Isothermal amplification-based microfluidic devices for detecting foodborne pathogens: a review.

The gold standard for nucleic acid amplification-based diagnosis is the polymerase chain reaction (PCR). The PCR recognizes the targets such as foodborne pathogens by amplifying their specific genes. The integration of nucleic acid amplification-based assays on microfluidic platforms represents a highly promising solution for convenient, cheap, and effective control of foodborne pathogens. However, the application of the PCR is limited to on-site detection because the method requires sophisticated equipment for temperature control, which makes it complicated for microfluidic integration. Alternatively, isothermal amplification methods are promising tools for integrating microfluidic platforms for on-site detection of foodborne pathogens. This review summarized advances in isothermal amplification-based microfluidic devices for detecting foodborne pathogens. Different nucleic acid extraction approaches and the integration of these approaches in microfluidic platforms were first reviewed. Microfluidic platforms integrated with three common isothermal amplification methods including loop-mediated isothermal amplification, recombinase polymerase amplification, and recombinase-aided amplification were then described and discussed.

Droplet-Based Microfluidics: Applications in Pharmaceuticals.

Droplet-based microfluidics offer great opportunities for applications in various fields, such as diagnostics, food sciences, and drug discovery. A droplet provides an isolated environment for performing a single reaction within a microscale-volume sample, allowing for a fast reaction with a high sensitivity, high throughput, and low risk of cross-contamination. Owing to several remarkable features, droplet-based microfluidic techniques have been intensively studied. In this review, we discuss the impact of droplet microfluidics, particularly focusing on drug screening and development. In addition, we surveyed various methods of device fabrication and droplet generation/manipulation. We further highlight some promising studies covering drug synthesis and delivery that were updated within the last 5 years. This review provides researchers with a quick guide that includes the most up-to-date and relevant information on the latest scientific findings on the development of droplet-based microfluidics in the pharmaceutical field.

Colorimetric detection of viable antibiotic resistant Enterococcus mediated by cordless operation of reverse transcription loop-mediated isothermal amplification.

In this study, we applied a tube-based reverse transcription loop-mediated isothermal amplification technique using preloaded amplification and detection reagents for simple screening of viable vancomycin-resistant Enterococcus in a cordless manner. We adopted an mRNA-based approach to detect live Enterococcus in vancomycin-treated cultures. We used agarose to preload and store all reagents for amplification and detection inside the tube, which could achieve on-site isothermal nucleic acid amplification and detection in less than 1 h without using sophisticated instruments. Moreover, the use of a portable insulated water tumbler eliminated the need for electricity, which is usually important in nucleic acid amplification-based assays. The water tumbler acted as a heat source to supply a stable heat required for the amplification reaction, which could last up to 45 min. In addition, colorimetric detection was realized using pH-based methods. The detection was triggered by shaking the tube so that the amplified solution was reacted with phenolphthalein embedded in the tube cap. The introduced one-pot strategy has many advantages such as easy and cordless operation, low cost, disposability, and less chance of contamination because the amplification and detection occur in a closed system. The system could have a great impact on nucleic acid analyses in instrument-free and low-resource areas.

Spinning and Fully Integrated Microdevice for Rapid Screening of Vancomycin-Resistant Enterococcus.

This study introduces a spinning and fully integrated paper-based microdevice that can perform multiple functions, including DNA extraction, amplification, and colorimetric detection, for monitoring two major vancomycin-resistant Enterococci (VREs), which carry the vanA and vanB genes. The spinning microdevice is composed of a stationary part and a spinning part. The square-shaped stationary part has two zones: the lysis and reaction zones. The spinning part, which has a spin wheel-like shape, was inserted perpendicularly into the stationary part so that its two semicircles remained on the upper and lower parts. Sodium hydroxide-treated glass microfiber filter discs, inserted in the upper semicircle, were soaked in the lysis chambers by folding them toward the lysis zone to capture DNA in the lysis chambers. The captured DNA was transferred to the reaction chambers by folding the discs toward the reaction chambers. Water was added to the sodium hydroxide-treated glass microfiber filter discs to elute purified DNA into the reaction chambers. The upper semicircle was then unfolded, and the reaction chambers were sealed for subsequent loop-mediated isothermal amplification (LAMP) for 45 min. After the reaction, the spinning part was spun in the lysis zone direction to bring the lower semicircle, inserted with phenolphthalein-treated glass microfiber filter discs, toward the upper part of the stationary part. By folding it toward the reaction chambers, the lower semicircle came into contact with them and the phenolphthalein-treated glass microfiber filter discs were soaked in the reaction chambers and expressed color after 30 s. Based on the pH change during the LAMP reaction, the phenolphthalein-treated discs remained pink in the absence of target DNA, while those in contact with the positive samples turned colorless. A sensitive detection with a VRE limit of detection of 102 CFU/mL for tap water spiked with VRE carrying the vanA gene was achieved using this microdevice. Both VREs, carrying vanA and vanB genes, were successfully identified from tap water and contaminated equipment surfaces within 75 min. The introduced microdevice demonstrated a rapid, accurate, and sensitive performance for the environmental assessment of VRE contamination in resource-limited regions.

Nucleic acid amplification-based microfluidic approaches for antimicrobial susceptibility testing.

Because of the global spread of antimicrobials, there is an urgent need to develop rapid and effective tools for antimicrobial susceptibility testing to help clinicians prescribe accurate and appropriate antibiotic doses sooner. The conventional methods for antimicrobial susceptibility testing are usually based on bacterial culture methods, which are time-consuming, complicated, and labor-intensive. Therefore, other approaches are needed to address these issues. Recently, microfluidic technology has gained significant attention in infection management due to its advantages including rapid detection, high sensitivity and specificity, highly automated assay, simplicity, low cost, and potential for point-of-care testing in low-resource areas. Microfluidic advances for antimicrobial susceptibility testing can be classified into phenotypic (usually culture-based) and genotypic tests. Genotypic antimicrobial susceptibility testing is the detection of resistant genes in a microorganism using methods such as nucleic acid amplification. This review (with 107 references) surveys the different forms of nucleic acid amplification-based microdevices used for genotypic antimicrobial susceptibility testing. The first section reviews the serious threat of antimicrobial-resistant microorganisms and the urgent need for fast check-ups. Next, several conventional antimicrobial susceptibility testing methods are discussed, and microfluidic technology as a promising candidate for rapid detection of antimicrobial-resistant microorganisms is briefly introduced. The next section highlights several advancements of microdevices, with an emphasis on their working principles and performance. The review concludes with the importance of fully integrated microdevices and a discussion on future perspectives.

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.

Fully integrated and slidable paper-embedded plastic microdevice for point-of-care testing of multiple foodborne pathogens.

This study presents a slidable paper-embedded plastic microdevice fully integrated with DNA extraction, loop-mediated isothermal amplification (LAMP), and colorimetric detection functionalities. The developed microdevice consists of three layers that allow a sliding movement to mix the sample and reagents for DNA purification, amplification, and detection in a sequential manner. An FTA card was employed in the main chamber for DNA extraction and purification from intact bacterial cells. Subsequently, LAMP reagents and fuchsin-stored chambers were pulled toward the main chambers for DNA amplifications at 65 °C. After 30 min, the detection reagents-stored chambers were then moved to main chambers for result analysis. For the detection of LAMP amplicons, a novel colorimetric fuchsin-based method was employed. The wide applicability of the integrated microdevice was demonstrated by successfully screening three major foodborne pathogens, namely Salmonella spp., Staphylococcus aureus, and Escherichia coli O157:H7 in food, enabling highly sensitive detection of 3.0 × 101 CFU/sample of Gram-negative bacteria (Salmonella spp. and Escherichia coli O157:H7) and 3.0 × 102 CFU/sample of Gram-positive bacteria (Staphylococcus aureus) within 75 min. The portable and integrated microdevice presented in this study holds significant promise for point-of-care applications to accurately and rapidly diagnose and control diseases.

A foldable isothermal amplification microdevice for fuchsin-based colorimetric detection of multiple foodborne pathogens.

In this study, we have developed a foldable microdevice fully integrating DNA purification, amplification, and detection processes for detecting multiple foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with a fuchsin-based direct DNA colorimetric detection method. The microdevice was composed of three parts: a sample zone, reaction zone, and detection zone. A sealing film attached to the sample, reaction, and detection zones served as a bottom layer to make the microdevice foldable. The detection zone was made up of paper strips attached to the sticky side of the sealing film, and fuchsin-stained lines were drawn on the paper strips. The microdevice can be folded to directly transfer the DNA template solution from the sample chambers to the reaction chambers. In this manner, fluid manipulation was readily realized and the use of a bulky instrument such as a pump or rotator was completely dispensed with. After the LAMP reaction, the detection zone was folded so that the fuchsin-stained lines were completely soaked into the reaction chambers. Genomic DNAs of Salmonella spp. and Escherichia coli O157:H7 were first successfully purified from thermally-lysed milk using polydopamine-coated paper, amplified by LAMP, and directly identified by the naked eye using fuchsin within 65 min. Using this microdevice, approximately 102 CFU per mL of Salmonella spp. was detected. These results indicate the significant potential of this microdevice for the sample-in-answer-out genetic analysis of multiple foodborne pathogens in resource-limited environments.

A rapid and eco-friendly isothermal amplification microdevice for multiplex detection of foodborne pathogens.

In this study, a plastic microdevice based on loop-mediated isothermal amplification (LAMP) was fabricated for the amplification and on-chip fluorescence detection of multiple pathogens. Papers infused with LAMP reagents and specific primers were embedded inside the multiple reaction chambers of the microdevice. A solution containing the target pathogens was injected into the sample chamber, located in the center of the microdevice, and evenly distributed to the reaction chambers simultaneously via centrifugal force. For detection, fisetin, a plant-derived fluorophore, was used as the DNA-intercalating dye. Purified DNAs of Escherichia coli O157:H7 (E. coli O157:H7), Salmonella spp., Staphylococcus aureus (S. aureus), and Cochlodinium polykrikoides were successfully amplified and directly detected on the microdevice, where as low as 0.13 and 0.12 ng µL-1 of the DNA of E. coli O157:H7 and S. aureus, respectively, were identified. In addition, the potential of this microdevice for point-of-care testing was further examined by incorporating on-chip sample purification module and testing using a real sample - milk spiked with Salmonella spp. The thermally lysed milk sample was filtered using polydopamine-coated paper embedded inside a sample chamber and seamlessly transported into the reaction chambers by centrifugal force for subsequent LAMP followed by direct on-chip detection inside the reaction chambers in which fisetin-soaked papers were embedded. The limit of detection for Salmonella spp. was determined to be approximately 1.7 × 102 CFU mL-1 using the microdevice. This microdevice is safe, easy to use, selective, and sensitive enough for point-of-care testing to identify foodborne pathogens as well as environmentally harmful microorganisms.