Development of a Multiplex Colorimetric Genetic Analysis Microchip for Identifying Foodborne Pathogens Using 3D-Printer
Authors: Kim Yong Tae 
Published: December 2024

The rapid and accurate detection of foodborne pathogens is paramount for safeguarding public health and ensuring the safety of food supplies. In this study, we introduce a groundbreaking approach in the form of a 3D printed multiplex colorimetric genetic analysis microchip tailored specifically for point-of-care analysis of foodborne pathogens. This microchip represents a significant advancement in genetic analysis technology, offering a streamlined and efficient solution for on-site detection.

Conventional methods for fabricating genetic analysis chips often involve photolithography, which can be time-consuming, expensive, and technically demanding. In contrast, our microchip is fabricated using stereolithography-digital light processing (SL-DLP) technology, which offers several distinct advantages. SL-DLP is known for its convenience, reproducibility, and cost-effectiveness, making it an ideal choice for the rapid and scalable production of genetic analysis chips. By leveraging this innovative fabrication technique, we have overcome many of the limitations associated with traditional manufacturing methods, paving the way for enhanced accessibility and widespread adoption of genetic analysis technologies.

The genetic analysis microchip consists of several key components, each meticulously designed to facilitate efficient and accurate detection of foodborne pathogens. Central to its functionality is the inclusion of a cell sample solution inlet, which allows for the introduction of the sample into the microchip. This inlet is strategically positioned to ensure uniform distribution of the sample throughout the microchip, maximizing the efficiency of the detection process. Additionally, the microchip features four reaction chambers, each dedicated to the isothermal amplification of a different target pathogen. This multiplexing capability enables simultaneous detection of multiple pathogens, greatly enhancing the efficiency and throughput of the analysis process.

To further streamline the detection process, the microchip incorporates four loop-mediated isothermal reaction (LAMP) reagent inlets, allowing for the pre-loading of lyophilized reagents into each reaction chamber. This pre-loading step eliminates the need for manual addition of reagents, reducing the risk of contamination and human error. Furthermore, the microchip is fabricated using a photocurable resin composed of poly(ethylene glycol) diacrylate (PEG-DA) (MW=258), which offers several advantages. Notably, this resin enables the printing of high-resolution and transparent structures, facilitating clear observation of color changes indicative of successful gene amplification.

To enable colorimetric genetic analysis, Eriochrome Black T (EBT) is added to the LAMP reagent, inducing a distinctive color change upon amplification of the target gene. This colorimetric signal serves as a visual indicator of pathogen presence, allowing for rapid and intuitive interpretation of results. In our experiments, we successfully detected four common foodborne pathogens - Escherichia coli O157:H7, Salmonella enterica, Vibrio parahaemolyticus, and Listeria monocytogenes - within a remarkably short timeframe of 20 minutes. This rapid turnaround time is critical for timely intervention and mitigation of foodborne illness outbreaks.

To assess the sensitivity of our microchip, we conducted a limit of detection (LOD) test using samples containing varying concentrations of the target pathogen. Remarkably, we were able to detect as few as 10 cells of the pathogen with a high degree of accuracy, underscoring the sensitivity and reliability of our approach. Furthermore, in multiplex detection tests, each target gene was independently detected within dedicated LAMP chambers, demonstrating the microchip's exceptional specificity and its ability to accurately distinguish between different pathogens.

Importantly, the simplicity of fabrication and the readiness for on-site analysis make our microchip highly versatile and adaptable to a wide range of applications beyond food safety. In addition to foodborne pathogen detection, our microchip holds promise for use in clinical diagnosis, forensic analysis, and environmental monitoring. By enabling rapid and accurate genetic analysis in diverse settings, our microchip has the potential to revolutionize the field of molecular diagnostics and significantly improve public health outcomes.

In summary, we have developed a novel 3D printed multiplex colorimetric genetic analysis microchip that represents a significant advancement in point-of-care diagnostic technology. By combining state-of-the-art fabrication techniques with innovative design principles, we have created a powerful tool for the rapid and reliable detection of foodborne pathogens. Moving forward, we envision widespread adoption of our microchip as a cornerstone of food safety protocols, facilitating proactive measures to protect consumer health and prevent foodborne illness outbreaks.