TY - JOUR
T1 - Microfluidic System for Detection of Viral RNA in Blood Using a Barcode Fluorescence Reporter and a Photocleavable Capture Probe
AU - Du, Ke
AU - Park, Myeongkee
AU - Griffiths, Anthony
AU - Carrion, Ricardo
AU - Patterson, Jean
AU - Schmidt, Holger
AU - Mathies, Richard
N1 - Funding Information:
We would like to thank D. Wartmann, H. Li, H. Cai, and A. Hawkins for valuable discussions. This research was supported by the NIH under Grants 4R33AI100229 and 1R21AI100229 and the NSF under Grant CBET-1159453 (H.S.).
Funding Information:
This research was supported by the NIH under Grants 4R33AI100229 and 1R21AI100229 and the NSF under Grant CBET-1159453 (H.S.).
PY - 2017/11/21
Y1 - 2017/11/21
N2 - A microfluidic sample preparation multiplexer (SPM) and assay procedure is developed to improve amplification-free detection of Ebola virus RNA from blood. While a previous prototype successfully detected viral RNA following off-chip RNA extraction from infected cells, the new device and protocol can detect Ebola virus in raw blood with clinically relevant sensitivity. The Ebola RNA is hybridized with sequence specific capture and labeling DNA probes in solution and then the complex is pulled down onto capture beads for purification and concentration. After washing, the captured RNA target is released by irradiating the photocleavable DNA capture probe with ultraviolet (UV) light. The released, labeled, and purified RNA is detected by a sensitive and compact fluorometer. Exploiting these capabilities, a detection limit of 800 attomolar (aM) is achieved without target amplification. The new SPM can run up to 80 assays in parallel using a pneumatic multiplexing architecture. Importantly, our new protocol does not require time-consuming and problematic off-chip probe conjugation and washing. This improved SPM and labeling protocol is an important step toward a useful POC device and assay.
AB - A microfluidic sample preparation multiplexer (SPM) and assay procedure is developed to improve amplification-free detection of Ebola virus RNA from blood. While a previous prototype successfully detected viral RNA following off-chip RNA extraction from infected cells, the new device and protocol can detect Ebola virus in raw blood with clinically relevant sensitivity. The Ebola RNA is hybridized with sequence specific capture and labeling DNA probes in solution and then the complex is pulled down onto capture beads for purification and concentration. After washing, the captured RNA target is released by irradiating the photocleavable DNA capture probe with ultraviolet (UV) light. The released, labeled, and purified RNA is detected by a sensitive and compact fluorometer. Exploiting these capabilities, a detection limit of 800 attomolar (aM) is achieved without target amplification. The new SPM can run up to 80 assays in parallel using a pneumatic multiplexing architecture. Importantly, our new protocol does not require time-consuming and problematic off-chip probe conjugation and washing. This improved SPM and labeling protocol is an important step toward a useful POC device and assay.
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U2 - 10.1021/acs.analchem.7b03527
DO - 10.1021/acs.analchem.7b03527
M3 - Article
AN - SCOPUS:85034961799
VL - 89
SP - 12433
EP - 12440
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 22
ER -