ABSTRACT
During the COVID-19 pandemic, point-of-care genetic testing (POCT) devices were used for on-time and on-site detection of the virus, which helped to prevent and control the spread of the pandemic. Smartphones, which are widely used electronic devices with many functions, have the potential to be used as a molecular diagnostic platform for universal healthcare monitoring. Several integrated diagnostics platforms for the real-time and end-point detection of COVID-19 were developed using the functions of smartphones, such as the operating system, power, sound, camera, data storage, and display. These platforms use the 5V output power of smartphones, which can be amplified to power a micro-capillary electrophoresis system or a thin-film heater, and the CMOS camera of smartphones can capture the color change during a colorimetric loop-mediated isothermal amplification test and detect fluorescence signals. Smartphones can also be used with self-written web-based apps to enable automatic and remote pathogen analysis on POCT platforms. Our lab developed a handheld micro-capillary electrophoresis device for end-point detection of SARS-CoV-2, as well as an integrated smartphone-based genetic analyzer for the qualitative and quantitative colorimetric detection of foodborne pathogens with the help of a custom mobile app. © 2023 SPIE.
ABSTRACT
A portable, inexpensive, and easy-to-manufacture microfluidic device is developed for the detection of SARS-CoV-2 dsDNA fragments. In this device, four reaction chambers separated by carbon fiber rods are pre-loaded with isothermal amplification and CRISPR-Cas12a reagents. The reaction is carried out by simply pulling the rods, without the need for manual pipetting. To facilitate power-free pathogen detection, the entire detection is designed to be heated with a disposable hand warmer. After the CRISPR reaction, the fluorescence signal generated by positive samples is identified by naked eye, using an inexpensive flashlight. This simple and sensitive device will serve as a new model for the next-generation viral diagnostics in either hospital or resource-limited settings. © 2023 SPIE.
ABSTRACT
An accurate, convenient, and rapid diagnostic platform, which can be applied in facility-limited or point-of-care (POC) settings, is essential to help prevent the spread of infectious diseases and enable the most effective treatment to be selected. In this study, we describe the development of a new isothermal molecular diagnostic system named multipurpose advanced split T7 promoter-based transcription amplification (MASTER) for the rapid and ultrasensitive detection of various pathogens containing single-stranded RNA and double-stranded DNA. MASTER produces a large number of RNA amplicons in the presence of target pathogens, which generate fluorescence or colorimetric signals based on light-up RNA aptamers or lateral flow assays. Implementing MASTER at 37 °C for<1 h achieved the detection of a single copy per reaction without cross-reactivity. Moreover, the testing of 40 clinical samples revealed that MASTER exhibited excellent accuracy with 100% sensitivity and specificity for SARS-CoV-2 diagnosis. Furthermore, a one-pot MASTER system capable of accelerating practical applications was demonstrated, indicating that the MASTER system is a promising platform for the effective surveillance of various pathogens. © 2023 Elsevier B.V.
ABSTRACT
Visual detection of nucleic acids is important to diagnose the serious acute infectious diseases such as coronavirus disease 2019 (COVID-19). During this pandemic, reliable visual detection kits have been in high demand for screening and prevention of the virus. While developing these visual detection kits, a real-time monitoring platform is usually applied to study the amplification and detection processes of nucleic acids and optimize the detecting conditions. Herein, we developed a real-time monitoring platform of colorimetric loop-mediated isothermal amplification (LAMP) to investigate the amplification and detection processes of nucleic acids. Using this platform, we could obtain the real-time amplification curves, and optimize the reaction temperature, color change, and detection time. Based on the optimized conditions, a visual detection kit for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was successfully developed with a sensitivity of 102 copies µL−1 in 12 min. This real-time monitoring platform has advantages of simple construction, steady performance, high sensitivity, and outstanding anti-pollution capability, and could replace the traditional colorimetric methods by photographing and reading values. This platform would accelerate the development of visual detection kits for colorimetric LAMP, help to explore the amplification and transcription of nucleic acids, and provide support for the prevention of emerging biological threats. © 2023
ABSTRACT
In the ongoing COVID-19 pandemic, sensitive and rapid on-site detection of the SARS-CoV-2 coronavirus has been one of crucial objectives. A point-of-care (PoC) device called PATHPOD for quick, on-site detection of SARS-CoV-2 employing a real-time reverse-transcription loop-mediated isothermal amplification (RT-rLAMP) reaction on a polymer cartridge. The PATHPOD consists of a standalone device (weighing under 1.2 kg) and a cartridge, and can identify 10 distinct samples and 2 controls in less than 50 minutes. The PATHPOD PoC system is fabricated and clinically validated for the first time in this work © 2023 IEEE.
ABSTRACT
Here, we developed a copper sulfate (CuSO4)-initiated diphenylamine (DPA)-based colorimetric strategy coupled with loop-mediated isothermal amplification (LAMP) for rapid detection of two critical contagious pathogens, SARS-CoV-2 and Enterococcus faecium. To detect the DNA, acid hydrolysis of LAMP amplicons was executed, enabling the development of a blue color. In the LAMP amplicons, the bond between the purines and deoxyribose is extremely labile. It can be broken using 70% sulfuric acid followed by phosphate group elimination, which generates a highly active keto aldehyde group. CuSO4 plays an imperative role inducing DPA to rapidly react with the keto aldehyde group, producing an intense blue color within 5 min. Moreover, low quantities such as 103 copies μL-1 of SARS-CoV-2 RNA and 102 CFU mL-1 of E. faecium were successfully detected, revealing the advantages of the introduced method. To confirm practical applicability, multiplex detection of pathogens was performed using a foldable microdevice comprising reaction and detection zones. Various reactions such as DNA extraction, LAMP, and acid hydrolysis occurred in the reaction zone. Then, colorimetric reagents (DPA, CuSO4, and ethylene glycol) contained in the detection zone were mixed with the keto aldehyde group by simply folding the microdevice, which was heated at 65 °C for 5 min for colorimetric detection. An intense blue color was developed where the target DNA was present. These results indicate that the method proposed in this study is highly suitable for point-of-care applications, especially in resource-limited settings for the rapid detection of harmful pathogens. © 2023 American Chemical Society.
ABSTRACT
We report a point-of-care (POC) device for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A viruses. The device carries out sample preparation using ball-based valves for sequential delivery of reagents. A microfluidic paper-based analytical device (µPAD) in the detection unit enables RNA isolation and enrichment, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and colorimetric detection. The device integrates all the necessary steps for the sample preparation, including virus lysis, RNA enrichment and purification of two virus samples. The device enabled simultaneous detection of SARS-CoV-2 and Influenza A N1H1 viruses in 50 min., with limit of detection of 2 and 6 genome equivalents (GEs), respectively. The device was also capable of detecting environmental sample of the two viruses. Copyright © 2022 by ASME.
ABSTRACT
Here, we developed a copper sulfate (CuSO4)-initiated diphenylamine (DPA)-based colorimetric strategy coupled with loop-mediated isothermal amplification (LAMP) for rapid detection of two critical contagious pathogens, SARS-CoV-2 and Enterococcus faecium. To detect the DNA, acid hydrolysis of LAMP amplicons was executed, enabling the development of a blue color. In the LAMP amplicons, the bond between the purines and deoxyribose is extremely labile. It can be broken using 70% sulfuric acid followed by phosphate group elimination, which generates a highly active keto aldehyde group. CuSO4 plays an imperative role inducing DPA to rapidly react with the keto aldehyde group, producing an intense blue color within 5 min. Moreover, low quantities such as 103 copies μL-1 of SARS-CoV-2 RNA and 102 CFU mL-1 of E. faecium were successfully detected, revealing the advantages of the introduced method. To confirm practical applicability, multiplex detection of pathogens was performed using a foldable microdevice comprising reaction and detection zones. Various reactions such as DNA extraction, LAMP, and acid hydrolysis occurred in the reaction zone. Then, colorimetric reagents (DPA, CuSO4, and ethylene glycol) contained in the detection zone were mixed with the keto aldehyde group by simply folding the microdevice, which was heated at 65 °C for 5 min for colorimetric detection. An intense blue color was developed where the target DNA was present. These results indicate that the method proposed in this study is highly suitable for point-of-care applications, especially in resource-limited settings for the rapid detection of harmful pathogens. © 2023 American Chemical Society
ABSTRACT
Rolling Circle Amplification (RCA) has shown significant potential for pathogen diagnostics providing high specificity and sensitivity combined with relatively low temperature (<37 °C) isothermal amplification. In the context of the ongoing COVID-19 pandemic, we report the development of an RCA-based method allowing direct detection of SARS-CoV-2 RNA in microfluidics. The viral RNA was hybridized to biotinylated oligos and L-probes in solution, enriched in a microchannel and subsequently amplified in situ using padlock probes against the L-probes. This method allowed the detection of 1x103 viral copies/μL within 90 minutes of amplification, demonstrating an alternative approach to current isothermal amplification methods. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
We present a nucleic acid-based point-of-care diagnostic for the detection of the SARS-CoV-2 from saliva using an additively manufactured microfluidic cartridge. The assay uses reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) to detect the presence of SARS-CoV-2 RNA on-cartridge in a point-of-care optical detection system based on a smartphone. We show positive results within the 10-30 minutes range and integrated biological controls on the cartridge. We demonstrate the microfluidic diagnostic with human patient samples, with results that are consistent with the off-cartridge validation. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
We report the development of an electrochemical sensor platform for ultrasensitive and rapid detection of SARS-CoV-2 viral RNA that integrates loop-mediated isothermal amplification (LAMP), CRISPR-based detection, and anti-fouling nanocomposite coating. By integrating LAMP amplification with CRISPR, we achieved ultrasensitive detection of SARS-CoV-2 RNA at levels as low as 5 copies µL-1. Data from this electrochemical diagnostic platform was comparable to traditional RT-PCR methodology in a fraction of the time, at low cost, and without requiring laboratory space. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
Silica fibers and capillaries offer opportunities for compact integration of optics with microfluidics while adding advantages such as;flexibility within a high aspect ratio format, uniaxial arrangements, and measurement-at-a-distance. Here, we describe droplet microfluidics-based nucleic acid detection of SARS-CoV-2 in a lab-in-a-fiber platform. The fiber component integrates three modules with key functions: droplet generation, incubation, and fluorescence detection. Within the scope of this work, we developed the component specifically to target the quantification of SARS-CoV-2 viral RNA through reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The all-fiber component could successfully generate uniform droplets and differentiate pre-amplified positive LAMP reaction from negative sample. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
Since the early reports of SARS-CoV-2 in Wuhan, China in the winter of 2019, the virus spread has resulted in the most socially-crippling pandemic of the last century. Here, we report the development of a rapid, molecular COVID-19 test utilizing for the first time a loop-mediated isothermal amplification (LAMP) assay on Lab-on-Printed Circuit Board (Lab-on-PCB) to exploit the established integration and up-scaling advantages the latter offers. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
The result readouts of loop-mediated isothermal amplification (LAMP) still remain challenging because current techniques require bulky equipment and could not give clear visualization. In this study, we developed a paper device to integrate LAMP and a novel strategy for power-free and naked-eye readout of result relied on polydopamine aggregation. The introduced paper device was used to detect DNA extracted from Escherichia coli O157:H7 (E. coli O157:H7), Enterococcus faecium (E. faecium), and SARS-CoV-2 plasmid. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
This paper presents a newly developed microfluidic flow control theory for autonomous sample dispensing into an array of reaction microchambers. The theoretical predictions for the possible dispensing number and maximum flow rate were validated by comparison to experimental results. Moreover, we successfully demonstrated the rapid genetic detection of multiple infectious viruses including SARS-CoV-2 in fabricated polydimethylsiloxane (PDMS)-based microfluidic devices based on the loop-mediated isothermal amplification (LAMP) method. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
We report a point-of-care (POC) testing platform for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus. The POC device integrates sample preparation using ball-based valves for sequential delivery of reagents, viral RNA isolation and enrichment by paper-based filtration, with reverse transcription loop-mediated isothermal amplification (RT-LAMP) and colorimetric detection. The device is capable of detecting both viruses, showing high sensitivity and specificity. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
Early and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the point-of-care (POC) is crucial for reducing the transmission of coronavirus disease 2019 (COVID-19). To address this need, we have developed a valve-enabled lysis, paper-based RNA enrichment, and RNA amplification device (VLEAD) for detecting SARSCoV-2. We have combined VLEAD with a smart coffee mug for sample preparation, nucleic acid isothermal amplification, and colorimetric detection using a smartphone camera or a naked eye. VLEAD enables two critical functions required for POC testing: sample preparation and detection. Since the reagents can be pre-packaged in the device, all operational steps can be carried out at POC. We have demonstrated the functions of the device by analyzing samples spiked with heat-inactivated SARSCoV-2, which were obtained from BEI Resources. This rapid and highly sensitive POC platform for SARS-CoV-2 detection has a potential to help reduce COVID-19 transmission. Copyright © 2021 by ASME