Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
enzymatic amplification techniques such as RAKE assay, 17 bioluminescence-enzyme<br />
labeling, 18 and rolling-circle amplification<br />
(RCA) 19 are also reported for sensitivity enhancement. These<br />
reported detection methods bring great benefit and improvement<br />
to the sensitivity of miRNA profiling to certain extends. Nevertheless,<br />
sample pretreatment and the modifications may result in loss<br />
of samples throughout the multiple pretreatment steps such as<br />
sample enrichment, labeling, and purification. The detection<br />
sensitivity and reliability are hence hindered. Since cellular miRNA<br />
concentration can be as low as 1000 molecules per cell, 20<br />
deficiencies in sensitivity may result in unsuccessful quantification<br />
of low-abundance miRNAs and, thus, false diagnostic result.<br />
Therefore, an ultrasensitive miRNA-profiling assay without the<br />
need of pretreatment is demanded.<br />
Single-molecule detection (SMD) technique has been widely<br />
applied in the behavioral study of individual biomolecules. 21,22<br />
Scientific issues such as monitoring enzymatic kinetics, 23,24 DNA<br />
adsorption/desorption behavior, 25,26 verification of nucleic acid<br />
hybridization, 27 DNA mismatch discrimination, 28 protein and DNA<br />
conformation dynamics, 29,30 and DNA mapping 31,32 have successfully<br />
been accomplished with SMD. Besides, several groups have<br />
also demonstrated the competence of SMD in the quantitation of<br />
biomolecules such as proteins 33 and viral DNA 34,35 by singlemolecule<br />
counting. Recently, quantitation of miRNA in singlemolecule<br />
level was displayed by Neely and co-workers. 36 The<br />
novel miRNA detection assay is free of sample enrichment and<br />
(15) Liang, R. Q.; Li, W.; Li, Y.; Tan, C. Y.; Li, J. X.; Jin, Y. X.; Ruan, K. C. Nucleic<br />
Acids Res. 2005, 33.<br />
(16) Yang, W. J.; Li, X. B.; Li, Y. Y.; Zhao, L. F.; He, W. L.; Gao, Y. Q.; Wan,<br />
Y. J.; Xia, W.; Chen, T.; Zheng, H.; Li, M.; Xu, S. Q. Anal. Biochem. 2008,<br />
376, 183–188.<br />
(17) Nelson, P. T.; Baldwin, D. A.; Scearce, L. M.; Oberholtzer, J. C.; Tobias,<br />
J. W.; Mourelatos, Z. Nat. Methods 2004, 1, 155–161.<br />
(18) Cissell, K. A.; Rahimi, Y.; Shrestha, S.; Hunt, E. A.; Deo, S. K. Anal. Chem.<br />
2008, 80, 2319–2325.<br />
(19) Cheng, Y.; Zhang, X.; Li, Z.; Jiao, X.; Wang, Y.; Zhang, Y. Angew. Chem.,<br />
Int. Ed. 2009, 48, 3268–3272.<br />
(20) Lim, L. P.; Lau, N. C.; Weinstein, E. G.; Abdelhakim, A.; Yekta, S.; Rhoades,<br />
M. W.; Burge, C. B.; Bartel, D. P. Genes Dev. 2003, 17, 991–1008.<br />
(21) Joo, C.; Balci, H.; Ishitsuka, Y.; Buranachai, C.; Ha, T. Annu. Rev. Biochem.<br />
2008, 77, 51–76.<br />
(22) Weiss, S. Science 1999, 283, 1676–1683.<br />
(23) Li, H. W.; Yeung, E. S. Anal. Chem. 2005, 77, 4374–4377.<br />
(24) Li, J. W.; Yeung, E. S. Anal. Chem. 2008, 80, 8509–8513.<br />
(25) Kang, S. H.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2001, 73, 1091–<br />
1099.<br />
(26) Li, H. W.; Park, H. Y.; Porter, M. D.; Yeung, E. S. Anal. Chem. 2005, 77,<br />
3256–3260.<br />
(27) Kang, S. H.; Kim, Y. J.; Yeung, E. S. Anal. Bioanal. Chem. 2007, 387,<br />
2663–2671.<br />
(28) Gunnarsson, A.; Jonsson, P.; Marie, R.; Tegenfeldt, J. O.; Hook, F. Nano<br />
Lett. 2008, 8, 183–188.<br />
(29) Cohen, A. E.; Moerner, W. E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,<br />
12622–12627.<br />
(30) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995,<br />
374, 555–559.<br />
(31) Chan, E. Y.; Goncalves, N. M.; Haeusler, R. A.; Hatch, A. J.; Larson, J. W.;<br />
Maletta, A. M.; Yantz, G. R.; Carstea, E. D.; Fuchs, M.; Wong, G. G.; Gullans,<br />
S. R.; Gilmanshin, R. Genome Res. 2004, 14, 1137–1146.<br />
(32) Xiao, M.; Phong, A.; Ha, C.; Chan, T. F.; Cai, D. M.; Leung, L.; Wan, E.;<br />
Kistler, A. L.; DeRisi, J. L.; Selvin, P. R.; Kwok, P. Y. Nucleic Acids Res.<br />
2007, 35, e16.<br />
(33) Tessler, L. A.; Reifenberger, J. G.; Mitra, R. D. Anal. Chem. 2009, 81, 7141–<br />
7148.<br />
(34) Lee, J. Y.; Li, J. W.; Yeung, E. S. Anal. Chem. 2007, 79, 8083–8089.<br />
(35) Li, J. W.; Lee, J. Y.; Yeung, E. S. Anal. Chem. 2006, 78, 6490–6496.<br />
(36) Neely, L. A.; Patel, S.; Garver, J.; Gallo, M.; Hackett, M.; McLaughlin, S.;<br />
Nadel, M.; Harris, J.; Gullans, S.; Rooke, J. Nat. Methods 2006, 3, 41–46.<br />
6912 <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
amplification, but continuous sample flow is needed for improvement<br />
in sensitivity. This microfluidic-assisted fluorescence correlation<br />
spectroscopy platform is also comparatively sophisticated.<br />
In this article, we present a quantitative single-molecule<br />
detection of miRNAs using total internal reflection fluorescence<br />
microscopy (TIRFM). For the proof of concept, we have chosen<br />
hsa-miR-21 (miR-21) as our detection target. The miR-21 is known<br />
as one of the most significant miRNAs elevated in at least six types<br />
of cancers including breast, colon, lung, pancreas, prostate, and<br />
stomach cancers. 7 Studies have indicated the miR-21 mediated<br />
tumor growth by serving as an oncogene 37 and targeting the<br />
tumor suppressor genes such as TPM1 and PDCD 4 in invasion<br />
and metastasis. 38-40 Compared to the conventional methods, the<br />
developed assay here is straightforward because no pretreatment<br />
steps are involved. Both the probe and target oligonucleotides<br />
are free of chemical modifications. The YOYO-1 labeled miRNA<br />
hybrids diffuse freely on unmodified coverslips and are monitored<br />
by electron-multiplying charge-coupled device (EMCCD) under<br />
TIRFM. TIRFM is a highly sensitive microscopic technique that<br />
has been used for SMD in solution. The total internal reflection<br />
(TIR) generates evanescent field layer that has a penetration depth<br />
of about 100-300 nm depending on the incident angle of the<br />
excitation laser beam. The excitation of fluorophores is confined<br />
within the evanescent field layer such that background signal from<br />
the bulk is greatly suppressed. Herein, the diffusing hybrids are<br />
observed as single fluorescent spots when they enter the excitation<br />
volume and are excited. Image of fluorescent molecules are<br />
acquired for single-molecule counting. The counted number is<br />
found to be proportional to the quantity of miRNAs in bulk<br />
solution. The developed assay was also employed for the determination<br />
of miR-21 in normal and cancerous cell lines and the<br />
results were validated with that of qRT-PCR detection.<br />
EXPERIMENTAL SECTION<br />
Slide Pretreatment. All coverslips were prewashed prior to<br />
experiments. Briefly, No. 1 22-mm square cover glasses (Gold<br />
Seal, Electron Microscopy System, Hatfield, PA) were sequentially<br />
sonicated for 30 min in household detergent, 30 min in acetone<br />
(AR grade, Labscan), and 30 min in absolute ethanol. The slides<br />
were then successively soaked for 30 min in Piranha solution<br />
(H2SO4/30% H2O2) (v/v 1:1), rinsed with distilled water<br />
extensively, sonicated for 30 min in HCl/30% H2O2/H2O (v/<br />
v/v 1:1:1) solution, sonicated in distilled water for 15 min,<br />
further sonicated for 30 min in Piranha solution, and finally<br />
sonicated for 15 min in distilled water twice. The slides were<br />
stored in distilled water and blow-dried with nitrogen before<br />
use.<br />
Preparation of Hybridization Buffers. A1× Tris-NaCl-EDTA<br />
(TNE) buffer containing 20 mM pH 8.0 Tris-HCl (Invitrogen,<br />
Carlsbad, CA), 1 mM EDTA, and various concentration (0, 50,<br />
150, 250, and 500 mM) of sodium chloride was prepared with<br />
DEPC-treated water (Ambion, Austin, TX) accordingly as the<br />
(37) Si, M. L.; Zhu, S.; Wu, H.; Lu, Z.; Wu, F.; Mo, Y. Y. Oncogene 2007, 26,<br />
2799–2803.<br />
(38) Lu, Z.; Liu, M.; Stribinskis, V.; Klinge, C. M.; Ramos, K. S.; Colburn, N. H.;<br />
Li, Y. Oncogene 2008, 27, 4373–4379.<br />
(39) Zhu, S. M.; Si, M. L.; Wu, H. L.; Mo, Y. Y. J. Biol. Chem. 2007, 282, 14328–<br />
14336.<br />
(40) Zhu, S. M.; Wu, H. L.; Wu, F. T.; Nie, D. T.; Sheng, S. J.; Mo, Y. Y. Cell<br />
Res. 2008, 18, 350–359.