BRET Nano Q-Body: A Nanobody-Based Ratiometric Bioluminescent Immunosensor for Point-of-Care Testing.

We developed a nanobody-based homogeneous bioluminescent immunosensor to achieve a one-pot detection for point-of-care testing (POCT). This immunosensor was named BRET nano Q-body as its emission color changes via bioluminescence resonance energy transfer (BRET) upon antigen addition. NanoLuc luciferase and a cysteine-containing tag were fused to the N-terminus of the nanobody, which was labeled with a fluorescent dye via thiol-maleimide Michael addition.

Customizable OpenGUS immunoassay: A homogeneous detection system using β-glucuronidase switch and label-free antibody.

We developed a customizable OpenGUS immunoassay that enables rapid and sensitive detection of analytes without requiring antibody modification. This immunoassay employs label-free whole antibodies, an antibody-binding Z domain (ZD) derived from Staphylococcal protein A, and a β-glucuronidase (GUS) switch mutant, allowing for easy replacement of antibodies to tailor the immunoassays for various targeted antigens. The working principle is that the OpenGUS probe, the fusion protein of ZD and a GUS switch, converts the antibody-antigen interaction into GUS activation in a one-pot reaction.

Efficient and rapid linker optimization with heterodimeric coiled coils improves the response of fluorescent biosensors comprising antibodies and protein M.

We developed a coiled Q-probe (CQ-probe), a fluorescent probe containing a coiled-coil peptide pair E4/K4, to convert antibodies into biosensors for homogeneous immunoassays. This probe consists of an antibody-binding protein, protein M (PM) with the E4 peptide and the K4 peptide with a fluorescent dye. Compared to PM Q-probes, which are generated by modifying the C-terminus of PM with a fluorescent dye, CQ-probe variants with various linkers are easy to prepare and therefore enable the establishment of biosensors with a significant fluorescence response by localizing the fluorescent dye at the optimal position for quenching and antigen-dependent release.

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions.

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed.

The Patrol Yeast: A new biosensor armed with antibody-receptor chimera detecting a range of toxic substances associated with food poisoning.

Baker's yeast is an attractive host with established safety and stability characteristics. Many yeast-based biosensors have been developed, but transmembrane signal transduction has not been used to detect membrane-impermeable substances using antigen-antibody interactions. Therefore, we created Patrol Yeast, a novel yeast-based immunosensor of various targets, particularly toxic substances in food.

Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface.

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required.

Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess.

With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O, etc.).

Rapid construction of fluorescence quenching-based immunosensor Q-bodies using α-helical coiled-coil peptides.

Here, we report a rapid and efficient method to fabricate Quenchbodies (Q-bodies) that can detect targets with antigen-dependent fluorescence augmentation using a stable coiled-coil peptide pair, E4 and K4 (coiled Q-body, CQ-body). The CQ-body allowed antigen detection not only in buffer but also in 50% plasma. Furthermore, we describe FRET-type CQ-bodies using a dual-coloured K4 peptide, which allowed a more precise antigen quantification.

BRET Q-Body: A Ratiometric Quench-based Bioluminescent Immunosensor Made of Luciferase-Dye-Antibody Fusion with Enhanced Response.

A quenchbody (Q-body) is an immunosensor comprising an antibody fragment containing an antigen-binding site that is site-specifically labeled with a fluorescent dye. The fluorescent dye of a Q-body is quenched in the absence of an antigen; however, its fluorescence recovers in the presence of an antigen, offering simple and rapid systems for antigen detection. In this study, we fused luciferase NanoLuc to a Q-body to construct a new immunosensor termed the "BRET Q-body" that can detect antigens based on the bioluminescence resonance energy transfer (BRET) principle.

PM Q-probe: A fluorescent binding protein that converts many antibodies to a fluorescent biosensor.

Quenchbody (Q-body) is a fluorescent biosensor in which a fluorescent dye is tagged near the antigen binding site of an antibody. The fluorescence of the dye is quenched by the tryptophan residues present in the variable region of the antibody, and is recovered when the antigen binds. Q-bodies have been prepared using recombinant DNA technology by introducing one or more tag sequence(s) at either the N-terminal of the Fab or the single chain variable region fragment of the antibody, and labeling the tag with a fluorescent dye.