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

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.

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

Therapeutic drug monitoring (TDM) of tumor necrosis factor-α (TNFα)-inhibitors adalimumab and infliximab is important to establish optimal drug dose and maximize treatment efficacy. Currently, TDM is primarily performed with ELISA techniques in clinical laboratories, resulting in a long sample-to-result workflow. Point-of-care (POC) detection of these therapeutic antibodies could significantly decrease turnaround times and allow for user-friendly home-testing. Here, we adapted the recently developed bioluminescent dRAPPID (dimeric Ratiometric Plug-and-Play Immunodiagnostics) sensor platform to allow POC TDM of infliximab and adalimumab. We applied the two best performing dRAPPID sensors, with limit-of-detections of 1 pM and 17 pM, to measure the infliximab and adalimumab levels in 49 and 40 patient serum samples, respectively. The analytical performance of dRAPPID was benchmarked with commercial ELISAs and yielded Pearson's correlation coefficients of 0.93 and 0.94 for infliximab and adalimumab, respectively. Furthermore, a dedicated bioluminescence reader was fabricated and used as a readout device for the TDM dRAPPID sensors. Subsequently, infliximab and adalimumab patient serum samples were measured with the TDM dRAPPID sensors and bioluminescence reader, yielding Pearson's correlation coefficients of 0.97 and 0.86 for infliximab and adalimumab, respectively, and small proportional differences with ELISA (slope was 0.97 ± 0.09 and 0.96 ± 0.20, respectively). The adalimumab and infliximab dRAPPID sensors, in combination with the dedicated bioluminescence reader, allow for ease-of-use TDM with a fast turnaround time and show potential for POC TDM outside of clinical laboratories.

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

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.

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation.

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/µL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum and Intracellular Free Zn2.

Fluorescent Zn2+ sensors play a pivotal role in zinc biology, but their application in complex media such as blood serum or plate reader-based cellular assays is hampered by autofluorescence and light scattering. Bioluminescent sensor proteins provide an attractive alternative to fluorescent sensors for these applications, but the only bioluminescent sensor protein developed so far, BLZinCh, has a limited sensor response and a suboptimal Zn2+ affinity. In this work, we expanded the toolbox of bioluminescent Zn2+ sensors by developing two new sensor families that show a large change in the emission ratio and cover a range of physiologically relevant Zn2+ affinities. The LuZi platform relies on competitive complementation of split NanoLuc luciferase and displays a robust, 2-fold change in red-to-blue emission, allowing quantification of free Zn2+ between 2 pM and 1 nM. The second platform was developed by replacing the long flexible GGS linker in the original BLZinCh sensor by rigid polyproline linkers, yielding a series of BLZinCh-Pro sensors with a 3-4-fold improved ratiometric response and physiologically relevant Zn2+ affinities between 0.5 and 1 nM. Both the LuZi and BLZinCh-Pro sensors allowed the direct determination of low nM concentrations of free Zn2+ in serum, providing an attractive alternative to more laborious and/or indirect approaches to measure serum zinc levels. Furthermore, the genetic encoding of the BLZinCh-Pro sensors allowed their use as intracellular sensors, where the sensor occupancy of 40-50% makes them ideally suited to monitor both increases and decreases in intracellular free Zn2+ concentration in simple, plate reader-based measurements, without the need for fluorescence microscopy.

Bioluminescent RAPPID Sensors for the Single-Step Detection of Soluble Axl and Multiplex Analysis of Cell Surface Cancer Biomarkers.

Early diagnosis of cancer is essential for the efficacy of treatment. Our group recently developed RAPPID, a bioluminescent immunoassay platform capable of measuring a wide panel of biomarkers directly in solution. Here, we developed and systematically screened different RAPPID sensors for sensitive detection of the soluble fraction of Axl (sAxl), a cell surface receptor that is overexpressed in several types of cancer. The best-performing RAPPID sensor, with a limit of detection of 8 pM and a >9-fold maximal change in emission ratio, was applied to measure Axl in three different contexts: clinically relevant sAxl levels (∼0.5 and ∼1 nM) in diluted blood plasma, proteolytically cleaved Axl in the cell culture medium of A431 and HeLa cancer cells, and Axl on the membrane of A431 cells. We further extended the sensor toolbox by developing dual-color RAPPID for simultaneous detection of Axl and EGFR on A431 and HeLa cells, as well as an AND-gate RAPPID that measures the concurrent presence of these two cell surface receptors on the same cell. These new RAPPID sensors provide attractive alternatives for more laborious protein detection and quantification methods such as FACS and immunostainings, due to their simple practical implantation and low intrinsic background signal.

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

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.

Bottom-up de novo design of functional proteins with complex structural features.

De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs. We applied the bottom-up strategy to successfully design five folds for four distinct binding motifs, including a bifunctionalized protein with two motifs. Crystal structures confirmed the atomic-level accuracy of the computational designs. These de novo proteins were functional as components of biosensors to monitor antibody responses and as orthogonal ligands to modulate synthetic signaling receptors in engineered mammalian cells. Our work demonstrates the potential of bottom-up approaches to accommodate complex structural motifs, which will be essential to endow de novo proteins with elaborate biochemical functions, such as molecular recognition or catalysis.