TY - JOUR
T1 - Smartphone clip-on instrument and microfluidic processor for rapid sample-to-answer detection of Zika virus in whole blood using spatial RT-LAMP
AU - Jankelow, Aaron M.
AU - Lee, Hankeun
AU - Wang, Weijing
AU - Hoang, Trung Hieu
AU - Bacon, Amanda
AU - Sun, Fu
AU - Chae, Seol
AU - Kindratenko, Victoria
AU - Koprowski, Katherine
AU - Stavins, Robert A.
AU - Ceriani, Dylann D.
AU - Engelder, Zachary W.
AU - King, William P.
AU - Do, Minh N.
AU - Bashir, Rashid
AU - Valera, Enrique
AU - Cunningham, Brian T.
N1 - Funding Information:
The following reagents were obtained through BEI Resources, NIAID, NIH: (1) Genomic RNA from Zika Virus, PRVABC59, NR-50244; (2) Genomic RNA from Zika Virus, R103451, NR-50358; (3) Zika Virus, PRVABC59, Infected Cell Lysate, Gamma-Irradiated, NR-50547. The authors are grateful for financial support from the National Institutes of Health (R01AI139401) and from the National Science Foundation Partnership for Innovation program (PFI-TT 1919015).
Publisher Copyright:
© 2022 The Royal Society of Chemistry.
PY - 2022/6/15
Y1 - 2022/6/15
N2 - Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent “bloom” events for positive samples, in an approach called “Spatial LAMP” (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17-32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 102 copies per μl, and a gamma-irradiated virus of 103 virus particles in a single 12.5 μl droplet blood sample.
AB - Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent “bloom” events for positive samples, in an approach called “Spatial LAMP” (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17-32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 102 copies per μl, and a gamma-irradiated virus of 103 virus particles in a single 12.5 μl droplet blood sample.
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U2 - 10.1039/d2an00438k
DO - 10.1039/d2an00438k
M3 - Article
C2 - 35726910
AN - SCOPUS:85132533453
SN - 0003-2654
VL - 147
SP - 3838
EP - 3853
JO - Analyst
JF - Analyst
IS - 17
ER -