Understanding ATP Bioluminescence in Food Safety

Robin Rijal

Adenosine triphosphate (ATP) bioluminescence assay works by detecting ATP produced by pathogens through a physicochemical reaction. ATP, often referred to as the energy currency of cells, is a key organic molecule responsible for storing and transferring energy in all living organisms. The presence of ATP in food and on food preparation surfaces indicates possible microbial contamination, as well as the presence of enzymes and allergens (Dunn & Grider, 2021; Masia et al., 2021).

Figure 1: Simplified reaction process illustrating ATP and luciferin as reactants for luciferase to produce light.

This assay, also known as an energy measurement technique, relies on ATP’s role in oxidizing luciferin into oxyluciferin, producing light (Bioluminescence) with the help of the enzyme luciferase (which facilitates bioluminescence) (Figure 1). During this process, ATP is converted into AMP, releasing PPi and emitting a chemiluminescent signal within the wavelength range of 470-700 nm (Eed et al., 2016; Singh et al., 2018; Masia et al., 2021). The reaction is shown in the equation below.

The level of bioluminescence is measured using a device called a luminometer and is expressed in relative light units (RLU). RLU results are indirectly related to CFU, as RLU is proportional but not equivalent to CFU. Since the amount of luminescence is directly proportional to ATP concentration (Figure 2), RLU can be converted into RLU per mole of ATP, providing insights into total cell viability in food samples and/or food contact surfaces (Singh et al., 2018; Ali et al., 2020).

Figure 2: The relationship between contamination, ATP production, and the RLU reading on the luminometer.

This method is highly sensitive, capable of detecting as little as 0.8–100 amol (1 amol = 1 × 10⁻¹⁸ mol) of intracellular ATP from living microbial cells within an hour, without requiring a culture step (Ishimaru et al., 2021). Kim et al. (2018) demonstrated that ATP bioluminescence measurement after photothermal lysis of targeted bacteria, using gold nanorods and NIR irradiation, is a highly selective and sensitive detection technique. This approach delivers results within 36 minutes, with only a short incubation period (30 minutes) and a 6-minute NIR irradiation step.

Figure 3: Relationship Between ATP and RLU as Indicators of Cleanliness or Contamination Levels.

In fact, higher RLU in an ATP test correspond to higher levels of ATP, which serve as an indicator of greater contamination, and vice-versa (Figure 3). In addition, high efficiency, accuracy, sensitivity, and specificity are crucial for obtaining precise and reliable results in ATP bioluminescence testing. Sensitivity can be further enhanced by increasing ATP levels through heat treatment, which helps release bound ATP from microbial cells and organic residues. This process improves the detection of low levels of contamination, ensuring more accurate hygiene monitoring. For example, heating food samples at 95°C for 10 minutes improves detection, while exposing bacterial samples to 50°C for 10 minutes increases RLU values by 2 to 3 times (Lee et al., 2017).

Table 1: An overview of commercial ATP Bioluminescence assay devices coupled with luminometers for food and beverages analysis (Source: Rijal, 2023).

Supplier Name	Luminometer Type	Method	Suitability	Application	LOD/ Detection Range	Incubation Time
3M	3M™ Clean-Trace™ Luminometer	Surface swab, liquid and solid foods (Single use)	Foodborne pathogens and enzymes	Fruits, vegetables, grain, pet foods, processed foods and seafoods	10 RLU (1.3 fmoles ATP)	4 hours
Charm Sciences Inc	NovaLUM® II Luminometer	Swab	Coliforms and hydrogen sulfide producing EB, allergens and pesticides	Dairy products, foods, grains, feed, hospitality and water treatment	1.0 fmoles ATP	30 minutes
Neogen	Neogen® AccuPoint Luminometer	Surface Swab, food and beverages	EB, TVC, Saccharomyces cerevisiae, Pseudomonas aeruginosa, Lactobacillus plantarum, Enzymes, and allergens	Surfaces, beverages, food and liquid samples	10 fmoles ATP	Real time testing
Hygiena™	EnSURE™ Touch	Surface swab, liquid, and solid foods (Single use)	Coliforms, EB, TVC, Enzymes, allergens and mycotoxins	Surfaces, beverages, and food samples	Ultrasnap kit-1.0 fmols ATP	6-8 hours
Hygiena™	EnSURE™ Touch	Surface swab, liquid, and solid foods (Single use)	Coliforms, EB, TVC, Enzymes, allergens and mycotoxins	Surfaces, foods and beverages	SuperSnap kit- 0.17 fmols ATP	6-8 hours
R-Biopharm	Lumitester™ PD-30	Surface swab, liquid and solid foods (Single use)	Salmonella, E. coli, Campylobacter, allergens, viruses, and bacterial toxins	Fluids, dry and moist surfaces	10 RLU (0 to 999999 RLUs)	15 minutes
Pall Corporation	Pall Pallchek Luminometer Trans Illuminator	liquid and solid foods (Multiwell-plates)	EB, P. aeruginosa S. aureus, and Enzymes	Food and beverages	10 – 100 CFU	2-3 hours
ThermoFisher Scientific	Fluoroskan™ FL Microplate Luminometer	Multiwell-plates	Cell proliferation, cytotoxicity, nucleic acid quantitation, gene assays, immunoassays, & enzyme activity.	Food, beverages, and surfaces	10 amol ATP/well using flash reaction	Real time testing

A variety of rapid ATP bioluminescence assay kits are available on the market, designed for easy and portable use in quickly screening food samples (Table 1). These kits enable high-throughput, automated detection of foodborne pathogens based on ATP bioluminescence. In this method, food and beverage samples can be tested directly in detection tubes, either with or without dilution. However, when monitoring surface hygiene, food production surfaces, tools, and equipment must first be swabbed before processing with the luciferin-luciferase complex. For the ATP bioluminescence test of food contact surface areas, swab a 10 x 10 cm (4 x 4 in) area. Move the swab side to side and up and down while rotating the tip to ensure thorough collection and complete coverage of the targeted area.

Several companies have developed highly specific and sensitive luminometers and detection tools. For instance, 3M offers ATP-based test devices such as the 3M™ Clean-Trace™ luminometer, 3M™ Clean-Trace™ total ATP test device, and surface ATP test device, which detect ATP levels with a limit of detection (LOD) of 1.3 femtomoles (fmol) of ATP (1 fmol = 10⁻¹⁵ moles). Similarly, Neogen® AccuPoint Luminometer uses an advanced ATP testing tube with a half-inch diameter, capable of measuring both total ATP and microbial ATP depending on the swab type, with an LOD of about 10 fmols ATP.

Figure 4: Recommended Cleaning and Corrective Action Procedures (Source: Hygiene Monitoring Guide).

Hygiena™ provides Ultrasnap and Supersnap swab kits, with LODs of 1.0 fmol and 0.17 fmol ATP, respectively. These kits can even detect as low as 1 CFU/ml after sample incubation and are AOAC-certified (Bottari et al., 2015). Hygiena™ also offers the Hygiena™ EnSURE™ Touch, a portable, handheld device with Wi-Fi and cloud storage access. This system measures both total ATP and microbial ATP levels, with low variability (5-12%) across different ATP concentrations (20-2,000 fmol). A low coefficient of variation (CV%) indicates more consistent and reliable results, with values closer to 0% is desirable. Refer to Figure 4 for the recommended cleaning and corrective action procedures outlined in the Hygiene Monitoring Guide.

These commercial ATP bioluminescence assay kits are widely used in the food and beverage industry, healthcare sector, canteens, and service industries due to their ability to provide immediate on-site results. However, a key limitation of this technique is that it cannot distinguish whether the detected ATP originates from harmful or beneficial bacteria (Kim et al., 2018).

Applications and benefits of ATP Bioluminescence rapid test kits.

The ATP Bioluminescence rapid test system provides fast and accurate results, making it an ideal point-of-care test for industries and developing countries. Using a luminometer, it delivers results in a readable format within few minutes to hours, significantly faster than traditional enumeration methods (Dilek, 2019; Zheng et al., 2019). Thanks to its compact and portable setup—including an incubator, luminometer, and test devices—this system is easy to transport and can be used for detecting pathogenic contamination in food, seeds, and food production surfaces. It can also detect high contamination levels without the need for serial dilutions, saving both time and sample quantity during analysis. With proper training, the equipment is simple to operate, making it especially valuable during food poisoning outbreaks. It can quickly identify total viable count, coliforms, allergens, and other harmful foodborne bacteria, allowing for immediate preventive actions (Nayak, 2014).

Traditional enumeration methods assume that a single bacterial colony originates from one cell, but this is not always accurate—some bacteria naturally form pairs, chains, or clusters, leading to an underestimation of the actual population (Nieto et al., 2022). In such cases, the MicroSnap device offers a more reliable alternative for bacterial detection.

Factors Contributing to False Results in ATP Bioluminescence Detection.

It is not uncommon for the system to produce false positive or false negative results. Several factors can influence variations in cell count, including differences in microbial cell size, cell development stages, background noise from the detection device, and ATP naturally present in food. Other factors such as chemical interference, contamination during sample preparation, and enumeration time can also contribute to discrepancies. The ATP content in food may impact the total ATP detected by the luciferase enzyme, leading to an overestimated count (Aon et al., 2014; Nayak, 2014; Arroyo et al., 2017). Additionally, background noise from the system, including electrical and chemical interference from food impurities and surfaces, may also result in an inflated cell count (Meighan, 2014; Meighan et al., 2016).

To conclude, The presence of ATP on surfaces signals inadequate cleaning and potential contamination from organic debris and bacteria. Food residues not only indicate poor sanitation but also create environments where bacteria can thrive, interfere with disinfectants, and contribute to biofilm formation. Additionally, allergenic food residues increase the risk of cross-contact. However, ATP tests cannot directly detect bacteria or allergenic proteins. Effective use of these tests requires a thorough understanding of their applications, pass-fail limits, and performance variations. Traditional ATP tests face limitations due to ATP breakdown into ADP and AMP, making residue detection challenging. To address this, the total adenylate (A3) test was developed, which detects ATP, ADP, and AMP, improving the identification of food residues. When combined with microbial culture and allergen testing, the A3 test enhances contamination detection and supports better hygiene management (Bakke, 2022).

References

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