Fully Integrated and Foldable Microdevice Encapsulated with Agarose for Long-Term Storage Potential for Point-of-Care Testing of Multiplex Foodborne Pathogens.

In this study, we fabricated a fully integrated and foldable microdevice encapsulated with 2-hydroxyethyl agarose for long-term storage of reagents for the integration of isothermal amplification and subsequent colorimetric detection for the monitoring of multiplex foodborne pathogens. The microdevice comprises a reaction zone and a detection zone. Both zones were made of a thin polycarbonate film and sealed by an adhesive film to make the microdevice foldable. The 2-hydroxyethyl agarose with loop-mediated isothermal amplification (LAMP) reagents and silver nitrate were deposited in the reaction and detection chambers, respectively, for long-term maintenance of reagent activity. A thin graphene-based heater associated with a handheld battery was employed to supply a constant temperature for on-chip amplification for 30 min. To simplify the sample manipulation process, a folding motion was adopted to allow the loading of LAMP amplicons from the reaction to the detection chambers and a colorimetric strategy was used for simple visual read-out of the results on-site. Using the agarose, the reagents were successfully stored and the reagent activity was maintained for at least 45 days. Prior to performing multiplex detections, the spiked juice was thermally lysed and purified with polydopamine-coated paper. The amplifications of Salmonella spp. and Escherichia coli O157:H7 (E. coli O157:H7) were successfully demonstrated based on the stable isothermal condition attained by the heater. The microdevice can detect the low concentration of E. coli O157:H7 at 2.5 × 102 copies per mL. The introduced microdevice acts as a simple and user-friendly platform for the identification of foodborne pathogens, paving the way for the construction of a truly portable, read-out microdevice for use as a public healthcare monitoring device.

Fabrication of a polycarbonate microdevice and boronic acid-mediated surface modification for on-chip sample purification and amplification of foodborne pathogens.

In this study, we integrated sample purification and genetic amplification in a seamless polycarbonate microdevice to facilitate foodborne pathogen detection. The sample purification process was realized based on the increased affinity of the boronic acid-modified surface toward the cis-diol group present on the bacterial outer membrane. The modification procedure was conducted at room temperature using disposable syringe. The visible color and fluorescence signals of alizarin red sodium were used to confirm the success of the surface modification process. Escherichia coli O157:H7 containing green fluorescence protein (GFP) and Staphylococcus aureus were chosen as the microbial models to demonstrate the nonspecific immobilization using the microdevice. Bacterial solutions of various concentrations were injected into the microdevice at three flow rates to optimize the operation conditions. This microdevice successfully amplified the 384-bp fragment of the eaeA gene of the captured E. coli O157:H7 within 1 h. Its detection limit for E. coli O157:H7 was determined to be 1 × 103 colony-forming units per milliliter (CFU mL-1). The proposed microdevice serves as a monolithic platform for facile and on-site identification of major foodborne pathogens.