Single-molecule tracing on a fluidic microchip for quantitative detection of low-abundance nucleic acids.
Here, we report a method capable of quantitative detection of low-abundance DNA/RNA molecules by incorporating confocal fluorescence spectroscopy, molecular beacons, and a molecular-confinement microfluidic reactor. By using a combination of ac and dc fields via a trio of 3-D electrodes in the microreactor, we are able to precisely direct the transport of individual molecules to a minuscule laser-focused detection volume ( approximately 1 fL). A burst of fluorescence photons is detected whenever a molecular beacon-target hybrid flows through the detection region, and the amount of targets can be directly quantified according to the number of recorded single-molecule flow-through events. This assay consumes only attomoles of molecular probes and is able to quantitatively detect subpicomolar DNA targets. A measurement time of less than 2 min is sufficient to complete the detection.
Wang
Tza-Huei
TH
Department of Mechanical Engineering and Whitaker Biomedical Engineering Institute, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA. thwang@jhu.edu
Peng
Yahui
Y
Zhang
Chunyang
C
Wong
Pak Kin
PK
Ho
Chih-Ming
CM
D004247:DNA / Q000032:analysis*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D012313:RNA / Q000032:analysis*; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes; D012313:RNA; D004247:DNA