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
The demand for scalable, rapid and sensitive COVID-19 diagnostics is particularly pressing at present to help contain the spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. We report the development of an integrated modular centrifugal microfluidic platform costing less than 250 USD to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The platform was validated with a panel of 131 nasopharyngeal swab samples collected from symptomatic COVID-19 patients. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
ABSTRACT
The rapid and specific detection of nucleic acid sequences is highly demanded for several applications including pathogen diagnostics, quality control of biopharmaceutical products and forensics. Nucleic acid amplification methods based on mixtures of primers and polymerase enzymes such as polymerase chain reaction (PCR) and other isothermal methods are typically the standard approach. Here, using SARS-CoV-2 ORF1ab sequence as a model, we report the development of a simple enzyme-free and single-step competitive hybridization method allowing the specific detection of any type of nucleic acid sequence (ss/dsDNA or RNA) within 15 min with 89% sequence homology and sensitivity in the pM-range. © 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
In this work, we present the design and fabrication of a fiber device that performs digital droplet microfluidics for molecular diagnostics. A variety of fibers and capillaries were used to build three connected modules dedicated to droplet generation, incubation, and fluorescence detection which enables a uniaxial arrangement. This is in contrast to the traditional 2-dimensional lab-on-a-chip architecture. We characterize our fiber device using a fluorescein dilution series. Our observed detection limit is on the order of 10 nM fluorescein. We demonstrate our all-fiber device for the fluorescence readout after loop-mediated isothermal amplification (LAMP) of synthetic SARS-CoV-2. Our results suggest that this fiber device can successfully distinguish between positive and negative samples in molecular diagnostics. We propose that our fiber device offers benefits over microfluidic chip techniques such as easier optical integration, much simpler sample loading, and faster diagnosis with high specificity and sensitivity. Keywords: All-fiber device, microfluidics, optofluidics, loop-mediated isothermal amplification (LAMP), molecular diagnostics, SARS-CoV2. © 2021 SPIE.