·         Prof. Taek Lee (Department of Chemical Engineering) and His Research Team

Develop an Integrated On-site DNA Pre-treatment Device and High-speed Biosensor System for Detecting E. coli in Milk

- Simplification of on-site DNA pre-treatment methods for E. coli and integration with a high-speed detection biosensor system -

- Demonstration of the field applicability of a biosensor using DNA extracted with the developed pre-treatment device -

- Published in the renowned international journal Chemical Engineering Journal (IF: 13.3, JCR: 3.8%) -

The research team of Professor Taek Lee from the Department of Chemical Engineering, along with graduate student Ye-Jin Yoon, developed an integrated on-site DNA pre-treatment device and high-speed detection biosensor system. The developed pre-treatment device could rapidly extract DNA from E. coli in milk within 20 minutes, and when applied to an AC electrothermal flow (ACEF)-based biosensor, it established an integrated pre-treatment-biosensor detection system capable of detecting within 10 minutes.

E. coli is a common and dangerous foodborne pathogen that causes various diseases in humans and animals, leading to bloody diarrhea, kidney failure, and even death in severe cases. It accounts for 20% of food poisoning cases globally. E. coli-related food poisoning often occurs due to the consumption of contaminated water or food, with contaminated dairy products being the leading cause of hospitalization due to foodborne illnesses. Since milk, which contains various nutrients, provides an ideal environment for the proliferation of even small amounts of E. coli, a system capable of rapidly detecting and monitoring E. coli during milk processing is essential. In response, Professor Lee’s team developed an integrated DNA pre-treatment and electrochemical biosensor system that can quickly detect E. coli in milk.

Single-stranded DNA (ssDNA), the fundamental material of biological genetics, can be designed with a specific sequence that targets a substance, allowing for sensitive and selective detection when applied as a receptor in biosensors. ssDNA can be easily functionalized and immobilized on biosensors using various covalent bonding technologies. Additionally, it provides high sensitivity in electrochemical analysis when binding to target DNA, enabling quantitative detection of the target using electrochemical methods. However, the pre-treatment step required to extract DNA from E. coli in complex matrices like milk can take a long time due to its various procedures.

To establish a rapid detection platform for on-site detection of E. coli, Professor Lee's team developed a pre-treatment device that controls reagents with valves to enable sequential reactions, which was then integrated with a DNA-based electrochemical biosensor. The pre-treatment device consists of a chamber filled with glass beads coated with tetraethyl orthosilicate (TEOS) and valves to control the reagents, effectively removing E. coli residues. Milk containing E. coli passes through the chamber, and after controlling the valves to extract DNA, nucleic acids are released and fragmented through subsequent physical treatments. The extracted target DNA of E. coli is applied to the biosensor for electrochemical performance evaluation. The ssDNA, designed using a highly variable genetic sequence identified through genetic analysis of E. coli, was applied as a receptor in the biosensor, showing sensitive electrochemical signal changes upon binding to the DNA extracted by the pre-treatment device.

To shorten the target binding time for the biosensor, the ACEF method was introduced, reducing detection time to 10 minutes and demonstrating 97.334% accuracy compared to natural diffusion over 24 hours, proving the reliability of rapid detection. The sensor was evaluated using electrochemical impedance spectroscopy (EIS), and a more sensitive signal change was observed with the developed pre-treatment device compared to nucleic acids extracted using a commercial pre-treatment kit. The biosensor operated linearly in the range of 10E-04 to 1 ng/μL, with detection limits of 9.883 × 10E-5 ng/μL in DIW and 9.235 × 10E-5 ng/μL in real milk, demonstrating sensitive detection performance. Additionally, the matrix effect due to the presence of Salmonella typhimurium, another common foodborne pathogen, was 7.425%, indicating sensitive signal changes corresponding to E. coli concentration despite the presence of interfering species. A false-positive test conducted on 20 negative and 20 positive samples in real spoiled milk demonstrated a high accuracy of 92.5% in distinguishing false positives. The critical signal value for negative/positive detection was 24.360 kΩ, and the sensor exhibited high sensitivity and selectivity, with 90% sensitivity and 85% selectivity. As a result, the developed pre-treatment-integrated E. coli detection biosensor successfully detected E. coli in real milk with high sensitivity and accuracy within 30 minutes, providing an effective platform for E. coli detection. This study is significant as the first to integrate a pre-treatment device and biosensor for rapid E. coli detection, proving the potential for real-world application and suggesting the sensor’s use as a highly effective pathogen detection platform. The pre-treatment-biosensor system developed by Professor Lee's team shows broad potential as a platform for detecting not only E. coli but also various harmful bacteria.

This study was supported by the Ministry of Environment of Korea (MOE) through the Korea Environmental Industry & Technology Institute (2020003030001) and by the National Research Foundation of Korea (2021R1C1C1005583), as well as by the National Research Council of Science & Technology (CRC22021-200). The research results were published in the renowned international journal Chemical Engineering Journal (IF: 13.3, JIF ranking: 96.2%) under the title "Construction of on-site DNA pre-treatment device and rapid electrochemical biosensor set for Escherichia coli detection in milk" (DOI: https://doi.org/10.1016/j.cej.2024.155898). (DOI: https://doi.org/10.1016/j.cej.2024.155898)