Towards quantitative point-of-care testing with novel bioluminescent immunosensor

Immunosensors have become indispensable tools in the fields of biochemistry and medical science, providing reliable methods for detecting specific biomolecules. They work by exploiting the interactions between antibodies and their target antigens, making them crucial in applications like clinical diagnostics, food safety monitoring, and environmental assessments. As demand grows for more effective and cost-efficient testing methods, researchers continue to innovate in immunosensor technology. Notably, the pursuit of homogeneous immunosensors that eliminate time-consuming washing steps has become a key focus.

A particularly promising class of homogeneous immunosensors, known as Quenchbodies (Q-bodies), work by emitting fluorescence in response to antigen binding. In their "quenched" state, Q-bodies remain inactive, but upon antigen binding, they undergo structural changes, leading to the emission of light by canceling of quenched state. Despite their potential, Q-bodies face significant limitations: they cannot function effectively in undiluted biological fluids such as blood or milk, and their storage and application in paper-based devices for point-of-care testing (POCT) on undiluted biofluids or those with reducing agents for safety handling remain challenging.

Against this backdrop, a research team from Institute of Science Tokyo, Japan, set out to develop a new immunosensor without such limitations. Led by Associate Professor Tetsuya Kitaguchi, they engineered a new type of Q-body that leverages the robustness of nanobodies and simple detection of bioluminescence. Published on 11 November 2024 in ACS Sensors, their innovative design is expected to revolutionize how immunosensor technology is used in clinical and environmental applications.

The researchers combined the nanobodies and NanoLuc with a well-known fluorescent dye, TAMRA. At the core of this design are nanobodies -- small, highly stable antibody fragments derived from camels. Nanobodies offer remarkable resistance to denaturation in harsh conditions, making them ideal for use in POCT. Moreover, the immunosensor also includes NanoLuc, a luciferase enzyme that emits blue light when it reacts with its substrate. Due to its exceptional brightness and less light scattering than fluorescence, the immunosensor shows great promise for applications in undiluted biological fluids such as blood or milk.

The process works as follows: when the target antigen binds to the nanobody, structural changes occur, which move the TAMRA dye away from the nanobody and closer to the NanoLuc enzyme. This change recovers the quenching of TAMRA and facilitates energy transfer between the two molecules, changing emission color from blue of the NanoLuc-catalyzed reaction to red of TAMRA. The ratio of emission intensities at different colors is then correlated with the concentration of the target antigen, enabling precise and sensitive quantification even using portable devices, including smartphones, for signal detection.

The researchers tested this new design through a series of experiments. "The proposed immunosensor, which we dubbed BRET nano Q-body, offers superior thermostability and endurance in organic solvents, reducing agents, and detergents due to the robust structure of the nanobody, and is equally suitable for use in biological fluids, such as milk, serum, and whole blood, without dilution, due to large emission ratio change derived from bioluminescence," remarks Kitaguchi.

Taking things one step further, the team also tested whether the BRET nano Q-bodies had potential for POCT by implementing them in paper devices to measure the concentration of a chemotherapeutic drug for a model experiment. The results were very favorable, as Kitaguchi highlights: "The paper devices also performed appropriately after long-term storage without temperature control, and in biological fluids and environmental samples without dilution, which makes them useful for detection at bedside, in the field, and at home. We expect paper-based platforms to be transformative for in situ detection in therapeutic, diagnostic, and environmental applications."

Overall, this study illustrates how various tools and concepts from analytical biochemistry can be combined into an exceptional piece of technology -- one that could improve our lives and help us preserve the environment.