Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
solution under appropriate incubation conditions. Compared with<br />
surface-based hybridization that involves washing of excess<br />
reagents, solution-based hybridization offers the advantage of<br />
higher hybridization efficiency. The hybridized duplexes were<br />
labeled with fluorescent dye YOYO-1 iodide (YOYO) for the singlemolecule<br />
fluorescence detection. YOYO is an intercalating fluorescent<br />
dye which electrostatically binds to the backbone of<br />
oligonucleotide. 41-43 The binding affinity of YOYO dyes on<br />
double-stranded oligonucleotide is very high (∼6 × 10 8 M -1 ) and<br />
there is a fluorescence intensity enhancement of approximately<br />
400-fold upon binding to DNA. 42 Since the binding mechanism<br />
is based on geometrical insertion, neither dye molecules nor<br />
the oligonucleotides have to be chemically modified during the<br />
labeling process. The detection of miRNA is, thus, straightforward.<br />
To improve the fluorescence signal intensity, the hybrids<br />
(∼20 bp) were labeled with YOYO in the ratio of 1 dye<br />
molecule/1 bp. After direct labeling of hybrids with YOYO,<br />
microliters of sample solution were sandwiched in a pair of<br />
precleaned coverslips with a solution depth of approximately<br />
20 µm, and observed under EMCCD-TIRF microscope. The<br />
cleanness of the coverslips was found to be very significant in<br />
the assay as scattering and autofluorescence of any dirt and<br />
stains on glass surface may result in false positive signals. It is<br />
crucial to clean coverslips extensively before use.<br />
YOYO-labeled miRNA hybrids were visualized by a singlemolecule<br />
TIRFM imaging system (Figure S1A). A 488 nm laser<br />
was used to excite the bound YOYO dyes. The laser beam with<br />
an incident angle of approximately 66° was total-internal-reflected<br />
at the glass/solution interface. The thickness of evanescent field<br />
layer (EFL) generated by total internal reflection was calculated<br />
to be ∼190 nm by d ) λ/(2π(η2 2 sin 2 θ - η1 2 ) 1/2 ), where d is the<br />
penetration depth of the field, λ is the wavelength of the<br />
excitation light in vacuum, η1 and η2 are the refraction indices<br />
of the solution and glass slides, and θ is the angle of incidence.<br />
For more homogeneous exciting laser intensity, a central<br />
region of 200 × 200 pixels (53 × 53 µm 2 ) of the EMCCD image<br />
was selected as the sampling area, and thus, the probe volume<br />
is estimated to be 0.54 pL. When 100 pM of miRNA hybrids<br />
was loaded on the coverslip, the theoretical number of observed<br />
hybrid molecules existing in the sampling region was 100 pM<br />
× (53 µm × 53 µm) × 190 nm × 6.02 × 10 23 molecules/mol )<br />
33. 44 Figure S1B shows a typical TIRFM image of miRNA hybrids<br />
acquired in single-molecule level. It is noted that molecules<br />
undergo random diffusional motion in a nonimmobilized system. 45<br />
The diffusion coefficient of the miRNA hybrids was calculated as<br />
76 µm 2 s -1 in bulk solution. 46 However, it was showed that<br />
molecular diffusion rate is much slower at the glass/solution<br />
interface compared to the bulk because of the electrostatic<br />
interaction between molecules and macroscopic glass surface<br />
in microsized domain. 45,47-49 The fluorescence signal generated<br />
(41) Cosa, G.; Focsaneanu, K. S.; McLean, J. R. N.; McNamee, J. P.; Scaiano,<br />
J. C. Photochem. Photobiol. 2001, 73, 585–599.<br />
(42) Gurrieri, S.; Wells, K. S.; Johnson, I. D.; Bustamante, C. Anal. Biochem.<br />
1997, 249, 44–53.<br />
(43) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.;<br />
Mathies, R. A.; Glazer, A. N. 1992, 20, 2803–2812.<br />
(44) He, Y.; Li, H. W.; Yeung, E. S. J. Phys. Chem. B 2005, 109, 8820–8832.<br />
(45) Xu, X. H.; Yeung, E. S. Science 1997, 275, 1106–1109.<br />
(46) Zhdanov, V. P. Mol. BioSyst. 2009, 5, 638–643.<br />
(47) Xu, X. H. N.; Yeung, E. S. Science 1998, 281, 1650–1653.<br />
Figure 2. Correlation between the number of observed miR-21<br />
molecules and expected number of miR-21 in the sampling volume<br />
(0.54 pL). The number of observed miR-21 was corrected as the net<br />
number of hybrids (number of observed molecules - number of<br />
observed in blank). The slope of the correlation curve is 0.80, which<br />
indicates the assay has a hybridization efficiency of approximately<br />
80%.<br />
from the single molecules is spreaded as they diffuse and the size<br />
of the fluorescence spots in the image is larger than the physical<br />
size of molecules of interest (see also movie file in Supporting<br />
Information). 45 Although molecules interact with the glass surface,<br />
nonspecific adsorption of hybrid molecules on the surface of glass<br />
slide was insignificant as both the oligonucleotides and the glass<br />
surface are highly negative-charged at pH 7.4. Figure S2 shows<br />
the histogram of residence time for each miRNA hybrids in 8<br />
consecutive frames. Among the 114 molecules detected in the 8<br />
frames, ∼ 80% of the molecules appeared and then disappeared<br />
in a single frame; 11%, 4%, and 4% of the molecules stayed at the<br />
same position for 2, 3, and 4 frames, respectively, and less than<br />
1% retained for 7 frames and eventually desorbed from the surface.<br />
In the TIRFM image, each fluorescent spot was regarded as a<br />
single molecule as a linear correlation on the number of counted<br />
fluorescence spots and the expected number of miR-21 calculated<br />
from the corresponding concentration was established (R 2 )<br />
0.991). Herein, hybridization is the main factor attributed to<br />
the differences in observed and expected number of molecules<br />
and its efficiency was approximated to be 80% from the slope<br />
of the plot as shown in Figure 2.<br />
Optimization of Hybridization Conditions. The stringency<br />
of hybridization governs the detection sensitivity and selectivity.<br />
It is crucial to optimize the hybridization conditions before<br />
performing further detection. The effects of ionic strength,<br />
selection of probes, and incubation time on the hybridization<br />
efficiency were evaluated.<br />
First, the effect of buffer ionic strength on hybridization<br />
efficiency was studied (Figure 3A). In general, hybridization<br />
affinity is improved at higher ionic strength as the electrostatic<br />
repulsions between the negatively charged oligonucleotides can<br />
be effectively shielded in the presence of salt. However, high ionic<br />
strength may also result in aggregation of molecules. The<br />
aggregates will be misinterpreted as an individual in SMD, and<br />
(48) Lyon, W. A.; Nie, S. M. Anal. Chem. 1997, 69, 3400–3405.<br />
(49) Isailovic, S.; Li, H. W.; Yeung, E. S. J. Chromatogr., A 2007, 1150, 259–<br />
266.<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
6915