Recent Advances in Molecular and Immunological Diagnostic Platform for Virus Detection: A Review.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an ongoing coronavirus disease (COVID-19) outbreak and a rising demand for the development of accurate, timely, and cost-effective diagnostic tests for SARS-CoV-2 as well as other viral infections in general. Currently, traditional virus screening methods such as plate culturing and real-time PCR are considered the gold standard with accurate and sensitive results. However, these methods still require sophisticated equipment, trained personnel, and a long analysis time. Alternatively, with the integration of microfluidic and biosensor technologies, microfluidic-based biosensors offer the ability to perform sample preparation and simultaneous detection of many analyses in one platform. High sensitivity, accuracy, portability, low cost, high throughput, and real-time detection can be achieved using a single platform. This review presents recent advances in microfluidic-based biosensors from many works to demonstrate the advantages of merging the two technologies for sensing viruses. Different platforms for virus detection are classified into two main sections: immunoassays and molecular assays. Moreover, available commercial sensing tests are analyzed.

Quercetin-Mediated Silver Nanoparticle Formation for the Colorimetric Detection of Infectious Pathogens Coupled with Loop-Mediated Isothermal Amplification.

Here, quercetin-mediated silver nanoparticle (AgNP) formation combined with loop-mediated isothermal amplification (LAMP) was introduced to colorimetrically detect two major infectious pathogens, SARS-CoV-2 and Enterococcus faecium, using a foldable PMMA microdevice. The nitrogenous bases of LAMP amplicons can readily form a complex with Ag+ ions, and the catechol moiety in quercetin, which acted as a reducing agent, could be chelated with Ag+ ions, resulting in the easy electron transfer from the oxidant to the reductant and producing brown-colored AgNPs within 5 min. The introduced method exhibited higher sensitivity than agarose gel electrophoresis due to more active redox centers in quercetin. The detection limit was attained at 101 copies µL-1 and 101 CFU mL-1 for SARS-CoV-2 RNA and E. faecium, respectively. A foldable microdevice made of two pieces of PMMA that fully integrates DNA extraction, amplification, and detection processes was fabricated to establish practical applicability. On one PMMA, DNA extraction was performed in a reaction chamber inserted with an FTA card, and then LAMP reagents were added for amplification. Silver nitrate was added to the reaction chamber after LAMP. On the other PMMA, quercetin-soaked paper discs loaded in the detection chamber were folded toward the reaction chamber for colorimetric detection. An intense brown color was produced within 5 min when heated at 65 °C. The introduced colorimetric assay, which is highly favorable for laboratory and on-site applications, could be a valuable alternative to conventional methods for detecting infectious diseases, given its unique principle, simplicity, and naked-eye detection.

Dual-mode visual detection strategies of viable pathogens for point-of-care testing

Here, we introduce a power-free foldable poly(methyl methacrylate) (PMMA) microdevice fully integrating DNA extraction, amplification, and visual detection, realized in novel dual modes–colorimetric and aggregate formation–using 4-aminoantipyrine (4-AP) for the first time for monitoring pathogens. The microdevice contains two parts: reaction and detection zones. A sealing film was utilized to connect the two zones and make the device foldable. The FTA card was deposited in the reaction zone for DNA extraction, followed by loop-mediated isothermal amplification (LAMP) at 65 °C for 45 min. When the detection zone is folded toward the reaction zone, paper discs modified with 4-AP placed in the detection zone are delivered to the reaction zone. Specifically, in the presence of LAMP amplicons, 4-AP is oxidized into red antipyrine or generates aggregates by interacting with copper sulfate, forming copper hybrid nanostructure (Cu-hNs). In the absence of LAMP amplicons, 4-AP is not oxidized and maintains yellow color or fails to form aggregates. Furthermore, we introduced the ethidium homodimer-1 (EthD-1) to identify viable bacteria. EthD-1 penetrated the compromised membranes of nonviable cells and prevented further DNA amplification by intercalating with the DNA. In this way, only samples containing viable cells displayed color change or formed aggregates upon reaction with 4-AP. Using this method, SARS-CoV-2 RNA and Enterococcus faecium were identified by naked eye, with the limit of detection of 103 copies/μL and 102 CFU/mL, respectively, within 60 min. The introduced microdevice can be used for rapidly monitoring viable pathogens and controlling outbreaks of infectious disease in resource-limited settings.

Recent advances in airborne pathogen detection using optical and electrochemical biosensors.

The world is currently facing an adverse condition due to the pandemic of airborne pathogen SARS-CoV-2. Prevention is better than cure; thus, the rapid detection of airborne pathogens is necessary because it can reduce outbreaks and save many lives. Considering the immense role of diverse detection techniques for airborne pathogens, proper summarization of these techniques would be beneficial for humans. Hence, this review explores and summarizes emerging techniques, such as optical and electrochemical biosensors used for detecting airborne bacteria (Bacillus anthracis, Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pneumoniae) and viruses (Influenza A, Avian influenza, Norovirus, and SARS-CoV-2). Significantly, the first section briefly focuses on various diagnostic modalities applied toward airborne pathogen detection. Next, the fabricated optical biosensors using various transducer materials involved in colorimetric and fluorescence strategies for infectious pathogen detection are extensively discussed. The third section is well documented based on electrochemical biosensors for airborne pathogen detection by differential pulse voltammetry, cyclic voltammetry, square-wave voltammetry, amperometry, and impedance spectroscopy. The unique pros and cons of these modalities and their future perspectives are addressed in the fourth and fifth sections. Overall, this review inspected 171 research articles published in the last decade and persuaded the importance of optical and electrochemical biosensors for airborne pathogen detection.

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.

Advances in Nucleic Acid Amplification-Based Microfluidic Devices for Clinical Microbial Detection

Accurate and timely detection of infectious pathogens is urgently needed for disease treatment and control of possible outbreaks worldwide. Conventional methods for pathogen detection are usually time-consuming and labor-intensive. Novel strategies for the identification of pathogenic nucleic acids are necessary for practical application. The advent of microfluidic technology and microfluidic devices has offered advanced and miniaturized tools to rapidly screen microorganisms, improving many drawbacks of conventional nucleic acid amplification-based methods. In this review, we summarize advances in the microfluidic approach to detect pathogens based on nucleic acid amplification. We survey microfluidic platforms performing two major types of nucleic acid amplification strategies, namely, polymerase chain reaction (PCR) and isothermal nucleic acid amplification. We also provide an overview of nucleic acid amplification-based platforms including studies and commercialized products for SARS-CoV-2 detection. Technologically, we focus on the design of the microfluidic devices, the selected methods for sample preparation, nucleic acid amplification techniques, and endpoint analysis. We also compare features such as analysis time, sensitivity, and specificity of different platforms. The first section of the review discusses methods used in microfluidic devices for upstream clinical sample preparation. The second section covers the design, operation, and applications of PCR-based microfluidic devices. The third section reviews two common types of isothermal nucleic acid amplification methods (loop-mediated isothermal amplification and recombinase polymerase amplification) performed in microfluidic systems. The fourth section introduces microfluidic applications for nucleic acid amplification-based detection of SARS-CoV-2. Finally, the review concludes with the importance of full integration and quantitative analysis for clinical microbial identification.

Fabrication of Wearable PDMS Device for Rapid Detection of Nucleic Acids via Recombinase Polymerase Amplification Operated by Human Body Heat.

Pathogen detection by nucleic acid amplification proved its significance during the current coronavirus disease 2019 (COVID-19) pandemic. The emergence of recombinase polymerase amplification (RPA) has enabled nucleic acid amplification in limited-resource conditions owing to the low operating temperatures around the human body. In this study, we fabricated a wearable RPA microdevice using poly(dimethylsiloxane) (PDMS), which can form soft-but tight-contact with human skin without external support during the body-heat-based reaction process. In particular, the curing agent ratio of PDMS was tuned to improve the flexibility and adhesion of the device for better contact with human skin, as well as to temporally bond the microdevice without requiring further surface modification steps. For PDMS characterization, water contact angle measurements and tests for flexibility, stretchability, bond strength, comfortability, and bendability were conducted to confirm the surface properties of the different mixing ratios of PDMS. By using human body heat, the wearable RPA microdevices were successfully applied to amplify 210 bp from Escherichia coli O157:H7 (E. coli O157:H7) and 203 bp from the DNA plasmid SARS-CoV-2 within 23 min. The limit of detection (LOD) was approximately 500 pg/reaction for genomic DNA template (E. coli O157:H7), and 600 fg/reaction for plasmid DNA template (SARS-CoV-2), based on gel electrophoresis. The wearable RPA microdevice could have a high impact on DNA amplification in instrument-free and resource-limited settings.

Fabrication of a fully integrated paper microdevice for point-of-care testing of infectious disease using Safranin O dye coupled with loop-mediated isothermal amplification.

In this study, we introduce a paper microdevice fully integrating DNA extraction, loop-mediated isothermal amplification (LAMP), and Safranin O-based colorimetric detection of two major infectious pathogens, namely SARS-CoV-2 and Enterococcus faecium. The paper microdevice is composed of two parts: sample and reaction chambers. A sealing film acted as a bottom layer to allow foldable motion for transferring DNA from sample chamber to reaction chamber in a seamless manner. An FTA card was employed in the sample chamber for DNA extraction and purification from bacteria-spiked milk. After LAMP reaction at 65 °C for 30 min, a novel aggregation-based DNA detection was obtained by Safranin O polymerization in the reaction chamber. Specifically, Safranin O underwent polymerization by addition of oxidant to form Safranin O oligomers. The electrostatic interaction between the positively charged Safranin O oligomers and the negatively charged DNA comprising LAMP amplicons resulted in the aggregation with a dark red color. Meanwhile, in the absence of LAMP amplicons, Safranin O oligomers were well dispersed and displayed their original red color. By using Safranin O-based detection, SARS-CoV-2 and E. faecium were successfully identified by naked eye within 60 min, and the limits of detection were 10-4 ng/µL and 102 CFU/mL, respectively. These results indicate that a fully integrated paper microdevice plays an important role in sample-in-answer-out format in the genetic analyses of infectious disease and serves as a rapid tool for controlling the spread of diseases.

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.

Ultraviolet-induced in situ gold nanoparticles for point-of-care testing of infectious diseases in loop-mediated isothermal amplification.

The present study investigated ultraviolet-induced in situ gold nanoparticles (AuNPs) coupled with loop-mediated isothermal amplification (LAMP) for the point-of-care testing (POCT) of two major infectious pathogens, namely, Coronavirus (COVID-19) and Enterococcus faecium (E. faecium spp.). In the process, gold ions in a gold chloride (HAuCl4) solution were reduced using trisodium citrate (Na3Ct), a reducing agent, and upon UV illumination, red-colored AuNPs were produced in the presence of LAMP amplicons. The nitrogenous bases of the target deoxyribonucleic acid (DNA) acted as a physical support for capturing gold ions dissolved in the sample. The high affinity of gold with the nitrogenous bases enabled facile detection within 10 min, and the detection limit of COVID-19 plasmid DNA was as low as 42 fg µL-1. To ensure POCT, we designed a portable device that contained arrays of reagent chambers and detection chambers. In the portable device, colorimetric reagents such as HAuCl4 and Na3Ct were contained in the reagent chambers; these reagents were subsequently transferred to the detection chambers where LAMP amplicons were present and thus allowed convenient sample delivery and multiplex detection. Owing to the high sensitivity of the in situ AuNPs, simplicity of portable device fabrication, and rapid colorimetric detection, we strongly believe that the fabricated portable device could serve as a kit for rapid POCT for instantaneous detection of infectious diseases, and could be readily usable at the bedside.