Analytical Chemistry Chemical Cytometry Quantitates Superoxide

solution under appropriate incubation conditions. Compared with
surface-based hybridization that involves washing of excess
reagents, solution-based hybridization offers the advantage of
higher hybridization efficiency. The hybridized duplexes were
labeled with fluorescent dye YOYO-1 iodide (YOYO) for the singlemolecule
fluorescence detection. YOYO is an intercalating fluorescent
dye which electrostatically binds to the backbone of
oligonucleotide. 41-43 The binding affinity of YOYO dyes on
double-stranded oligonucleotide is very high (∼6 × 10 8 M -1 ) and
there is a fluorescence intensity enhancement of approximately
400-fold upon binding to DNA. 42 Since the binding mechanism
is based on geometrical insertion, neither dye molecules nor
the oligonucleotides have to be chemically modified during the
labeling process. The detection of miRNA is, thus, straightforward.
To improve the fluorescence signal intensity, the hybrids
(∼20 bp) were labeled with YOYO in the ratio of 1 dye
molecule/1 bp. After direct labeling of hybrids with YOYO,
microliters of sample solution were sandwiched in a pair of
precleaned coverslips with a solution depth of approximately
20 µm, and observed under EMCCD-TIRF microscope. The
cleanness of the coverslips was found to be very significant in
the assay as scattering and autofluorescence of any dirt and
stains on glass surface may result in false positive signals. It is
crucial to clean coverslips extensively before use.
YOYO-labeled miRNA hybrids were visualized by a singlemolecule
TIRFM imaging system (Figure S1A). A 488 nm laser
was used to excite the bound YOYO dyes. The laser beam with
an incident angle of approximately 66° was total-internal-reflected
at the glass/solution interface. The thickness of evanescent field
layer (EFL) generated by total internal reflection was calculated
to be ∼190 nm by d ) λ/(2π(η2 2 sin 2 θ - η1 2 ) 1/2 ), where d is the
penetration depth of the field, λ is the wavelength of the
excitation light in vacuum, η1 and η2 are the refraction indices
of the solution and glass slides, and θ is the angle of incidence.
For more homogeneous exciting laser intensity, a central
region of 200 × 200 pixels (53 × 53 µm 2 ) of the EMCCD image
was selected as the sampling area, and thus, the probe volume
is estimated to be 0.54 pL. When 100 pM of miRNA hybrids
was loaded on the coverslip, the theoretical number of observed
hybrid molecules existing in the sampling region was 100 pM
× (53 µm × 53 µm) × 190 nm × 6.02 × 10 23 molecules/mol )
33. 44 Figure S1B shows a typical TIRFM image of miRNA hybrids
acquired in single-molecule level. It is noted that molecules
undergo random diffusional motion in a nonimmobilized system. 45
The diffusion coefficient of the miRNA hybrids was calculated as
76 µm 2 s -1 in bulk solution. 46 However, it was showed that
molecular diffusion rate is much slower at the glass/solution
interface compared to the bulk because of the electrostatic
interaction between molecules and macroscopic glass surface
in microsized domain. 45,47-49 The fluorescence signal generated
(41) Cosa, G.; Focsaneanu, K. S.; McLean, J. R. N.; McNamee, J. P.; Scaiano,
J. C. Photochem. Photobiol. 2001, 73, 585–599.
(42) Gurrieri, S.; Wells, K. S.; Johnson, I. D.; Bustamante, C. Anal. Biochem.
1997, 249, 44–53.
(43) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.;
Mathies, R. A.; Glazer, A. N. 1992, 20, 2803–2812.
(44) He, Y.; Li, H. W.; Yeung, E. S. J. Phys. Chem. B 2005, 109, 8820–8832.
(45) Xu, X. H.; Yeung, E. S. Science 1997, 275, 1106–1109.
(46) Zhdanov, V. P. Mol. BioSyst. 2009, 5, 638–643.
(47) Xu, X. H. N.; Yeung, E. S. Science 1998, 281, 1650–1653.
Figure 2. Correlation between the number of observed miR-21
molecules and expected number of miR-21 in the sampling volume
(0.54 pL). The number of observed miR-21 was corrected as the net
number of hybrids (number of observed molecules - number of
observed in blank). The slope of the correlation curve is 0.80, which
indicates the assay has a hybridization efficiency of approximately
80%.
from the single molecules is spreaded as they diffuse and the size
of the fluorescence spots in the image is larger than the physical
size of molecules of interest (see also movie file in Supporting
Information). 45 Although molecules interact with the glass surface,
nonspecific adsorption of hybrid molecules on the surface of glass
slide was insignificant as both the oligonucleotides and the glass
surface are highly negative-charged at pH 7.4. Figure S2 shows
the histogram of residence time for each miRNA hybrids in 8
consecutive frames. Among the 114 molecules detected in the 8
frames, ∼ 80% of the molecules appeared and then disappeared
in a single frame; 11%, 4%, and 4% of the molecules stayed at the
same position for 2, 3, and 4 frames, respectively, and less than
1% retained for 7 frames and eventually desorbed from the surface.
In the TIRFM image, each fluorescent spot was regarded as a
single molecule as a linear correlation on the number of counted
fluorescence spots and the expected number of miR-21 calculated
from the corresponding concentration was established (R 2 )
0.991). Herein, hybridization is the main factor attributed to
the differences in observed and expected number of molecules
and its efficiency was approximated to be 80% from the slope
of the plot as shown in Figure 2.
Optimization of Hybridization Conditions. The stringency
of hybridization governs the detection sensitivity and selectivity.
It is crucial to optimize the hybridization conditions before
performing further detection. The effects of ionic strength,
selection of probes, and incubation time on the hybridization
efficiency were evaluated.
First, the effect of buffer ionic strength on hybridization
efficiency was studied (Figure 3A). In general, hybridization
affinity is improved at higher ionic strength as the electrostatic
repulsions between the negatively charged oligonucleotides can
be effectively shielded in the presence of salt. However, high ionic
strength may also result in aggregation of molecules. The
aggregates will be misinterpreted as an individual in SMD, and
(48) Lyon, W. A.; Nie, S. M. Anal. Chem. 1997, 69, 3400–3405.
(49) Isailovic, S.; Li, H. W.; Yeung, E. S. J. Chromatogr., A 2007, 1150, 259–
266.
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