Illuminating Life: The Frontier of Bioluminescent and Chemiluminescent Biosensors

The Dawn of Biological Illumination

In the modern era of biomedical research, light has emerged as one of the most powerful tools to understand the intricacies of life. Fluorescent reporters and biosensors have long been indispensable, but their reliance on external light sources limits their application in live organisms, especially in dense tissues where light scattering and autofluorescence obscure clarity. This is where bioluminescence—a naturally occurring phenomenon in organisms such as fireflies and certain marine species—offers a revolutionary alternative. By generating light through biochemical reactions without requiring external excitation, bioluminescent systems promise a sensitive, non-invasive, and dynamic approach to probing biological processes.

The unique attributes of bioluminescence, such as its low phototoxicity and compatibility with in vivo imaging, have propelled its use in applications ranging from gene regulation to whole-animal imaging. Meanwhile, chemiluminescence, which generates photons through synthetic chemical reactions, has introduced a complementary toolset, further broadening the horizons of luminescent biosensors.

Bioluminescent Reporters: Engineering Natural Light for Science

The Mechanics of Bioluminescence

Bioluminescence originates from the oxidation of a substrate, such as luciferin, by an enzyme, luciferase. This reaction produces an excited-state intermediate that emits photons as it transitions to its ground state. Unlike fluorescence, bioluminescence generates light against a dark background, eliminating the need for excitation light and reducing autofluorescence. This advantage is particularly valuable for live-cell and in vivo imaging, where the signal-to-noise ratio is critical.

Firefly luciferase (FLuc) is one of the most widely used bioluminescent systems, catalyzing a reaction between its native substrate, d-luciferin, and adenosine triphosphate (ATP). Advances in protein engineering have further refined FLuc, yielding mutants with enhanced stability, brightness, and compatibility with synthetic luciferin analogs. For example, Akaluc, paired with AkaLumine, has demonstrated the remarkable ability to visualize single cells in live animals, pushing the boundaries of bioluminescence resolution.

Expanding the Palette: Coelenterazine and Its Variants

Marine-derived luciferases, such as those utilizing coelenterazine (CTZ) as a substrate, offer unique advantages over ATP-dependent systems like FLuc. These luciferases, including Renilla luciferase (RLuc) and NanoLuc, do not require cofactors like ATP, making them ideal for environments where ATP availability is limited. NanoLuc, in particular, stands out for its compact size and extreme brightness, enabling applications such as tracking viral infections and studying protein-protein interactions.

Synthetic chemistry has further expanded the utility of CTZ systems by introducing analogs that improve brightness, shift emission wavelengths, or increase in vivo stability. Pairing these luciferases with tailored CTZ derivatives has unlocked new possibilities in multiplexed imaging and biosensing.

Chemiluminescent Sensors: The Power of Synthetic Reactions

Chemiluminescence shares a conceptual similarity with bioluminescence but relies on purely chemical reactions to generate light. This approach offers several advantages, including the ability to design highly specific probes for detecting reactive molecules or enzymatic activities.

As was mentioned, chemiluminescent sensors harness chemical reactions to emit light, offering several advantages over traditional detection methods. These sensors exhibit high sensitivity and specificity, enabling the detection of low analyte concentrations with minimal interference. The absence of an external light source reduces background noise, enhancing signal clarity. Additionally, chemiluminescent assays often have a wide dynamic range and rapid response times, making them suitable for various analytical applications.

A notable advancement in chemiluminescent technology is the development of Schaap’s dioxetane derivatives. Introduced by A. Paul Schaap and colleagues in the late 1980s, these compounds are stable dioxetane derivatives that emit light upon activation by specific triggers, such as enzymatic reactions. This property has been utilized in various assays, including those for detecting alkaline phosphatase activity.

Recent research has focused on enhancing the emission wavelengths of chemiluminescent probes into the near-infrared (NIR) spectrum. NIR-emitting probes are advantageous for in vivo imaging due to their deeper tissue penetration and reduced scattering. Modifications to the dioxetane scaffold, such as conjugation with NIR fluorophores or incorporation of extended π-conjugated systems, have been explored to achieve direct NIR chemiluminescence. enhancing its suitability for in vivo imaging by minimizing tissue absorption and scattering. These advances, coupled with improvements in chemiluminescence intensity and stability, position chemiluminescent sensors as a powerful complement to bioluminescent systems. They hold promise for improved biomedical imaging and diagnostic applications

Bioluminescent Biosensors: Bridging Biology and Light

Functional Assays with Chemically Modified Luciferins

To transform bioluminescent reporters into biosensors, researchers have chemically modified luciferins to respond to specific biochemical changes. These “caged” luciferins remain inactive until triggered by enzymes, metabolites, or reactive molecules, enabling precise monitoring of biological events. For example, luciferin derivatives have been developed to detect caspase activity, hydrogen peroxide, and metal ions, among other targets.

This approach extends the reach of bioluminescent biosensors into areas such as drug screening, disease diagnosis, and real-time monitoring of cellular responses. The simplicity and versatility of luciferin modifications have made them a cornerstone of functional assays.

The Emergence of BRET-Based Sensors

Bioluminescence resonance energy transfer (BRET) has revolutionized the design of biosensors by enabling the detection of molecular interactions and conformational changes. BRET occurs when the energy emitted by a bioluminescent donor, such as RLuc or NanoLuc, is transferred to a fluorescent acceptor, generating a secondary emission. This phenomenon is highly sensitive to distance changes, making it ideal for studying dynamic protein interactions.

NanoLuc-based BRET sensors, in particular, have set new benchmarks for brightness and sensitivity. These systems have been adapted to monitor intracellular ATP levels, calcium signaling, and neuronal activity, among other processes. Their ability to operate without external light sources makes them especially valuable for live-cell and in vivo applications.

Overcoming Challenges in Bioluminescence and Chemiluminescence

Despite their promise, luminescent systems face technical challenges that require continued innovation. For bioluminescence, low photon flux and limited emission wavelengths in the near-infrared region hinder tissue penetration and sensitivity in vivo. Efforts to engineer brighter luciferases and red-shifted luciferin analogs are addressing these limitations, paving the way for multicolor imaging and deeper tissue visualization.

Chemiluminescent systems, while powerful, often suffer from low reaction rates and limited stability in biological environments. The development of more robust chemiluminescent scaffolds, such as modified dioxetanes, has significantly improved their performance, expanding their applicability to complex biological systems.

Toward the Next Generation of Luminescent Tools

The future of luminescent biosensors lies in their integration with emerging technologies, such as gene editing and optogenetics. Genetically encoded bioluminescent systems are already enabling the creation of transgenic animals and cell lines for longitudinal studies. Meanwhile, the combination of luminescence with machine learning and advanced microscopy techniques promises to unlock new insights into cellular and molecular biology.

As researchers continue to refine and expand the luminescent toolbox, these systems are poised to transform not only the biomedical field but also fundamental biology. By illuminating life at its most intricate levels, bioluminescent and chemiluminescent technologies are guiding us toward a brighter future in science and medicine.

Study DOI: https://doi.org/10.1146/annurev-anchem-061318-115027

Engr. Dex Marco Tiu Guibelondo, B.Sc. Pharm, R.Ph., B.Sc. CpE

Editor-in-Chief, PharmaFEATURES