Paper-Based All-in-One Origami Microdevice for Nucleic Acid Amplification Testing for Rapid Colorimetric Identification of Live Cells for Point-of-Care Testing

Abstract

We report herein the colorimetric identification of live cells based on a nucleic acid amplification testing (NAAT) methodology using an all-in-one origami paper microdevice integrated with DNA purification, loop-mediated isothermal amplification (LAMP), and on-site colorimetric detection. First, origami paper was partially embossed to create microchannel networks and chambers. Subsequently, hydrophobic polydimethylsiloxane prepolymer was coated onto the embossed paper to stabilize the structures on paper and provide fluid barriers. The paper microdevice was composed of splitting, purification, wicking, reaction, and dye pads folded alternatively to accomplish sensitive and specific NAAT. For the viability assay, propidium monoazide (PMA) was employed to penetrate dead cells and form covalent bonds with necrotic cell DNA; thus, amplification can be solely performed with DNA obtained from live bacterial cells. Purification functionality was implemented into the microdevice using chitosan to electrostatically capture DNA. Herein, methylene blue, which is typically used for electrochemical detection, is introduced for the first time for colorimetric detection of LAMP amplicons. This origami paper microdevice was successfully applied to determine the viability of foodborne pathogens, such as Escherichia coli O157:H7 and Salmonella spp., in which amplification was performed for 30 min followed by the execution of the colorimetric method for 10 min, thereby demonstrating tremendous potential for multiplexing and versatility for point-of-care applications. The introduced origami paper microdevice could be an attractive substitute as an instantaneous and convenient screening tool for the identification of viable pathogens in the control and monitoring of foodborne outbreaks in low-resource environments.

Paper-based microfluidics for rapid diagnostics and drug delivery

“…Recently, LAMP-based paper devices have been widely applied in food safety and foodborne pathogen detection. These pathogens including Escherichia coli (E. coli) [22,46,[79][80][81][82][83], Salmonella [22,46,81,82], Cochlodinium polykrikoides [46], Staphylococcus aureus (SA) [84], Vibrio parahaemolyticus (VP) [84], food allergens [83], Sus scrofa (porcine) [85], and Bacillus subtilis [85] have also been detected using these devices. We introduced several examples of the different foodborne pathogen detection methods mentioned above.…”

“…We introduced several examples of the different foodborne pathogen detection methods mentioned above. In particular, we highlighted a study from Lee's group because they made a great contribution to foodborne pathogen detection using a LAMP-based paper device [22,46,81,82].…”

“…4. (A) A rapid and eco-friendly isothermal amplification microfluidic device for the multiplex detection of foodborne pathogens (Reprinted with permission from ref [46]); (B) A foldable isothermal amplification microfluidic device for the fuchsin-based colorimetric detection of multiple foodborne pathogens (Reprinted with permission from ref [81]); (C) Fully integrated and foldable microfluidic device encapsulated with agarose for long-term storage that has potential for POC testing of multiplex foodborne pathogens (Reprinted with permission from ref [82]); (D) Paper-based all-in-one origami microfluidic device for nucleic acid amplification testing for rapid colorimetric identification of live cells for POC testing (Reprinted with permission from ref [22]).…”

“…Although many diagnostic applications have been used in developing countries due to limited resources and expertise, there are also many applications in developed countries [21]. These advantages promote the development of this field as paper-based microfluidics is used in portable devices for diagnostics and drug delivery [15,22].…”

Point-of-Need Diagnostics for Foodborne Pathogen Screening

“…Origami-based devices present another promising paper-based platform that has been utilized for microbiological testing of food. 110,111 Origami is the traditional Japanese art of paper folding, which has been utilized for several centuries to construct 3D geometries using a flat piece of paper. 112 These foldable devices can be fabricated on a single sheet of paper and assembled via simple folding, which reduces production costs and complexity.…”