Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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enzymatic amplification techniques such as RAKE assay, 17 bioluminescence-enzyme labeling, 18 and rolling-circle amplification (RCA) 19 are also reported for sensitivity enhancement. These reported detection methods bring great benefit and improvement to the sensitivity of miRNA profiling to certain extends. Nevertheless, sample pretreatment and the modifications may result in loss of samples throughout the multiple pretreatment steps such as sample enrichment, labeling, and purification. The detection sensitivity and reliability are hence hindered. Since cellular miRNA concentration can be as low as 1000 molecules per cell, 20 deficiencies in sensitivity may result in unsuccessful quantification of low-abundance miRNAs and, thus, false diagnostic result. Therefore, an ultrasensitive miRNA-profiling assay without the need of pretreatment is demanded. Single-molecule detection (SMD) technique has been widely applied in the behavioral study of individual biomolecules. 21,22 Scientific issues such as monitoring enzymatic kinetics, 23,24 DNA adsorption/desorption behavior, 25,26 verification of nucleic acid hybridization, 27 DNA mismatch discrimination, 28 protein and DNA conformation dynamics, 29,30 and DNA mapping 31,32 have successfully been accomplished with SMD. Besides, several groups have also demonstrated the competence of SMD in the quantitation of biomolecules such as proteins 33 and viral DNA 34,35 by singlemolecule counting. Recently, quantitation of miRNA in singlemolecule level was displayed by Neely and co-workers. 36 The novel miRNA detection assay is free of sample enrichment and (15) Liang, R. Q.; Li, W.; Li, Y.; Tan, C. Y.; Li, J. X.; Jin, Y. X.; Ruan, K. C. Nucleic Acids Res. 2005, 33. (16) Yang, W. J.; Li, X. B.; Li, Y. Y.; Zhao, L. F.; He, W. L.; Gao, Y. Q.; Wan, Y. J.; Xia, W.; Chen, T.; Zheng, H.; Li, M.; Xu, S. Q. Anal. Biochem. 2008, 376, 183–188. (17) Nelson, P. T.; Baldwin, D. A.; Scearce, L. M.; Oberholtzer, J. C.; Tobias, J. W.; Mourelatos, Z. Nat. Methods 2004, 1, 155–161. (18) Cissell, K. A.; Rahimi, Y.; Shrestha, S.; Hunt, E. A.; Deo, S. K. Anal. Chem. 2008, 80, 2319–2325. (19) Cheng, Y.; Zhang, X.; Li, Z.; Jiao, X.; Wang, Y.; Zhang, Y. Angew. Chem., Int. Ed. 2009, 48, 3268–3272. (20) Lim, L. P.; Lau, N. C.; Weinstein, E. G.; Abdelhakim, A.; Yekta, S.; Rhoades, M. W.; Burge, C. B.; Bartel, D. P. Genes Dev. 2003, 17, 991–1008. (21) Joo, C.; Balci, H.; Ishitsuka, Y.; Buranachai, C.; Ha, T. Annu. Rev. Biochem. 2008, 77, 51–76. (22) Weiss, S. Science 1999, 283, 1676–1683. (23) Li, H. W.; Yeung, E. S. Anal. Chem. 2005, 77, 4374–4377. (24) Li, J. W.; Yeung, E. S. Anal. Chem. 2008, 80, 8509–8513. (25) Kang, S. H.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2001, 73, 1091– 1099. (26) Li, H. W.; Park, H. Y.; Porter, M. D.; Yeung, E. S. Anal. Chem. 2005, 77, 3256–3260. (27) Kang, S. H.; Kim, Y. J.; Yeung, E. S. Anal. Bioanal. Chem. 2007, 387, 2663–2671. (28) Gunnarsson, A.; Jonsson, P.; Marie, R.; Tegenfeldt, J. O.; Hook, F. Nano Lett. 2008, 8, 183–188. (29) Cohen, A. E.; Moerner, W. E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12622–12627. (30) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995, 374, 555–559. (31) Chan, E. Y.; Goncalves, N. M.; Haeusler, R. A.; Hatch, A. J.; Larson, J. W.; Maletta, A. M.; Yantz, G. R.; Carstea, E. D.; Fuchs, M.; Wong, G. G.; Gullans, S. R.; Gilmanshin, R. Genome Res. 2004, 14, 1137–1146. (32) Xiao, M.; Phong, A.; Ha, C.; Chan, T. F.; Cai, D. M.; Leung, L.; Wan, E.; Kistler, A. L.; DeRisi, J. L.; Selvin, P. R.; Kwok, P. Y. Nucleic Acids Res. 2007, 35, e16. (33) Tessler, L. A.; Reifenberger, J. G.; Mitra, R. D. Anal. Chem. 2009, 81, 7141– 7148. (34) Lee, J. Y.; Li, J. W.; Yeung, E. S. Anal. Chem. 2007, 79, 8083–8089. (35) Li, J. W.; Lee, J. Y.; Yeung, E. S. Anal. Chem. 2006, 78, 6490–6496. (36) Neely, L. A.; Patel, S.; Garver, J.; Gallo, M.; Hackett, M.; McLaughlin, S.; Nadel, M.; Harris, J.; Gullans, S.; Rooke, J. Nat. Methods 2006, 3, 41–46. 6912 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 amplification, but continuous sample flow is needed for improvement in sensitivity. This microfluidic-assisted fluorescence correlation spectroscopy platform is also comparatively sophisticated. In this article, we present a quantitative single-molecule detection of miRNAs using total internal reflection fluorescence microscopy (TIRFM). For the proof of concept, we have chosen hsa-miR-21 (miR-21) as our detection target. The miR-21 is known as one of the most significant miRNAs elevated in at least six types of cancers including breast, colon, lung, pancreas, prostate, and stomach cancers. 7 Studies have indicated the miR-21 mediated tumor growth by serving as an oncogene 37 and targeting the tumor suppressor genes such as TPM1 and PDCD 4 in invasion and metastasis. 38-40 Compared to the conventional methods, the developed assay here is straightforward because no pretreatment steps are involved. Both the probe and target oligonucleotides are free of chemical modifications. The YOYO-1 labeled miRNA hybrids diffuse freely on unmodified coverslips and are monitored by electron-multiplying charge-coupled device (EMCCD) under TIRFM. TIRFM is a highly sensitive microscopic technique that has been used for SMD in solution. The total internal reflection (TIR) generates evanescent field layer that has a penetration depth of about 100-300 nm depending on the incident angle of the excitation laser beam. The excitation of fluorophores is confined within the evanescent field layer such that background signal from the bulk is greatly suppressed. Herein, the diffusing hybrids are observed as single fluorescent spots when they enter the excitation volume and are excited. Image of fluorescent molecules are acquired for single-molecule counting. The counted number is found to be proportional to the quantity of miRNAs in bulk solution. The developed assay was also employed for the determination of miR-21 in normal and cancerous cell lines and the results were validated with that of qRT-PCR detection. EXPERIMENTAL SECTION Slide Pretreatment. All coverslips were prewashed prior to experiments. Briefly, No. 1 22-mm square cover glasses (Gold Seal, Electron Microscopy System, Hatfield, PA) were sequentially sonicated for 30 min in household detergent, 30 min in acetone (AR grade, Labscan), and 30 min in absolute ethanol. The slides were then successively soaked for 30 min in Piranha solution (H2SO4/30% H2O2) (v/v 1:1), rinsed with distilled water extensively, sonicated for 30 min in HCl/30% H2O2/H2O (v/ v/v 1:1:1) solution, sonicated in distilled water for 15 min, further sonicated for 30 min in Piranha solution, and finally sonicated for 15 min in distilled water twice. The slides were stored in distilled water and blow-dried with nitrogen before use. Preparation of Hybridization Buffers. A1× Tris-NaCl-EDTA (TNE) buffer containing 20 mM pH 8.0 Tris-HCl (Invitrogen, Carlsbad, CA), 1 mM EDTA, and various concentration (0, 50, 150, 250, and 500 mM) of sodium chloride was prepared with DEPC-treated water (Ambion, Austin, TX) accordingly as the (37) Si, M. L.; Zhu, S.; Wu, H.; Lu, Z.; Wu, F.; Mo, Y. Y. Oncogene 2007, 26, 2799–2803. (38) Lu, Z.; Liu, M.; Stribinskis, V.; Klinge, C. M.; Ramos, K. S.; Colburn, N. H.; Li, Y. Oncogene 2008, 27, 4373–4379. (39) Zhu, S. M.; Si, M. L.; Wu, H. L.; Mo, Y. Y. J. Biol. Chem. 2007, 282, 14328– 14336. (40) Zhu, S. M.; Wu, H. L.; Wu, F. T.; Nie, D. T.; Sheng, S. J.; Mo, Y. Y. Cell Res. 2008, 18, 350–359.
dilution and hybridization buffer. The pH of the TNE buffers was adjusted to 7.4 by addition of 1 M HCl dropwisely. The buffer solution was then filtered through a 0.22 µm nylon membrane filter, autoclaved, and photobleached with UV-C lamp overnight prior to use. Preparation of Probe and Target MicroRNA Oligonucleotides. Commercial available LNA-modified oligonucleotide probe (miRCURYLNAmicroRNADetectionProbe,Productno:38102-00) (3′-TCAACATCAGTCTGATAAGCTA-5′) specific to hsa-miR-21 was purchased from Exiqon (Denmark). Two HPLC-purified synthetic DNA oligonucleotides having the complementary sequence to hsa-miR-21 (3′-TCAACATCAGTCTGATAAGCTA-5′) and the mature sequence of hsa-miR-214 (3′-ACAGCAGGCACA- GACAGGCAGU-5′) were custom-designed and obtained from Invitrogen (Hong Kong) as the DNA probe and the negative control of the experiments. Two Anti-miR miRNA inhibitors of miR-21 (AM17000, Product ID: AM10206, 3′-CAACACCAGUC- GAUGGGCUGU-5′), and anti-miR-21 (AM17000, Product ID: AM12979, 3′-UAGCUUAUCAGACUGAUGUUGA-5′), were purchased from Ambion, acting as the RNA probe and target miR-21 strands, respectively. All oligonucleotides were suspended in 1 µL of DEPC-treated water (Ambion) and further diluted into appropriate concentration with 1× TNE buffer. The melting temperatures (Tm) of the oligonucleotides were predicted using the Probe Tm Predictor accessible online (www.exiqon.com) based on the thermodynamic nearest neighbor model. Optimization of Hybridization Conditions. Ionic Strength. For the determination of optimum hybridization ionic strength, sodium chloride concentrations from 0 to 500 mM in the TNE buffer were used throughout the oligonucleotides dilution and hybridization mixture preparation. Selection of Probe. For the selection of optimal probe with the best hybridization affinity toward miRNA, probes of DNA, RNA and LNA were prepared and hybridized with target miR-21 as described below. In addition, two control experiments including probes only and negative control miR-214 were also performed. Hybridization Time. For the determination of optimum hybridization time, hybrids of same concentration and ionic strength (250 mM NaCl) were incubated for 15 min, 30 min, 1 and 3 h respectively. Hybridization and Labeling of MicroRNA. The hybridization and fluorescence labeling of miRNAs were performed according to the following procedures. LNA probe, DNA probe, RNA probe, miRNA target, and DNA negative control oligonucleotides were diluted with 1× TNE buffers (pH 7.4) to 300 pM in concentration, respectively. The hybridization cocktail contained 15 µL of 300 pM probe strand, 15 µL of appropriate concentration of target miR-21 strand, and 14 µL of TNE buffer. The cocktail was incubated in form of free-diffusing solutions in dry bath (AccuBlock Digital Dry Bath D1100, Labnet, NJ) for 1 h. The hybridization temperature was set to be 20 °C below the T m of the probe, that is, 52, 47, and 36 °C for LNA, DNA, and RNA probe, respectively. After incubation, 1 µL of 100 nM YOYO-1 Iodide (YOYO) (Invitrogen) was added subsequently to label the hybrids. YOYO labels the hybrids in a dye to base pair (dye/bp) ratio of 1:1. The mixture was kept for 5 min in order to achieve equilibrium and 10 µL of solution was pipetted to the precleaned coverslips for TIRF imaging. External Calibration of MicroRNA. A calibration curve was established to correlate number of detected single fluorescent spots and concentration of miRNAs. Synthetic miRNA with final concentration of 100, 75, 50, 25, 10, 5, and 1 pM target miR-21 strand was hybridized with probe strand of final concentration of 100 pM under optimal conditions as mentioned above. Cell Culture. Human umbilical vein endothelial cells (HU- VEC) were purchased from Lonza (Walkersville, MD). M199 medium, heparin, gelatin, and endothelial cell growth supplement (ECGS) were purchased from Sigma. Fetal bovine serum (FBS) and penicillin-streptomycin (PS) were purchased from Invitrogen. Dulbecco’s modified Eagle medium (DMEM) and Roswell Park Memorial Institute medium (RPMI) were purchased from Gibco (Grand Island, NY). HUVEC were grown in M199 medium supplemented with 20% heat-inactivated FBS, 20 µg/mL ECGS, 90 µg/mL heparin, 1% PS in 75 cm 2 culture flasks coated with 0.1% gelatin. HUVEC from passage 2-6 were used in this study. In addition, human hepatocellular carcinoma (HepG2) was cultured in DMEM supplemented with 10% FBS and 1% PS, and invasive breast ductal carcinoma (MCF-7) was cultured in RPMI supplemented with 10% FBS and 0.5% PS. All cells were maintained in a humidified incubator at 37 °C with 5% CO2 and 80% relative humidity. Total RNA Isolation. Total RNA of HUVEC, HepG2, and MCF-7 were extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. Briefly, the sample was lysed and homogenized with TRIzol Reagent. Phase separation was followed by addition of chloroform and centrifugation. RNA in the aqueous phase was then recovered by precipitation with isopropyl alcohol. The RNA pellet was washed with 75% ethanol and finally redissolved in RNase-free water. The RNA quantity was determined by measuring optical density at 260 nm using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and the RNA quality was assessed by performing agarose gel electrophoresis. Quantification of miR-21 in Cells by Quantitative Real- Time PCR (qRT-PCR). Complementary DNA (cDNA) was generated from 10 ng of total RNA per 5 µL of gene specific reverse transcription (RT) reaction by using reagents from TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and mature hsa-miR-21-specific RT primer (5×) from TaqMan MicroRNA Assays (P/N: 4373090, Applied Biosystems). Each RT reaction contained 1 mM dNTPs, 50 U MultiScribe reverse transcriptase, 1× reverse transcription buffer, and 3.8 U RNase inhibitor as well as 1× specific RT primer. The reaction mixture was incubated in PTC-100 Programmable Thermal Controller (MJ Research, Inc., Waltham, MA) for 30 min at 16 °C, 30 min at 40 °C, 5 min at 85 °C, and then held at 4 °C. The cDNA sample was then amplified by PCR using TaqMan 2× Universal PCR Master Mix (No AmpErase UNG) (Applied Biosystems) and TaqMan Assay (20×) from TaqMan MicroRNA Assays (P/N: 4373090, Applied Biosystems). Each PCR reaction included 0.67 µL of RT product, 5 µL of TaqMan 2× Universal PCR Master Mix, No AmpErase UNG, and 0.5 µLof20× TaqMan Assay (a mix preformulated miRNA-specific forward PCR primer, specific reverse PCR primer and miRNA-specific TaqMan MGB probe). The reaction mixture was then run at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min in Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6913
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Anal. Chem. 2010, 82, 6745-6750 Let
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purchased from Invitrogen-Molecular
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Figure 3. Electropherograms of TPP-
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Anal. Chem. 2010, 82, 6751-6755 Res
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(pH 8.0) with cysteine and cystamin
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Figure 4. Ion mobility mass spectra
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GOx glucose + O298 gluconic acid +
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coater at 2000 rpm for 20 s, and th
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Figure 5. Comparison of cyclic volt
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in channels with either no grooves
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indicators of atmospheric processin
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Figure 1. GC/MS total ion chromatog
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Table 2. Concentrations and Stable
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on a substrate are preferred. 20-24
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Figure 4. SERS analysis of NAADP co
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Anal. Chem. 2010, 82, 6775-6781 Hig
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tion, 2 µL of proprionaldehyde wer
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Figure 4. Analysis of 2a by HPLC-MS
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eaction of 1a with PBH can be condu
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Numerous references had demonstrate
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Figure 1. TEM images of the prepare
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Figure 4. Schematic representation
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esult in a big SPR signal change wi
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also reduces chemical noise, which
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Table 1. Extraction Yields, Liquid
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scanning of AMPP amides of the anal
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Anal. Chem. 2010, 82, 6797-6806 δ
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Figure 1. Schematic view of the pre
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from the specimen and enclosed in a
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Figure 4. (A) IRMS mass-44 chromato
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Table 3. Ambient Measurement Result
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Anal. Chem. 2010, 82, 6807-6813 Dir
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Polymerase (1 U per sample). Reacti
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of which was constant for all ampli
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analytically useful signals at less
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carbon black and RP-C18 for the ext
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solution in an equal volume, and 1
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Table 2. Concentrations and Ratios
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Anal. Chem. 2010, 82, 6821-6829 Mac
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mg/mL protein, followed by separati
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Figure 2. Productivity of SEQUST an
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Figure 4. High-resolution MS/MS spe
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Figure 6. Characterization of PSMs
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containing T-T mismatches. 23 Based
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Figure 1. Extinction spectra of sol
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Figure 3. (A) The value of Ex650 nm
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Figure 5. Extinction spectra and co
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wished to explore the dehydration o
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constant medium for separation; we
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Figure 2. Standards of (Pi)n, n ) 1
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Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
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Figure 1. 226 Ra spectrum by liquid
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Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
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Figure 5. Volcano plots detailing t
Page 117 and 118: data as well. The 41 genes in Table
Page 119 and 120: corresponding compound if its chemi
Page 121 and 122: Figure 1. Schematic illustration of
Page 123 and 124: Table 1. Absolute Quantification Re
Page 125 and 126: ment. Therefore, the long-time drea
Page 127 and 128: Figure 1. Chemically actuated micro
Page 129 and 130: Figure 2. Influence of a surfactant
Page 131 and 132: Figure 5. Device to eject and mix s
Page 133 and 134: Anal. Chem. 2010, 82, 6877-6886 Imm
Page 135 and 136: NaCl, phosphate buffer saline (PBS)
Page 137 and 138: Figure 2. Product ion mass spectra
Page 139 and 140: -70 °C resolved the problem, givin
Page 141 and 142: Table 1. Intraday Precision and Acc
Page 143 and 144: Anal. Chem. 2010, 82, 6887-6894 Fer
Page 145 and 146: Figure 1. Infrared spectra of (A) u
Page 147 and 148: Figure 3. Cyclic voltammograms obta
Page 149 and 150: Figure 6. Calculated charge from ch
Page 151 and 152: Anal. Chem. 2010, 82, 6895-6903 Ele
Page 153 and 154: Table 1. Chemical Structure, pKa Va
Page 155 and 156: pH with a tilted baseline (Figure 1
Page 157 and 158: Table 2. Linearity and Detection Li
Page 159 and 160: ascorbic acid (AA), uric acid (UA),
Page 161 and 162: the multielement capabilities, the
Page 163 and 164: RESULTS AND DISCUSSION Sulfur Detec
Page 165 and 166: Table 2. Molecular Properties and C
Page 167: Anal. Chem. 2010, 82, 6911-6918 Dir
Page 171 and 172: solution under appropriate incubati
Page 173 and 174: Figure 4. Standardization curve for
Page 175 and 176: Anal. Chem. 2010, 82, 6919-6925 Ele
Page 177 and 178: the ×10 objective, to have a large
Page 179 and 180: Figure 2. With a suitable removal o
Page 181 and 182: the NB signal in a much better foot
Page 183 and 184: Scheme 1. Reactions of Selenium Rea
Page 185 and 186: Figure 2. (a) ESI-MS spectrum showi
Page 187 and 188: Figure 4. (a) ESI-MS spectrum showi
Page 189 and 190: Anal. Chem. 2010, 82, 6933-6939 Dif
Page 191 and 192: trode 28 by a finite element using
Page 193 and 194: Figure 4. Comparison between simula
Page 195 and 196: Figure 6. Comparison between simula
Page 197 and 198: educed in the vicinity of double bo
Page 199 and 200: Figure 2. Normalized product ion ab
Page 201 and 202: Figure 4. EID (a) and IRMPD (b) of
Page 203 and 204: Anal. Chem. 2010, 82, 6947-6957 Ide
Page 205 and 206: Figure 1. Schematic flowchart showi
Page 207 and 208: difference, ppm compound Table 1. I
Page 209 and 210: isoforms, its successful use, in th
Page 211 and 212: Figure 5. Extracted ion current ESI
Page 213 and 214: m/z 1172.935 was observed for Ser14
Page 215 and 216: linked products via affinity tags.
Page 217 and 218: Scheme 2. Fragmentation Mechanism o
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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Figure 5. (A) MALDI-TOF/TOF product
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
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allow the use of higher aptamer con
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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
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Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
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Table 2. Capillary Electrophoresis
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Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
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Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
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Anal. Chem. 2010, 82, 7015-7020 Sel
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Table 1. Effect of 1 D-LC Condition
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Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
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luciferase (T7 control vector) was
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Figure 3. Inhibitory effects of luc
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Anal. Chem. 2010, 82, 7027-7034 Pat
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Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
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Table 3. Film Thickness Measurement
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Anal. Chem. 2010, 82, 7035-7043 Qua
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Figure 1. (A) FL spectra of BSPOTPE
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Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
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Scheme 1. Proposed Mechanism for Fl
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one or more drawbacks including poo
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Figure 2. Free fluorophores do not
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Anal. Chem. 2010, 82, 7049-7052 Dev
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Figure 2. Comparative analysis of d
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