Have you ever experienced stomach pain after a gathering? It is likely that you have suffered from food poisoning. These illnesses are typically caused by the consumption of food or drinks accidentally contaminated with pathogens like bacteria, viruses, parasites, or fungi. With one in ten people affected globally every year, foodborne diseases remain a significant challenge for healthcare systems.
Salmonella is a bacterium that lives in the digestive system of warm-blooded animals like birds and mammals, including humans. “It is one of the most prevalent food contaminants,” explains Tohid Didar, one of the brains behind lab-in-a-package. “It causes gastroenteritis with symptoms including diarrhea, severe abdominal discomfort, fever, and vomiting.”
Governments perform sanitary checks at different points of the food production chain to reduce the risks of foodborne illnesses. The reference method to detect food pathogens is culture, which verifies pathogens’ presence by testing their ability to grow under optimal conditions. However, this method is laborious and slow (it can take a week for bacteria and longer for fungi) as it requires sampling at the production site, shipping samples to the laboratory, and analyzing them using specialized equipment and trained personnel.
To address these limitations, Associate Professor Tohid Didar and colleagues from McMaster University in Canada joined their efforts. They designed a smart device, which they named lab-in-a-package, that can sense dangerous levels of bacteria quickly and without even opening the package.
In this work, they designed a sensor specific for Salmonella Typhimurium (S. Typhimurium), a species of Salmonella most commonly associated with chicken and other poultry products. With lab-in-a-package, the team successfully detected S. Typhimurium in contaminated chicken on site, with high sensitivity and specificity, and in real-time.
Conceiving lab-in-a-package
To avoid shipping samples to the lab and the culture method, scientists have developed multiple on-site biosensing platforms for food monitoring; however, these newer techniques each have their own limitations.
Culture and modern monitoring technologies cause significant food waste as they need packages to be opened to collect the samples. To address this issue, the scientists developed a special tray with a pathogen-sensing window at the bottom to include in the packages. Besides serving as a container, the tray transports the fluids from the food to the sensor, sampling the entire product without requiring the parcel to be opened. Integrating the sensor into the box also ensures individual control of products, solving the problem of poor representation when controlling only a few batch items.
Didar adds, “modern food monitoring technologies often require complementary reagents and must be pretreated before detection […]”. But reagents can alter other properties of food usually controlled before reaching the consumer, such as flavor, smell, and color. To avoid this, the team incorporated the reagents for the detection step absorbed in a membrane above the sensor.
Choosing the right components
To make the lab-in-a-package, the scientists first set up the platform parts individually. As chicken is most often associated with Salmonella contaminations, in these experiments they used chicken purge, the fluids released by chicken when stored.
To begin with, they designed trays with different side inclinations (45°, 60°, and 90°) to test which was best at directing chicken purge to the sensing window. They chose the 45° side inclined tray for the platform as they found it localized the fluids in the sensing window more quickly and efficiently.
The team considered several candidate materials for the membrane, including cotton, cellulose, and polyester. After a thorough evaluation, they selected the cotton membranes as they showed a better porosity than the other candidates and performed well in liquid absorption and retention tests, crucial properties in preventing the reagents from diffusing into the food.
To build the sensor for S. Typhimurium, the team used an RNA molecule recognized and cleaved specifically by an enzyme of this bacterium. In the RNA cleavage site, there is a pair of molecules called fluorophore and quencher. “Prior to contact with the enzyme, the RNA is in its pre-cleavage state where the quencher molecule masks the bright fluorescent signal of the fluorophore”, explains Didar. “In the presence of S. Typhimurium, the enzyme cleaves the RNA molecule, causing the quencher molecule to separate from the fluorophore. This enables the bright fluorescent signal of the fluorophore, which indicates S. Typhimurium contamination has occurred.”
Proof of concept testing
Ready-to-eat products are particularly vulnerable to contamination episodes due to their longer production chain. Therefore, the scientists validated lab-in-a-package using cooked chicken.
To investigate lab-in-a-package sensitivity, the scientists contaminated ready-to-eat chicken with chicken purge containing growing amounts of S. Typhimurium. The platform detected as little as 1000 bacterium per milliliter and responded positively to bacterial concentration, meaning that a higher amount of microorganisms gives a brighter fluorescence. This response exceeds the requirements of current control methods that only look for a positive or negative answer.
A biosensing platform must only respond to the microorganism for which it was designed, a property called specificity. The results of lab-in-a-package in specificity testing were impressive: it was able to detect S. Typhimurium in chicken pieces contaminated with a mixture of this bacterium with other common foodborne bacteria. In addition, it did not respond to contamination with the mix containing only the non-target bacteria.
Food contamination can occur at any stage of the food chain due to low hygiene in the workplace and culinary tools. To evaluate the performance of lab-in-a-package upon different contamination sources, the team challenged it with chicken pieces that had been in contact with a contaminated knife, glove, and surface. In all the contamination means, lab-in-a-package effectively detected S. Typhimurium.
Products acquired in the supermarket may contain less bacteria than artificial contaminations in the lab due to fluctuations in ambient conditions of storage. Thus, Didar recognizes that evaluating products acquired directly in the supermarket is needed for a more realistic application of lab-in-a-package.
But he remains optimistic about the scope of this smart package. “We believe that the technology is quickly approaching commercialization. Our packaging platform represents a plug-and-play system that can be leveraged by other scientists for efficient in-package monitoring of packaged products using sensors they have developed, with little modification”.
Reference: Akansha Prasad and Shadman Khan et al., Advancing in situ Food Monitoring Through a Smart Lab-in-a-Package System Demonstrated by the Detection of Salmonella in Whole Chicken, Advanced Materials (2023). DOI: doi.org/10.1002/adma.202302641.
