Researchers from the Laboratory of Functional Biointerfaces of the Institute of Physics of the Czech Academy of Sciences have advanced the development of a compact, fully portable and fast biosensing device and demonstrated its effectiveness in detecting bacterial and viral pathogens in a wide range of food samples. Their accomplishment has been achieved by means of intense teamwork, innovative thinking and the involvement of young researchers who together have created a technology with the potential to improve diagnostics in the fields of food safety and medicine. An important part in this success has also been played by effective collaboration with potential end users, e.g. the Protection Service of the Police of the Czech Republic and other entities.
An operator collects a sample of potentially contaminated food on site, puts it into the device that takes no longer than 30 minutes to analyse the sample and show whether or not the food contains a certain type of pathogen and thus whether it is safe to eat (with respect to that pathogen). A task that normally requires several days including lengthy cultivation in a laboratory environment now takes less than an hour. This is exactly what the practical use of biosensors from the Laboratory of Functional Biointerfaces of the Division of Optics developed by the team led by Hana Lísalová offers.
Success story: From idea to implementation
At the beginning of this research and development, the vision was to create a biosensor that could detect pathogens quickly and efficiently directly on site not requiring complicated sample preparation and laboratory equipment and conditions. "The story of our biosensors is one of efficient teamwork, with each member contributing in their own unique way to achieve our goals," says Hana Lísalová, laboratory head.
What is unique is the breadth of the research, which is not focused on the development of a specific part, but of a complex device. The results thus include not only the work of a multidisciplinary team, but also collaboration with a number of other research institutions in the Czech Republic and abroad (e.g. Masaryk University or Mendel University in Brno, the Extreme Light Infrastructure ERIC, or Johannes Kepler University in Linz).
Earlier this year, the Laboratory of Functional Biointerfaces’ team published a paper in which they verified the performance of a system for detecting S. aureus in unprocessed industrial dairy product achieving comparable sensitivity to the standard culture method, while reducing the detection time from days to just 30 minutes [1]. We have already written about this success on the FZU website.
Recently, together with scientists from MUNI and ELI ERIC they demonstrated the detection of the dangerous E. coli O 157:H7 bacteria directly in milk and a homogenized dumpling and hamburger[2]. The results also showed high stability of the functional surface and the ability to analyse high numbers of samples while even after analysing up to 70 hamburger samples on a single biochip, the desired surface properties and ability to capture the bacterium were maintained.
Polymer layer alone is not enough
The most important element of the biochip is the polymeric antifouling nanolayer, which ensures that impurities contained in untreated samples do not adhere to its surface, but only the pathogens that the biochip is designed to identify. However, developing a biochip based on it alone would not be enough, so the team have also been considering and including other aspects. These include the development of analytical software that incorporates an algorithm for immediate qualitative evaluation of the analysis [3]. The research results suggest that this robust method could be used as a simple tool for automating sample classification, easily adaptable to label-free methods, supporting its wider applicability. Another important aspect for its use on site and for the analysis of large numbers of samples is the long-term stability of the developed layers, which was demonstrated in further research by the team. The biochips retain their antifouling properties and receptor anchoring ability even after several months of storage not only in aqueous environments but also in the dried state, at room temperature, in the cold and after freezing[4].
Microfluidics to increase biosensor sensitivity
An integral part of the biosensor is a well-designed microfluidics system, i.e. a system that ensures the transport of the sample and other reagents to the surface of the biochip. In parallel with reliability testing and speeding up the process for the end user, the team is working on making the device easier to use and more sensitive.
In doing so, they are focusing on the development of a new microfluidic system and flow cell. The team, led by Nicholas Scott Lynn, has demonstrated that reducing the height of the flow cell and using radial flow has led to a significant increase in detection sensitivity. The research showed that simple modifications to the flow cell can significantly increase the sensitivity of the biosensor compared to commercially available cells[5].
The integration of the Quartz Crystal Microbalance method, the microfluidic system, the biochip with a functional nanolayer and the accompanying software into one portable device can thus have a positive impact on food safety. The results of the research are now being tested by the Police Protective Service, which was also involved in the development itself.
"The results of the biosensor research at the Laboratory of Functional Biointerfaces are a good example of how science can help solve practical problems if scientists ask the right questions and tenaciously seek answers. The results also reflect the stability of the research team and the ease with which the Institute of Physics manages to attract young people – from trainees to postdoctoral fellows – into biosensor research," adds Alexandr Dejneka, Head of the Division of Optics of the Institute of Physics of the Czech Academy of Sciences.
How does it all work?
The measurement principle combines established quartz crystal microbalance (QCM) technology with innovative polymer nanolayers on the quartz crystal surface. This densely packed nanolayer, which resembles a brush in structure, is composed of multiple components to allow specific receptors to bind, but also to resist non-specific fouling from complex food samples and possibly other types of concentrated biological samples. This layer makes it possible to analyse samples in their original form without the need for complex modifications and purifications which is highly impractical for use on site. Furthermore, based on the selection and the receptor bound to the nanolayer, the biosensor can be "tuned" to detect a specific pathogen.
"Research into biosensors for the detection of real and therefore potentially dangerous pathogens is very demanding, requiring long hours in the lab with tightened security controls, but the social dimension of this research, team support or space for new creative ideas are the main driving forces that help me overcome research obstacles," says Michala Forinová, a PhD student from Hana Lísalová's team who is significantly involved in this research.
"It would not be possible to conduct such extensive interdisciplinary research without the stable facilities and support that the Institute of Physics provides to scientists. The institute creates an inspiring environment that motivates excellence and provides the opportunity to focus on long-term basic research," adds Hana Lísalová.
The research continues with the aim to develop new generations of biosensors optimised for the specific needs of individual users. At the same time, it will focus even more on a deeper understanding of the mechanisms of molecular interactions on polymer nanolayers. This will be achieved using innovative approaches such as single-molecule microscopy methods and advanced electrochemical techniques.
1. Forinová, M., et al., A comparative assessment of a piezoelectric biosensor based on a new antifouling nanolayer and cultivation methods: Enhancing S. aureus detection in fresh dairy products.Current Research in Biotechnology, 2023. 6: p. 100166.
2. Forinová, M., et al., A reusable QCM biosensor with stable antifouling nano-coating for on-site reagent-free rapid detection of E. coli O157:H7 in food products. Food Control, 2024. 165: p. 110695.
3. Kunčák, J., et al., Automating data classification for label-free point-of-care biosensing in real complex samples. Sensors and Actuators A: Physical, 2024. 374: p. 115501.
4. Vrabcová, M., et al., Long-term stability of antifouling poly(carboxybetaine acrylamide) brush coatings. Progress in Organic Coatings, 2024. 188: p. 108187.
5. Lynn, N.S., et al., Radial flow enhances QCM biosensor sensitivity. Sensors and Actuators B: Chemical, 2024. 401: p. 134949.