Recent advances in biosensors for screening plant pathogens.

Worldwide, plant pathogens have been a considerably important cause of economic loss in agriculture especially in the decades of agricultural intensification. The increasing losses in agriculture due to biotic plant diseases have drawn attention towards the development of plant disease analyzing methods. In this context, biosensors have emerged as significantly important tools which help farmers in on-field diagnosis of plant diseases. Compared to traditional methods, biosensors have outstanding features such as being highly sensitive and selective, cost-effective, portable, fast and user-friendly operation, and so on. There are three common types of biosensors including electrochemical, fluorescent, and colorimetric biosensors. In this review, some common biotic plant diseases caused by fungi, bacteria, and viruses are first summarized. Then, current advances in developing biosensors are discussed.

Pipette-Free and Fully Integrated Paper Device Employing DNA Extraction, Isothermal Amplification, and Carmoisine-Based Colorimetric Detection for Determining Infectious Pathogens.

A pipette-free and fully integrated device that can be used to accurately recognize the presence of infectious pathogens is an important and useful tool in point-of-care testing, particularly when aiming to decrease the unpredictable threats posed by disease outbreak. In this study, a paper device is developed to integrate the three main processes required for detecting infectious pathogens, including DNA extraction, loop-mediated isothermal amplification (LAMP), and detection. All key reagents, including sodium dodecyl sulfate (SDS), NaOH, LAMP reagents, and carmoisine, are placed on the paper device. The paper device is operated simply via sliding and folding without using any bulky equipment, and the results can be directly observed by the naked eye. The optimized concentrations of sodium dodecyl sulfate (SDS), sodium hydroxide (NaOH), and carmoisine were found to be 0.1%, 0.1 M, and 0.5 mg/mL, respectively. The paper device was used to detect Enterococcus faecium at concentrations as low as 102 CFU/mL within 60 min. Also, E. faecium spiked in milk was successfully detected using the paper device, demonstrating the feasible application in real sample analysis.

Copper: DNA extraction and solid phase detection agent for all-in-one molecular diagnostic device coupled with isothermal amplification.

In this study, an all-in-one poly(methyl methacrylate) (PMMA) device integrating two novel techniques - DNA extraction employing a CuSO4/H2O2 system and DNA detection utilizing solid phase copper tape - coupled with loop-mediated isothermal amplification (LAMP) is developed for on-site pathogen detection. The CuSO4/H2O2 system, also known as Fenton-like reaction, is used to produce hydroxyl radicals, which can disrupt bacterial membranes via lipid peroxidation and release DNA at room temperature. The released DNA is subsequently amplified by LAMP reaction. The acidic environment resulting from the production of hydrogen ions in the presence of target DNA in the LAMP reaction can stimulate the color change on copper tape due to the corrosion, while the innate alkaline environment in a negative sample not containing target DNA cannot stimulate the corrosion. The fabricated PMMA device integrates all the functionalities necessary for molecular diagnostics such as DNA extraction, amplification, and detection, and a carbon paste-based heater is fabricated for LAMP reaction. Using the PMMA device, Enterococcus faecium was detected as low as 4.67 × 102 CFU/mL within 90 min. E. faecium spiked in milk was successfully detected using the all-in-one PMMA device. The equipment-free techniques for decentralized diagnostics and naked-eye readout of results coupled with the portable heater serves as a promising solution for point-of-care testing particularly in a resource-limited environment.

A Rotatable Paper Device Integrating Reverse Transcription Loop-Mediated Isothermal Amplification and a Food Dye for Colorimetric Detection of Infectious Pathogens.

In this study, we developed a rotatable paper device integrating loop-mediated isothermal amplification (RT-LAMP) and a novel naked-eye readout of the RT-LAMP results using a food additive, carmoisine, for infectious pathogen detection. Hydroxyl radicals created from the reaction between CuSO4 and H2O2 were used to decolor carmoisine, which is originally red. The decolorization of carmoisine can be interrupted in the presence of DNA amplicons produced by the RT-LAMP reaction due to how DNA competitively reacts with the hydroxyl radicals to maintain the red color of the solution. In the absence of the target DNA, carmoisine is decolored, owing to its reaction with hydroxyl radicals; thus, positive and negative samples can be easily differentiated based on the color change of the solution. A rotatable paper device was fabricated to integrate the RT-LAMP reaction with carmoisine-based colorimetric detection. The rotatable paper device was successfully used to detect SARS-CoV-2 and SARS-CoV within 70 min using the naked eye. Enterococcus faecium spiked in milk was detected using the rotatable paper device. The detection limits for the SARS-CoV-2 and SARS-CoV targets were both 103 copies/µL. The rotatable paper device provides a portable and low-cost tool for detecting infectious pathogens in a resource-limited environment.

Polydopamine aggregation: A novel strategy for power-free readout of loop-mediated isothermal amplification integrated into a paper device for multiplex pathogens detection.

Loop-mediated isothermal amplification (LAMP) has been widely used for detecting pathogens. However, power-free and clear visualization of results still remain challenging. In this study, we developed a paper device integrated with power-free DNA detection strategy realized by polydopamine aggregation. In the presence of DNA amplicons, the polymerization of dopamine into aggregated polydopamine was hindered, while in the absence of DNA amplicons, polydopamine aggregation is facilitated. The porosity of the paper enabled the capillary flow of dispersed polydopamine for positive sample, while aggregated polydopamine remained at the bottom of the paper strip due to large size of the aggregates for negative sample. Based on this mechanism, we fabricated a slidable paper device integrating LAMP with dopamine polymerization for the naked-eye detection, operated in a seamless manner. Moreover, the introduced paper device was successfully used to detect DNA extracted from Escherichia coli O157:H7 and SARS-CoV-2 within 25 min, as well as Enterococcus faecium within 35 min. The detection limits of both Escherichia coli O157:H7 and SARS-CoV-2 were 10-4 ng/µL. The introduced paper device can be used as a simple and sensitive tool for detecting multiple infectious pathogens, making it an ideal tool particularly for resource-limited environment.