4 °C until use.…”