Researchers have developed a new class of bioluminescent sensors capable of accurately tracking pH levels inside living mammalian cells, offering a stable, ratiometric alternative to traditional fluorescent probes.
Study: Selection and Engineering of Novel Brighter Bioluminescent Reporter Gene and Color- Tuning Luciferase for pH-Sensing in Mammalian Cells. Image Credit: unoL/Shutterstock.com
The study, published in Biosensors, introduces engineered firefly luciferases optimized for intracellular sensing. By fine-tuning the enzymes’ pH sensitivity and thermal stability, the team has created biosensors that perform reliably in live-cell environments, crucial for both research and clinical applications where real-time, non-invasive monitoring is needed.
From Light Emission to Live-Cell Sensing
Firefly luciferases are already widely used in molecular biology for their ability to emit light in the presence of luciferin. While traditionally employed as genetic reporters, their natural sensitivity to environmental factors like pH makes them strong candidates for use as optical biosensors.
However, native luciferases often fall short at mammalian body temperature (37 °C), limiting their application in live-cell studies. Previous work has mapped key amino acid sites that influence pH sensitivity, but few luciferases maintain performance under physiological conditions. This study addresses that challenge head-on by engineering luciferases specifically designed to function as stable pH sensors in mammalian cells.
Sensor Design and Methodology
The team focused on luciferases from two firefly species—Amydetes vivianii and Cratomorphus distinctus—and compared them to a luciferase from Macrolampis fireflies. After humanizing the gene sequences to ensure compatibility with mammalian systems, the researchers cloned them into pCDNA3 plasmid vectors and introduced them into COS-1 fibroblast cells.
To evaluate sensor performance, they measured bioluminescence across a pH range of 6.0 to 8.0 using a spectroluminometer. This allowed for ratiometric analysis based on the ratio of green to red light emission—an approach that helps normalize output and improve measurement accuracy. Temperature sensitivity was also tested, with a particular focus on maintaining performance at 37 °C.
Amy-Luc: A Standout Sensor
Of the three luciferases tested, the variant from Amydetes vivianii—dubbed Amy-Luc—emerged as the top performer. It produced significantly higher bioluminescence (up to five times more intense) than the other candidates and showed stable signal output at physiological temperature. Its emission spectrum, peaking at 548 nm at physiological temperature, exhibited distinct and resolvable pH-dependent shifts, making it well suited for ratiometric sensing.
By contrast, Cratomorphus and Macrolampis luciferases showed reduced signal intensity and red-shifted emission profiles under the same conditions, making them less effective for accurate intracellular sensing.
In addition to its strong pH sensitivity, Amy-Luc also showed pronounced temperature-dependent spectral shifts at acidic pH, pointing to its potential as a dual-function sensor for pH and temperature, particularly relevant in disease models or metabolic studies where both factors vary.
Applications and Outlook
With its strong signal output, thermal stability, and consistent pH responsiveness, Amy-Luc stands out as a versatile biosensing tool for live-cell applications. These features make it especially well-suited for high-resolution imaging and long-term intracellular monitoring—key needs in both research and diagnostic contexts.
Unlike fluorescent probes, luciferase-based sensors don’t require external light for excitation, which significantly reduces background noise and minimizes phototoxicity. This makes them ideal for continuous monitoring in delicate cell systems where conventional methods may interfere with cell health or behavior.
Notably, the study also points to the potential for integrating these luciferases with smartphone-based imaging platforms. This opens the door to portable, low-cost, and real-time sensing solutions that could be deployed in clinical or field settings, bringing lab-grade biosensing capabilities closer to point-of-care use.
Conclusion
This work marks a meaningful advancement in the development of genetically encoded, bioluminescent sensors for intracellular monitoring. By engineering luciferases that remain stable and sensitive under physiological conditions, the researchers have introduced a powerful tool for tracking pH in live mammalian cells with precision and ease.
Amy-Luc, in particular, shows strong promise as a next-generation biosensor for research and diagnostic use. It demonstrates how thoughtful enzyme design can overcome long-standing limitations in live-cell sensing.
Journal Reference
Bevilaqua V.R., Pelentir G.F. et al. (2025). Selection and Engineering of Novel Brighter Bioluminescent Reporter Gene and Color- Tuning Luciferase for pH-Sensing in Mammalian Cells. Biosensors 15(1):18. DOI: 10.3390/bios15010018, https://www.mdpi.com/2079-6374/15/1/18