Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification.
We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.
Department of Biomedical Engineering, Johns Hopkins University, Baltimore 21218, USA.|Department of Mechanical Engineering, Johns Hopkins University, Baltimore 21218, USA.
Weihua
W
Guan
Department of Mechanical Engineering, Johns Hopkins University, Baltimore 21218, USA.
Liben
L
Chen
Department of Biomedical Engineering, Johns Hopkins University, Baltimore 21218, USA.
Tushar D
TD
Rane
Department of Biomedical Engineering, Johns Hopkins University, Baltimore 21218, USA.|Department of Mechanical Engineering, Johns Hopkins University, Baltimore 21218, USA.
Tza-Huei
TH
Wang
D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018189:Immunomagnetic Separation / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods*
D012313:RNA