Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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of OH-TPP-E + in the whole cell lysate (cf. eq 13), on average each cell contains ∼7.7 amol. However, this bulk analysis is biased because it ignores that cells do not necessarily have the same membrane potential. 18-20 It is important to bear in mind that superoxide dismutases (SODs) are present in the matrix (Mn-SOD) and outside the mitochondria (Cu, Zn-SOD). 5 In general SODs transform superoxide into hydrogen peroxide (k ∼ 2 × 10 9 M -1 s -1 ), 21-23 outcompeting the relatively slow reaction of superoxide with the TPP-HE probe (i.e., k ∼ 4 × 10 6 M -1 s -1 ). 5 However, in the mitochondrial matrix, the mitochondrial membrane potential increases the TPP-HE concentration ∼100-fold relative to the cytosol 7 that partially compensates for its relative slow reaction rate constant with superoxide. The concentration of Mn-SOD in the matrix has been reported to be ∼20 µM. 24 On the basis of this value and the kinetic rate constants of TPP-HE and Mn- SOD with superoxide (and assuming that there are no other reactions consuming superoxide), the estimated rate of OH- TPP-E + accumulation is ∼10% of the formation rate of H2O2 by Mn-SOD. This simplified kinetic analysis argues that, at this relative rate of formation, the detected amounts of OH-TPP- E + are adequate to calculate [O2 - ]average,inside (eq 12), even in the presence of competing Mn-SOD. On the other hand, outside the mitochondria there is no enhanced accumulation of TPP-HE. Given a concentration of Cu, Zn-SOD ∼6 µM, 25 the rate of OH-TPP-E + accumulation outside the mitochondria is only ∼0.3% of the rate of H2O2 formed by Cu, Zn-SOD. This calculation further confirms that OH-TPP- E + produced outside the mitochondria does not contribute significantly to the total OH-TPP-E + detected (i.e., “x” ineq8 is small; cf. Supporting Information, Part I). Single Cell Superoxide Quantitative Analysis. We used chemical cytometry to analyze mitochondrial matrix superoxide levels in single cells (Figure 2). In order to correct for variations in mitochondrial membrane potentials observed among individual cells, cells were treated with TPP-HE and R123. The MEKC-LIF separation and detection of OH-TPP-E + and R123 in single myoblasts provides the measurements needed to determine superoxide at the single-cell level (cf. eq 12). The detection of mitochondrial matrix superoxide at the single cell level was further confirmed by treatment with tiron and CCCP. Tiron is an SOD mimetic that scavenges intracellular superoxide in cultured cells. 6,26 As expected, OH-TPP-E + was not detected in single myoblasts treated with tiron (Figure 2). The R123 peak was still detected showing that tiron treatment does not compro- (18) Johnson, L. V.; Walsh, M. L.; Bockus, B. J.; Chen, L. B. J. Cell Biol. 1981, 88, 526. (19) Ludovico, P.; Sansonetty, F.; Corte-Real, M. Microbiology 2001, 147, 3335. (20) Heerdt, B. G.; Houston, M. A.; Augenlicht, L. H. Cancer Res. 2005, 65, 9861. (21) Klug, D.; Rabani, J.; Fridovich, I. J. Biol. Chem. 1972, 247, 4839. (22) Pick, M.; Rabani, J.; Yost, F.; Fridovich, I. J. Am. Chem. Soc. 1974, 96, 7329. (23) Behar, D.; Czapski, G.; Rabani, J.; Dorfman, L. M.; Schwarz, H. A. J. Phys. Chem. 1970, 74, 3209. (24) Quijano, C.; Hernandez-Saavedra, D.; Castro, L.; McCord, J. M.; Freeman, B. A.; Radi, R. J. Biol. Chem. 2001, 276, 11631. (25) Chang, L. Y.; Slot, J. W.; Geuze, H. J.; Crapo, J. D. J. Cell Biol. 1988, 107, 2169. (26) Yamada, J.; Yoshimura, S.; Yamakawa, H.; Sawada, M.; Nakagawa, M.; Hara, S.; Kaku, Y.; Iwama, T.; Naganawa, T.; Banno, Y.; Nakashima, S.; Sakai, N. Neurosci. Res. 2003, 45, 1. 6748 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 2. Electropherograms of TPP-HE oxidation products and R123 in individual myoblasts under basal conditions and upon treatments with tiron and CCCP. Separations and detection conditions were same to those described for Figure 1. mise the mitochondrial membrane potential. CCCP is a proton ionophore that dissipates the mitochondrial membrane potential. 27,28 Upon CCCP treatment both R123 and TPP-HE concentrations in the matrix decrease ∼100-fold relative to untreated cells (Supporting Information, Parts II and III). The expected R123 amount in a CCCP-treated cell is ∼0.2 amol of R123 (Supporting Information, Part III), which is undetectable in the MEKC-LIF system used in this study (i.e., limit of detection ∼0.2 amol of R123). Furthermore, the low TPP-HE concentration in CCCP-treated cells significantly decreases the rate of formation of OH-TPP-E + inside the mitochondria (cf. eq 5; Supporting Information, Part II). The expected amount of OH-TPP-E + formed in a CCCP-treated myoblast is ∼0.4 amol, which is undetectable in the MEKC- LIF that was used in this study (i.e., limit of detection ∼2 amol of OH-TPP-E + ). Thus, the concomitant disappearance of the R123 and the OH-TPP-E + peaks in CCCP-treated myoblasts (Figure 2) demonstrates that the mitochondrial membrane potential is vital to the accumulation of TPP-HE and formation of OH-TPP-E + in the mitochondrial matrix. 5 On the basis of fluorescence microscopy, individual myoblasts display ∼39% variation in mitochondrial membrane potential (Supporting Information, Part V; Figure S-3). In MEKC-LIF, the ratio of the areas of OH-TPP-E + and R123 corrects for variations in mitochondrial membrane potential between individual cells (cf. eq 12). This calculation is based on the assumptions that both R123 and TPP-HE accumulate into the mitochondria according to a Nernstian behavior (cf. eqs 1 and 2) and that the amount of superoxide product outside the mitochondria is only a small fraction of that in the mitochondrial matrix (cf. eqs 7 and 8; Supporting Information, Part I). On the basis of MEKC-LIF, the steady state superoxide concentration in the mitochondrial matrix of single myoblasts under basal conditions was ∼(0.29 ± 0.10) × 10 -12 M with a RSD of 35% (n ) 12) (Figure 4). This variance is statistically different from that obtained if the signal of OH- (27) Heytler, P. G.; Prichard, W. W. Biochem. Biophys. Res. Commun. 1962, 7, 272. (28) Lim, M. L.; Minamikawa, T.; Nagley, P. FEBS Lett. 2001, 503, 69.
Figure 3. Electropherograms of TPP-HE oxidation products and R123 in individual myoblasts under basal conditions and upon treatments with rotenone and antimycin. Separations and detection conditions were same to those described for Figure 1. TPP-E + is not corrected by membrane potential (RSD ∼69%) (F-test: p < 0.05). In summary, the simultaneous monitoring of OH-TPP-E + and R123 provides a correction for variations in membrane potential and provides the means to quantitate mitochondrial matrix superoxide levels in single cells. Effects of Mitochondrial Respiratory Inhibitors. The mitochondria is considered a major source of superoxide in cells, where superoxide is mainly released by complex I and III in the mitochondrial electron transport chain (ETC). 1,2 Rotenone and antimycin A are two respiratory inhibitors that block electron transfer through complex I and III, respectively, thereby stimulating superoxide production. 29,30 Although recent studies have utilized the TPP-HE probe to investigate the effects of rotenone and antimycin A on intracellular superoxide release in single cells by fluorescent microscopy and/or flow cytometry, 5,10-12 these studies are qualitative because they have not considered the effect of mitochondrial membrane potential. Here we demonstrated that chemical cytometry is suitable to quantify mitochondrial matrix superoxide levels in individual myoblasts upon their treatment with either rotenone or antimycin A (Figure 3). Some studies have reported such inhibitors at elevated concentrations may alter the membrane potential of isolated mitochondria. 31-34 In this report, bulk analyses showed that the treatments with rotenone and antimycin A did appear to alter the mitochondria membrane potentials because there were no significant changes in the ratio of mOH-TPP-E + ,outside to mOH-TPP-E + ,inside compared to basal conditions (29) Grivennikova, V. G.; Vinogradov, A. D. Biochim. Biophys. Acta 2006, 1757, 553. (30) Muller, F. L.; Liu, Y. H.; Van Remmen, H. J. Biol. Chem. 2004, 279, 49064. (31) Petit, P. X.; O’Connor, J. E.; Grunwald, D.; Brown, S. C. Eur. J. Biochem. 1990, 194, 389. (32) Kataoka, M.; Fukura, Y.; Shinohara, Y.; Baba, Y. Electrophoresis 2005, 26, 3025. (33) Tzung, S. P.; Kim, K. M.; Basanez, G.; Giedt, C. D.; Simon, J.; Zimmerberg, J.; Zhang, K. Y.; Hockenbery, D. M. Nat. Cell Biol. 2001, 3, 183. (34) De Bari, L.; Atlante, A.; Guaragnella, N.; Principato, G.; Passarella, S. Biochem. J. 2002, 365, 391. Figure 4. Effects of rotenone and antimycin A on the superoxide levels detected in the mitochondrial matrix of single myoblasts. Data of individual cells as well as means ( SD are presented for each condition (n ) 12 cells for basal, 10 for rotenone, and 12 for antimycin A). * stands for p < 0.05 vs basal. (Supporting Information, Part I). Thus, eq 12 is adequate to determine mitochondrial matrix superoxide levels following inhibitor treatments. In single myoblasts treated with rotenone or antimycin A, the steady state superoxide concentrations in the mitochondrial matrix were (0.97 ± 0.61) × 10 -12 M(n ) 10) and (2.15 ± 1.20) × 10 -12 M(n ) 12), respectively (Figure 4). These values are ∼3.5- and 8.2-fold higher compared to basal levels. The magnitude of such changes are comparable with the ∼2 to 3-fold change observed in aged cells or cells associated with diabetic hyperglycemia relative to their respective controls. 35-37 Therefore, the use of inhibitors demonstrates that chemical cytometry is a suitable approach to quantitate mitochondrial superoxide levels expected in aging and disease related studies. CONCLUDING REMARKS Here we report the quantitation of mitochondrial matrix superoxide levels in single cells using a MEKC-based chemical cytometry method that is not biased by the mitochondrial membrane potential. On the basis of eq 12, the ratio of OH- TPP-E + and R123 signals corrects for variation in membrane potential of mitochondria among individual cells. According to this method, the mitochondrial matrix superoxide levels of single myoblasts are in the picomolar range. The method could also be extended to investigate other cellular systems such as cultured single skeletal muscle fibers, 38,39 whose superoxide levels were analyzed qualitatively in our earlier (35) Chen, H.; Cangello, D.; Benson, S.; Folmer, J.; Zhu, H.; Trush, M. A.; Zirkin, B. R. Exp. Gerontol. 2001, 36, 1361. (36) Klamt, F.; Gottfried, C.; Tramontina, F.; Dal-Pizzol, F.; Da Frota, M. L., Jr.; Moreira, J. C.; Dias, R. D.; Moriguchi, E.; Wofchuk, S.; Souza, D. O. Neuroreport 2002, 13, 1515. (37) Nishikawa, T.; Edelstein, D.; Du, X. L.; Yamagishi, S.-i.; Matsumura, T.; Kaneda, Y.; Yorek, M. A.; Beebe, D.; Oates, P. J.; Hammes, H.-P.; Giardino, I.; Brownlee, M. Nature 2000, 404, 787. (38) Pye, D.; Palomero, J.; Kabayo, T.; Jackson, M. J. J. Physiol. 2007, 581, 309. (39) Palomero, J.; Pye, D.; Kabayo, T.; Spiller, D. G.; Jackson, M. J. Antioxid. Redox Signaling 2008, 10, 1463. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6749
Page 1 and 2: Anal. Chem. 2010, 82, 6745-6750 Let
Page 3: purchased from Invitrogen-Molecular
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Page 9 and 10: (pH 8.0) with cysteine and cystamin
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
Page 15 and 16: coater at 2000 rpm for 20 s, and th
Page 17 and 18: Figure 5. Comparison of cyclic volt
Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
Page 23 and 24: Figure 1. GC/MS total ion chromatog
Page 25 and 26: Table 2. Concentrations and Stable
Page 27 and 28: on a substrate are preferred. 20-24
Page 29 and 30: Figure 4. SERS analysis of NAADP co
Page 31 and 32: Anal. Chem. 2010, 82, 6775-6781 Hig
Page 33 and 34: tion, 2 µL of proprionaldehyde wer
Page 35 and 36: Figure 4. Analysis of 2a by HPLC-MS
Page 37 and 38: eaction of 1a with PBH can be condu
Page 39 and 40: Numerous references had demonstrate
Page 41 and 42: Figure 1. TEM images of the prepare
Page 43 and 44: Figure 4. Schematic representation
Page 45 and 46: esult in a big SPR signal change wi
Page 47 and 48: also reduces chemical noise, which
Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
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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
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data as well. The 41 genes in Table
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corresponding compound if its chemi
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Figure 1. Schematic illustration of
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Table 1. Absolute Quantification Re
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ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
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Figure 5. Device to eject and mix s
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Anal. Chem. 2010, 82, 6877-6886 Imm
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NaCl, phosphate buffer saline (PBS)
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Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
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Table 1. Intraday Precision and Acc
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Anal. Chem. 2010, 82, 6887-6894 Fer
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
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Anal. Chem. 2010, 82, 6895-6903 Ele
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Table 1. Chemical Structure, pKa Va
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pH with a tilted baseline (Figure 1
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Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
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the multielement capabilities, the
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RESULTS AND DISCUSSION Sulfur Detec
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Table 2. Molecular Properties and C
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Anal. Chem. 2010, 82, 6911-6918 Dir
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
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Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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Figure 5. Extracted ion current ESI
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m/z 1172.935 was observed for Ser14
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linked products via affinity tags.
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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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