A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays

Abstract

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.

Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay

“…Binding elements are tethered to small (SmBiT) and large (LgBiT) fragments of the engineered luciferase NanoLuc, such that analyte binding induces fragment colocalization and reconstitution of active enzyme ( Figure 1 ). 12 − 14 This rapid, homogeneous, wash-free assay has a simple mix-and-read format, and the bioluminescent output can even be read with a camera, so it is well suited to adoption in POCTs. 14 The NanoBiT fragments have been engineered for weak background affinity and high reconstituted bioluminescent activity, maximizing assay sensitivity and dynamic range.…”

Engineering protein activity into off-the-shelf DNA devices

“…It is important for a biosensor to function in dirty environments. This is especially problematic for luciferase-based sensors, as many existing designs show diminished luminescence in serum due to absorbance by extraneous components ( Ni et al., 2021 ). In 10% serum, nLuc-AFF bound to AP-1 with similar affinity (K D = 37 ± 11 nM; Figures 2 B and 2D) as in buffer, as determined by a plate reader and a cell-phone camera.…”

A Bioluminescent Sensor for Rapid Detection of PPEP-1, a Clostridioides difficile Biomarker

“…Another key requirement for POC assays is an easily interpretable readout-ideally, a binary "on-off" signal. Standard digital cameras are useful in this context and capable of capturing Nluc emission [18,21,37,38]. To examine the feasibility of CgNluc imaging with a digital camera, we first established an optimal concentration of the sensor (10 nM) for facile detection (Figure S8).…”