Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 4. Standardization curve for the quantification of miR-21.
Different concentrations of miR-21 were hybridized with 100 pM LNA
probe in solution at 52 °C for 1 h. The data depict the averages of
three experiments, and the error bars are the standard error of mean
of the three trials.
Information, Figure S4). As a result, the hybridization time was
set to 60 min for later experiments in this study.
Quantification of Synthetic miR-21. Under the optimal
hybridization conditions of miRNAs as discussed above, a calibration
plot of molecules counts as a function of the target miR-21
concentration was constructed. Synthetic miRNA of 0-100 pM
was hybridized with 100 pM LNA probes in solution and labeled
with YOYO. By single-molecule counting on the series of images
acquired, a plot of the number of molecule counts as a function
of the concentration of synthetic miR-21 was obtained with a
coefficient of determination of 0.991 (Figure 4). The detection limit
of the assay was estimated to be 5 pM (i.e., 50 amol in 10 µL of
sample). The ultimate theoretic limit of single molecule detection
is to detect a sole molecule in the bulk sample solution sandwiched
between the glass slides. Ideally, the theoretic limit is calculated
to be 170 zM, with a single target molecule in 10-µL sample
solution. To achieve such an ultimate theoretic limit requires
sufficiently long sampling time until the single target molecule
enters the probe volume (0.54 pL) by chance and gets excited
and detected. Otherwise, regardless of the sampling time, the
concentration of sample solution should be ∼3 pM, so that a single
molecule always locates in the probe volume. To improve the
detection limit, several factors including the probe volume,
viscosity of sample solution, sampling time, photostability, and
Figure 5. Quantification of miR-21 contents in total RNA of (A) MCF-7, (B) HepG2, and (C) HUVEC cells by standard addition methods with
TIRFM. Synthetic miR-21 was spiked into matrix of total RNA and LNA probe. The data depict the averages of three experiments, and the error
bars are the standard error of mean of the three trials. (D) Comparison between the miR-21 contents in total RNA of HUVEC, HepG2, and
MCF-7 cells determined by qRT-PCR assay and single-molecule TIRFM assay.
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
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