What are luminescence assays

Crispr Cas9

Introduction to Luminescence Assays

Definition and Core Concept

Luminescence assays are a collection of analytical methods that detect analytes by measuring light produced by a chemical reaction. Unlike fluorescence assays, detection does not require external excitation, as the chemical generates light directly.1

Importance in Life Sciences

Luminescence assays are widely used in life sciences due to their advantages:

They are applicable in drug discovery, biochemical research and several cell-based assays.1

Mechanism and Core Components

How Luminescence Works

Luminescence occurs when a chemical reaction or enzymatic interaction produces photons. This intrinsic light emission allows for the detection and quantification of biological or chemical analytes without external light sources.1

Key Components

The main components of a luminescence assay include:2

Types of Luminescence Assays

Luminescence assays can be categorized based on the mechanism or the temporal profile of light emission.

SpectraMax® L Microplate Reader

SpectraMax® L Microplate Reader

Sensitive luminometer with programmable injector options for 96- and 384-well microplates

Learn More

Based on the Light Emission Mechanism

Emission Kinetics

Comparative Insights

Glow assays provide consistent, long-lasting signals, whereas flash assays offer higher peak intensity but shorter duration.6

The choice of the ideal luminescence assay depends on the level of sensitivity, reaction kinetics, the outcome being studied and the signal duration and intensity required.

Detection Technologies

Devices Used

Luminescence signals are measured using specialized instruments. Microplate readers and luminometers are designed to detect low-intensity light from samples in multi-well plates, making them suitable for high-throughput assays. Furthermore, white microplates are used to improve assay sensitivity through signal amplification.7

Detection Performance

Key parameters that determine luminescence detection performance include:8

These parameters guide researchers in optimization strategies. For example, adjusting substrate concentration and enzyme activity can maximize signal generation, while fine-tuning reaction conditions, such as pH and buffer composition, helps maintain enzyme stability and reaction efficiency. Finally, proper reagent selection and plate handling protocols are critical to minimizing background noise.1

Instrument-related considerations can facilitate further recalibration. Microplate reader settings, such as integration time, sensitivity and well geometry, can significantly affect signal detection. In particular, white or opaque plates enhance signal reflection, reducing crosstalk between wells. In addition, incubation conditions must be closely monitored to ensure consistency across samples.9

Keywords and Concepts

Luminescence vs Fluorescence vs Absorbance

Luminescence is one of the three major optical detection methods in biochemical and cell-based assays, along with fluorescence and absorbance. While all involve light-based measurements, they differ in how the signal is generated and detected.

The key differences between luminescence and fluorescence assays are the light source and the emission duration. The emission time for luminescence might vary from microseconds to hours, depending on the type of assay, whereas excitation is mostly short-lived in fluorescence.10

The intrinsic emission capacity makes luminescence assays more suitable for low-signal environments, quantitative enzyme activity and gene expression studies. On the other hand, fluorescence assays are favored for imaging, localization studies and multiplexed detection.10

Applications and Advantages

Luminescence assays are widely used in life sciences applications due to their sensitivity and non-invasive measurement capabilities.

In Cell Biology

In Molecular Biology

In Biochemistry

In Industry and Research

Advantages

The applications listed above are made possible by several advantages of luminescence assays:1

Protocol and Workflow

Luminescence Assay Protocol

Light emission: The photons are measured using a microplate reader or luminometer
Luminescence protocols are generally simpler and faster than absorbance and fluorescence assays, requiring no external excitation source or emission filters. Furthermore, they employ “add-and-read” formats that do not require washing or separation steps, which lends luminescence to high-throughput screening (HTS applications. The workflow can be automated via robotic liquid-handling deployment.20

Best Practices

Optimizing luminescence assays ensures accurate, consistent and high-sensitivity measurements. Common practices include:20

Commercial Kits and Tools

A wide range of commercial luminescence assay kits and detection systems is available for different applications:

Troubleshooting and Optimization

Common Issues and Solutions

While luminescence assays are robust and highly sensitive, certain technical factors can lead to inconsistent or weak signals. Common problems and strategies to resolve them include:20

Optimization

Fine-tuning assay parameters is essential for maximizing performance and ensuring reproducibility. Key strategies include:20

Conclusion and Future Directions

Summary

Luminescence assays are powerful, versatile and highly sensitive analytical tools that yield insights into biological samples without damaging their integrity. They generate strong, background-free signals without external excitation, facilitating precise detection of biochemical processes in cells. From traditional biochemical and molecular studies to advanced high-throughput drug screening and diagnostic applications, luminescence-based techniques play a central role in quantitative and mechanistic research.

Emerging trends continue to advance luminescence assay protocols, which open the doors for novel applications.

See how Danaher Life Sciences can help

Talk to an expert

FAQs

What are luminescence assays used for?

They detect and quantify biological or chemical analytes in applications, such as cell viability, enzyme activity, gene expression and drug screening.

How do luminescence assays work?

They measure light produced by a chemical or enzymatic reaction, typically involving luciferase or similar enzymes, without requiring external excitation.

What is the difference between fluorescence and luminescence assays?

Fluorescence requires external light to excite a fluorophore, while luminescence generates light intrinsically from a reaction, resulting in lower background and higher sensitivity.

What are the main types of luminescence assays?

Bioluminescence, chemiluminescence and BRET (bioluminescence resonance energy transfer).

How does glow luminescence differ from flash luminescence?

Glow produces a stable, long-lasting signal; flash gives a short, high-intensity burst.

What is a luciferase assay used for?

To monitor gene expression, enzyme activity and cellular ATP levels.

References

  1. Baljinnyam B, Ronzetti M, Simeonov A. Advances in luminescence-based technologies for drug discovery. Expert Opin Drug Discov 2023;18(1):25-35.
  2. Calabretta MM, Michelini E. Current advances in the use of bioluminescence assays for drug discovery: an update of the last ten years. Expert Opin Drug Discov 2024;19(1):85-95.
  3. Syed AJ, Anderson JC. Applications of bioluminescence in biotechnology and beyond. Chem Soc Rev 2021;50(9):5668-5705.
  4. Wang Z, Huang J, Huang J, Yu B, Pu K, Xu FJ. Chemiluminescence: From mechanism to applications in biological imaging and therapy. Aggregate 2021;2(6):e140.
  5. Kelly T, Yang X. Application of fluorescence-and bioluminescence-based biosensors in cancer drug discovery. Biosensors 2024;14(12):570.
  6. Schramm S, Lippold T, Navizet I. Chemiluminescent 2-Coumaranones: Synthesis, Luminescence Mechanism, and Emerging Applications. Molecules 2025;30(7):1459.
  7. Reyes SN, Rodriguez R, Murias A, Dikici E, Daunert S, Deo SK. Tandem Bioluminescent Tests for Rapid Detection of Urinary Tract Infections and Antimicrobial Resistance Using a Portable Luminometer. Anal Chem 2025;97(30):16628-16638.
  8. Bergua JF, Alvarez-Diduk R, Idili A, Parolo C, Maymo M, Hu L, et al. Low-cost, user-friendly, all-integrated smartphone-based microplate reader for optical-based biological and chemical analyses. Anal Chem 2022;94(2):1271-1285.
  9. Eid M, Barayeu U, Dick TP. Chemogenetic detection and quantitation of H2O2 in living cells. Nat Protoc 2025:1-24.
  10. Guilbault GG. Practical fluorescence: CRC Press, 2020.
  11. Nozhat Z, Khalaji MS, Hedayati M, Kia SK. Different methods for cell viability and proliferation assay: Essential tools in pharmaceutical studies. Anti-Cancer Agents Med Chem 2022;22(4):703-712.
  12. Campoccia D, Montanaro L, Ravaioli S, Mariani V, Bottau G, De Donno A, et al. Antibacterialactivity on orthopedic clinical isolates and cytotoxicity of the antimicrobial peptide dadapin-1. Int J Mol Sci 2023;24(1):779.
  13. Somiya M, Kuroda Si. Real-time luminescence assay for cytoplasmic cargo delivery of extracellular vesicles. Anal Chem 2021;93(13):5612-5620.
  14. Nishihara R, Dokainish HM, Kihara Y, Ashiba H, Sugita Y, Kurita R. Pseudo-luciferase activity of the SARS-CoV-2 spike protein for Cypridina luciferin. ACS Central Science 2024;10(2):283-290.
  15. Neefjes M, Housmans B, Van Den Akker G, Van Rhijn L, Welting T, Van Der Kraan P. Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity. Sci Rep 2021;11(1):1359.
  16. Efremenko E, Senko O, Stepanov N, Maslova O, Lomakina GY, Ugarova N. Luminescent analysis of ATP: Modern objects and processes for sensing. Chemosensors 2022;10(11):493.
  17. Hydorn M, Nagarajan SN, Fones E, Harwood CS, Dworkin J. Analysis of (p) ppGpp metabolism and signaling using a dynamic luminescent reporter. PLoS Genet 2025;21(8):e1011691.
  18. Feng R, Li G, Ko C-N, Zhang Z, Wan J-B, Zhang Q-W. Long‐lived second near‐infrared luminescent probes: An emerging role in time‐resolved luminescence bioimaging and biosensing. Small Structures 2023;4(2):2200131.
  19. Yuan H, Chen P, Wan C, Li Y, Liu B-F. Merging microfluidics with luminescence immunoassays for urgent point-of-care diagnostics of COVID-19. TrAC, Trends Anal Chem 2022;157:116814.
  20. Kim S-B. Bioluminescence: Methods and Protocols, Volume 1: Springer Nature, 2022.
  21. Riss TL, O’Brien MA, Moravec RA, Kupcho K, Niles AL. Apoptosis marker assays for HTS. AGM [Internet] 2021.
  22. Park C, Abafogi AT, Ponnuvelu DV, Song I, Ko K, Park S. Enhanced luminescent detection of circulating tumor cells by a 3D printed immunomagnetic concentrator. Biosensors 2021;11(8):278.
  23. Morrow RJ, Ernst M, Poh AR. Longitudinal quantification of mouse gastric tumor organoid viability and growth using luminescence and microscopy. STAR protocols 2023;4(1):102110.
  24. Ni Y, Rosier BJ, van Aalen EA, Hanckmann ET, Biewenga L, Pistikou A-MM, et al. A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays. Nat Commun 2021;12(1):4586.
  25. Mujawar A, Phadte P, Palkina KA, Markina NM, Mohammad A, Thakur BL, et al. Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase–Luciferin Pair. Sensors 2023;23(17):7313.