Remco Arts

Robust Red FRET Sensors Using Self-Associating Fluorescent Domains

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn(2+) sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn(2+) FRET sensors to image Zn(2+) over a broad concentration range in the same cellular compartment.

Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone

Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity

FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination. These sensors consist of the bright and stable luciferase NanoLuc and the fluorescent protein mNeonGreen, fused together via a linker containing a recognition site for caspase-3, -8, or -9. In vitro characterization showed that each caspase sensor displayed a robust 10-fold decrease in BRET ratio upon linker cleavage, with modest caspase specificity. Importantly, whereas scattering and background fluorescence precluded FRET-based detection of intracellular caspase activity in plate-reader assays, such measurements could be easily performed using our caspase BRET sensors in a high throughput format. The brightness of the BRET sensors also enabled long-term single-cell imaging, allowing BRET-based recording of cell heterogeneity in caspase activity in a heterogenic cell population.

Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution

Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.

Dual-Color Bioluminescent Sensor Proteins for Therapeutic Drug Monitoring of Antitumor Antibodies

Monitoring the levels of therapeutic antibodies in individual patients would allow patient-specific dose optimization, with the potential for major therapeutic and financial benefits. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection directly in blood plasma. In this study, we targeted four clinically important therapeutic antibodies, the Her2-receptor targeting trastuzumab, the anti-CD20 antibodies rituximab and obinutuzumab, and the EGFR-blocking cetuximab. A strong correlation was found between the affinity of the antibody binding peptide and sensor performance. LUMABS sensors with physiologically relevant affinities and decent sensor responses were obtained for trastuzumab and cetuximab using mimotope and meditope peptides, respectively, with affinities in the 10–7 M range. The lower affinity of the CD20-derived cyclic peptide employed in the anti-CD20 LUMABS sensor (Kd = 10–5 M), translated in a LUMABS sensor with a strongly attenuated sensor response. The trastuzumab and cetuximab sensors were further characterized with respect to binding kinetics and their performance in undiluted blood plasma. For both antibodies, LUMABS-based detection directly in plasma compared well to the analytical performance of commercial ELISA kits. Besides identifying important design parameters for the development of new LUMABS sensors, this work demonstrates the potential of the LUMABS platform for point-of-care detection of therapeutic antibodies.

Engineering BRET sensor proteins

FRET-sensors have become important tools for intracellular imaging, but their dependence on external illumination presents some limitations, such as photobleaching and phototoxicity, which limit measurements over extended periods of time. Fluorescence measurements also suffer from autofluorescence and light scattering, which hampers in vivo imaging and measurements in strongly absorbing and scattering media such as blood. In principle, these issues can be resolved by using sensors based on bioluminescence resonance energy transfer (BRET). The recent development of brighter and more stable luciferases and the concomitant improvement in luciferase substrates have substantially decreased the sensitivity gap between fluorescence and bioluminescence. As a result, the application of BRET-sensors is no longer restricted to measurements on cell populations, but they can also be used for imaging of single living cells, and BRET has started to emerge as an attractive sensor format for point-of-care diagnostics. The aim of this chapter is to first provide a brief overview of the basic design principles for BRET-sensors. Next, important design considerations will be discussed in more detail by describing the development of three different classes of BRET-sensors, both from our own work and that of others. These examples are all based on the NanoLuc luciferase, a bright and very stable blue light-emitting luciferase developed by Promega that has quickly risen to prominence in the bioluminescence field.

Paper‐Based Antibody Detection Devices Using Bioluminescent BRET‐Switching Sensor Proteins

Abstract This work reports on fully integrated “sample‐in‐signal‐out” microfluidic paper‐based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET‐based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume‐independent and fully reagent‐free operation, including on‐device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti‐HIV1, anti‐HA, and anti‐DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user‐friendly point‐of‐care testing in low‐resource environments.