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Absorbance Vs Luminescence Assays: 6 Key Factors to Help You Decide
When selecting the ideal detection technology for your research or application, you may need to choose between absorbance and luminescence assays. Both methods are commonly used in research, but they have key differences that are important to consider. Understanding these differences is crucial for achieving accurate and reliable results and effectively addressing your research questions.
In this article, we will explore the key factors to consider when selecting between absorbance and luminescence assays, helping you confidently navigate these options.
Principle of detection: Absorbance vs. luminescence
Absorbance assays quantify the amount of light absorbed by a sample at specific wavelengths. When light passes through the sample, some is absorbed, while the rest is transmitted (Fig. 1). According to Beer's Law, the measured absorbance of a solution is directly proportional to the concentration of the analyte present within the sample. Popular examples include colorimetric assays such as ELISA and Bradford assays.
Figure 1: Illustration highlights the key differences between chemiluminescence and absorbance processes.
On the other hand, Luminescence assays are based on the phenomenon where substances emit light after absorbing energy (1). This energy can come from various sources:
- Photoluminescence: Light is emitted after absorbing photons. This includes processes like fluorescence and phosphorescence.
- Chemiluminescence: Light is generated due to a chemical reaction (Fig. 1).
- Bioluminescence: Light is emitted through biochemical reactions.
Various luminescence assays provide valuable insights into a substance's properties, such as its interactions with other molecules and even its localization within a biological sample (2). Examples include Bioluminescence Resonance Energy Transfer (BRET), reporter gene assays, ATP assays, etc. While there are different types of luminescence, as mentioned above, in this article, we will specifically refer to chemiluminescence and bioluminescence whenever the term "luminescence" is used.
Critical Criteria for Assay Selection: Absorbance vs. Luminescence
1. Sensitivity and Detection Limit
Absorbance assays typically offer low to moderate sensitivity. They are prone to interference from background noise caused by impurities in the sample, sample color, or turbidity, which can impact the measurement (3). As a result, these factors can limit the effectiveness of absorbance assays in detecting low concentrations of analytes.
Luminescence assays are known for their high sensitivity. Since light is emitted directly from the sample without needing an external light source, there is minimal background interference (2), making luminescence assays highly effective for detecting very low concentrations of analytes.
Due to their superior sensitivity, luminescence assays are often preferred for research requiring the detection of very low analyte levels, such as femtomolar concentrations. For instance, Cao et al. utilized a chemiluminescent-based assay to detect platelet-derived growth factor B-chain (PDGF-BB) antigen with a detection limit of 10 fmol (4).
Conversely, absorbance assays may be adequate if only moderate sensitivity is required, such as in the nanomolar range to the micromolar range. A typical example is the Bradford assay (5).
2. Background Interference
Background interference refers to unwanted signals or substances that can distort the accurate measurement of the target substance. Absorbance measurements depend on transmitting a light beam through a sample, making them susceptible to background interference from any components in the light path. Impurities in samples, cell fragments, or other debris can scatter or absorb light, resulting in artifacts (6). Consequently, absorbance assays often exhibit a high background signal.
In contrast, Luminescence assays generally experience lower background interference. This is because they do not require an external light source for the sample to emit light, resulting in little to no autofluorescence and a high signal-to-noise ratio (2). Additionally, these assays are less sensitive to any obstructions in the light path than absorbance assays.
The reduced background interference of luminescence assays makes them particularly useful in applications requiring high sensitivity and accuracy, such as drug screening (2,7) and clinical diagnostics, such as the Chemiluminescent Immunoassay (CLIA) for detecting SARS-CoV-2 antibodies (8).
Fig. 2: A graph illustrating the relationship between equipment costs and detection parameters in absorbance and luminescence assays. These parameters are crucial in guiding the choice between the two assay types based on experimental requirements and budget constraints
3. Dynamic Range and Linearity
Dynamic range refers to the range of analyte concentrations that an assay can accurately and precisely quantify. It encompasses the lowest and highest concentration ranges at which the assay can reliably detect and measure the analyte. Linearity indicates how well the assay's response (e.g., signal intensity) is directly proportional to the concentration of the analyte.
Absorbance measurements are susceptible to background interference, which can impact the accuracy of the results. Additionally, the linear relationship between absorbance and concentration holds primarily at lower analyte concentrations (9). As a result, absorbance assays generally exhibit a lower dynamic range, typically limited to 2-3 orders of magnitude.
In comparision, luminescence assays offer a wider dynamic range of 6-7 orders of magnitude (4). This superior performance is due to their low background interference and high sensitivity, which allow them to accurately measure a broad spectrum of concentrations—from very low to very high—without compromising linearity. Luminescence assays are particularly effective for reporter gene or cell-based kinetic assays (10) due to their wide dynamic range, allowing tracking of a wide range of signal intensities over time.
4. Budget (Equipment & Kits)
Equipment: Absorbance assays require less specialized equipment and can be performed using standard spectrophotometers or absorbance plate readers. Absorbance plate readers tend to be less expensive. For example, an absorbance plate reader can cost approximately $3,000 to $20,000, depending on various factors such as wavelength range, footprint, and throughput capabilities. Many absorbance plate readers fall within the lower price range category in the market (3-10k) while offering varying levels of performance. For those seeking a cost-effective, compact plate reader to streamline their absorbance assay workflow, the Absorbance 96 could be an ideal choice, providing simultaneous readout from all 96 wells in just 3 seconds.
Luminescence assays are known for their higher sensitivity, but they require specialized equipment such as luminometers or multi-mode plate readers, which can be relatively expensive. Depending upon various specifications, such as detection modes, sensitivity, or integrated accessories, luminescence plate readers can cost between $3,500 and $40,000, with most high-performance readers typically falling within the higher price range. However, Luminescence 96 offers a more budget-friendly option without compromising performance. It offers a sensitivity of 100 fmol ATP/well and a linear dynamic range of 8 orders of magnitude.
Kits: The cost of kits for absorbance and luminescence assays can vary based on the type of assay and specific application. For example, an absorbance-based assay like the Pierce™ Coomassie (Bradford) Protein Assay Kit costs approximately $250 for 630 assays in tubes or 3,800 assays in microtiter plates. A luminescence-based reporter gene assay, such as the Revvity twinlite® dual luciferase reporter gene assay, is priced at around $280 for 100 assays (96-well) or 400 assays (384-well).
To compare similar applications, such as cell viability assays, the absorbance-based Invitrogen® CyQUANT® MTT Assay costs approximately $469 for 1,000 assays (in a 96-well format), while the luminescence-based CellTiter-Glo® Assay costs around $486 for the same quantity of assays. Although luminescence assays are generally more sensitive, their kit costs could be comparable to absorbance assay kits.
5. Specific Applications
Absorbance assays are suitable for applications that require moderate sensitivity. Examples include basic protein quantification (Bradford assays), DNA/RNA quantification, bacterial growth assays, and ELISAs.
Luminescence assays, however, are ideal for applications requiring high sensitivity and wide dynamic range. Examples include chemiluminescence ELISA (CLIA) for analyte quantification, protein-protein interactions (BRET), and real-time kinetic analyses such as cell-based assays (ATP or Kinase) or gene expression analysis (luciferase reporter assay).
6. Assay Workflow & Complexity
Absorbance assays are commonly used and regularly performed in life science laboratories. However, compared to luminescence assays, absorbance assays often involve more steps. These steps can include multiple reagent additions, washing procedures, and longer incubation times, which make them more time-consuming and complex, as well as more susceptible to experimental errors. Additionally, absorbance assays require plate readers that can measure absorbance at specific wavelengths according to the needs of the assay.
In contrast, luminescence assays are generally simpler and involve fewer steps. Reagents are added directly to the sample, and the incubation time is shorter, which enables rapid detection and quicker completion of the assay. For measurement, only plate readers equipped with luminescence detection capabilities are required.
Figure 3: Illustration demonstrating the differences in workflows between the colorimetric MTT assay and the luminescence-based cell viability assay.
For example, the colorimetric cell viability MTT assay involves several steps, including adding reagents, washing, a 4-hour incubation, and measuring absorbance at 570 nm. In contrast, a luminescence-based cell viability assay has a simpler and quicker workflow: the reagent is added directly to the cells, and the luminescence is measured after just a 10-minute incubation using a luminometer (Fig. 3). Both assays are widely used and provide reliable results for assessing cell viability. The choice between them depends on specific experimental needs, such as sensitivity and available equipment.
Conclusion
The choice between absorbance and luminescence assays often depends on various factors, including background interference, sensitivity, dynamic range, assay complexity, and budget limitations (Table 1). By evaluating these criteria, you can select the assay technology that best aligns with your research goals and ensures the accuracy and reliability of your results. Whether you choose absorbance or luminescence, making an informed decision is crucial for achieving optimal outcomes in your study.
Table 1: Absorbance vs Luminescence assays
References
1. Sasaki, A., Ohmiya, Y., (2022) Standardization of luminescence, fluorescence measurements, and light microscopy: Current situation and perspectives. Biophys Physicobiol;19.
2. Baljinnyam, B., Ronzetti, M., & Simeonov, A. (2022). Advances in luminescence-based technologies for drug discovery. Expert Opinion on Drug Discovery, 18(1).
3. Simeonov, A., & Davis, M. I. (2018). Interference with Fluorescence and Absorbance. Assay Guidance Manual.
4. Cao, Z. J., Peng, Q. W., Qiu, X., Liu, C. Y., & Lu, J. Z. (2011). Highly sensitive chemiluminescence technology for protein detection using aptamer-based rolling circle amplification platform. Journal of Pharmaceutical Analysis, 1(3), 159–165.
5. Zor, T., & Selinger, Z. (1996). Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Analytical Biochemistry, 236(2), 302–308.
6. Grela, E., Ząbek, A., Grabowiecka, A., (2015) Interferences in the Optimization of the MTT Assay for Viability Estimation of Proteus mirabilis. Avicenna J Med Biotechnol. 7(4):159-67.
7. Cho, E. J., & Dalby, K. N. (2021). Luminescence Energy Transfer–Based Screening and Target Engagement Approaches for Chemical Biology and Drug Discovery. SLAS Discovery, 26(8), 984–994.
8. Montesinos, I., Gruson, D., Kabamba, B., Dahma, H., van den Wijngaert, S., Reza, S., Carbone, V., Vandenberg, O., Gulbis, B., Wolff, F., & Rodriguez-Villalobos, H. (2020). Evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-SARS-CoV-2 antibodies. Journal of Clinical Virology, 128, 104413.
9. Mayerhöfer, T., Popp, J., (2019) Beer's Law - Why Absorbance Depends (Almost) Linearly on Concentration. Chemphyschem. 20(4):511-515
10. Neefjes, M., Housmans, B. A. C., van den Akker, G. G. H., L. W. van Rhijn., Welting, T. J. M., van der Kraan, P. M., (2021). Reporter Gene Comparison Demonstrates Interference of Complex Body Fluids with Secreted Luciferase Activity. SciReports: 1359.
