Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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micellar electrokinetic capillary chromatography with laserinduced fluorescence detection (MEKC-LIF) analysis. An intact cell was then introduced into a narrow-bore capillary and lysed therein. Subsequently, the R123 and OH-TPP-E + contents released from each cell were analyzed by MEKC-LIF. 7 Since both R123 and TPP-HE accumulate into mitochondria according to the mitochondrial membrane potential, the mole ratio of OH- TPP-E + /R123 is approximately proportional to the steady state superoxide concentration in the mitochondrial matrix of individual cells with polarized mitochondria. Thus, determination of this ratio make it possible to quantitate superoxide in single cells. In the future, this method could be used for investigating the role of superoxide in xenobiotic toxicity, aging, and disease. THEORY When cells are incubated with TPP-HE and R123, both probes accumulate in their mitochondria according to the mitochondrial membrane potential (∆ψ). 5,14 The relevant Nernst equations are Thus ∆ψ )- RT nF ln [TPP-HE] inside [TPP-HE] outside ∆ψ )- RT nF ln [R123] inside [R123] outside [TPP-HE] inside ) [R123] inside [TPP-HE] outside [R123] outside where [TPP-HE]inside and [R123]inside, and [TPP-HE]outside and [R123]outside are the concentrations of TPP-HE and R123 in the mitochondrial matrix and outside the mitochondria, respectively. In this study, [TPP-HE]outside is 10 µM and [R123]outside is 50 nM. The reaction of TPP-HE with superoxide and its relevant kinetics equations are k - TPP-HE + O2 f d[OH-TPP-E + ] dt OH-TPP-E + [OH-TPP-E + ] T ) k[TPP-HE]∫ 0 (1) (2) (3) - ) k[TPP-HE][O2 ] (4) T - [O2 ]dt (5) where k is the kinetic rate of the reaction of TPP-HE with superoxide (∼4 × 10 6 M -1 s -1 ), 5 T is the incubation time of the cells with TPP-HE (60 min), and ∫0 T [O2 - ]dt is the integral of steady state superoxide concentration over the entire incubation time. If [O2 - ] is constant during the incubation time, T - - ∫ [O 0 2 ]dt ) [O2 ]averageT (6) (14) Scaduto, R. C.; Grotyohann, L. W. Biophys. J. 1999, 76, 469. 6746 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 where [O2 - ]average is the average steady state superoxide concentration over the length of the incubation time. When a cell is lysed and analyzed by MEKC-LIF, the detected OH-TPP-E + is the total from both the matrix and outside the mitochondria of the cell. 7 Thus, the amount of OH-TPP-E + in each individual cell is m OH-TPP-E+ ) m OH-TPP-E+, inside + m OH-TPP-E+, outside where mOH-TPP-E + ,inside and mOH-TPP-E + ,outside are the amounts of OH- TPP-E + in the mitochondrial matrix and outside the mitochondria of a given cell, respectively. On the basis of the results from bulk analysis (Supporting Information, Part I; Figure S-1), the mOH-TPP-E + ,outside is a small fraction of the mOH-TPP-E + ,inside under basal conditions and upon treatments with rotenone and antimycin A. To simplify eq 7, the amount of OH-TPP-E + in one cell can be expressed as m OH-TPP-E+ ) (1 + x)m OH-TPP-E+, inside where x is the fraction of mOH-TPP-E + ,outside relative to mOH-TPP-E + ,inside. This fraction “x” represent the bias of the measurement due to the superoxide found outside the mitochondria. On the basis of bulk measurements, x is ∼0.048 under basal conditions, ∼0.063 upon treatment with rotenone, and ∼0.071 upon treatment with antimycin A (Supporting Information, Part I). Note that eq 8 is not applicable when mitochondria are depolarized by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment (Supporting Information, Part II). With the combination of eqs 5, 6, and 8, m ) OH-TPP-E+ (1 + x)k[TPP-HE] outside - mR123 [O2 ]average,insideT [R123] outside (9) where [O2 - ]average,inside is the average of steady state superoxide concentration in the mitochondrial matrix of single cells over the incubation time. On the basis of the MEKC-LIF calibration curves of OH-TPP- E + and R123, the following equations are yielded and (7) (8) m OH-TPP-E+ ) aA OH-TPP-E+ + m (10) m R123 ) bA R123 + n (11) where the respective slopes are a and b, and the respective intercepts are m and n, and the respective peak areas in the electropherograms are AOH-TPP-E + and AR123. Thus, - [O2 ]average,inside ) K 1 (aA + m) OH-TPP-E+ (1 + x) (bAR123 + n) (12) where K ) ([R123]outside)/(k[TPP-HE]outsideT) ≈ 3.47 × 10 -13 M. EXPERIMENTAL SECTION Chemicals. Rhodamine 123 (R123), TPP-HE (aka MitoSOX Red), and tetramethylrhodamine methyl ester (TMRM) were
purchased from Invitrogen-Molecular Probes (Eugene, OR). OH- TPP-E + was synthesized and purified following a previously described procedure. 15 Its purity was verified by MEKC-LIF. Its concentration was determined spectrophotometrically using the reported molar extinction coefficient at 478 nm. 5,15 OH- TPP-E + is stable for at least 1 month when stored at -20 °C. 15 All the other reagents were obtained from Sigma-Aldrich (St. Louis, MO). The MEKC running buffer contained 10 mM sodium borate and 1 mM cetyltrimethylammonium bromide (CTAB) (pH 9.3). All buffers were made with Milli-Q deionized water and filtered through 0.22 µm filters before use. Cell Culture. Rat muscle L6 myoblast cell line was obtained from the American Tissue Culture Collection (Manassas, VA). The cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% (v/v) calf serum and 10 µg/mL gentamicin at 37 °C and 5% CO2. The cells were maintained by splitting them every 3-4 days. Cell Treatments. The cultured myoblasts were incubated with 10 µM TPP-HE and 50 nM R123 in DMEM at 37 °C for 1 h. When needed, before incubation in the presence of TPP-HE and R123, cells were treated with either 1 mM tiron, 50 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 5 µM rotenone, or 5 µM antimycin A. After treatments, the cells were washed two times with DMEM, trypsinized, and resuspended in PBS for either whole cell lysate or single cell analysis. MEKC-LIF. To do the whole cell lysate analysis, ∼2 × 10 6 cells in PBS were centrifuged down at 600g for 5 min. The cell pellet was then dissolved in running buffer, treated with 2 mg/ mL proteinase K and 400U/mL DNase 1, and analyzed by MEKC-LIF following previously described procedures. 7 Briefly, separations were carried out at -400 V/cm in MEKC running buffer. 7 The 488 nm line (12 mW) of an argon-ion laser (Melles Griot, Irvine, CA) was used for excitation, and fluorescence was selected with a bandpass filter transmitting in the 607-662 nm range (Omega Optical, Brattleboro, VT). To do single-cell analysis, a cell was injected into a 150 µm o.d., 30 µm i.d. fused silica capillary (Polymicro Technologies, Phoenix, AZ) following previously reported procedures. 16,17 After the cell lysis in the capillary, the MEKC-LIF procedure was done as described for bulk analysis. Upon completion of the separation, the capillary was washed for 2 min with 0.1 M NaOH and 5 min with MEKC running buffer between runs. Separate calibration curves for OH-TPP-E + and R123, (area in electropherogram versus amount injected) were built by injecting standards of these compounds. The resulting calibration curves for OH-TPP-E + and R123 were m ) (122.4 ( 0.4)A + (1.0 ( 0.6); OH-TPP-E+ OH-TPP-E+ (R 2 ) 0.99) (13) mR123 ) (12.9 ( 0.0)AR123 + (0.3 ( 0.1); (R 2 ) 0.99) (14) where m is given in attomoles, A is the peak area in the electropherogram, and the errors are standard deviations. These (15) Zielonka, J.; Vasquez-Vivar, J.; Kalyanaraman, B. Nat. Protoc. 2008, 3, 8. (16) Krylov, S. N.; Starke, D. A.; Arriaga, E. A.; Zhang, Z.; Chan, N. W.; Palcic, M. M.; Dovichi, N. J. Anal. Chem. 2000, 72, 872. (17) Anderson, A. B.; Gergen, J.; Arriaga, E. A. J. Chromatogr., B: Anal. Technol Biomed. Life Sci. 2002, 769, 97. Figure 1. Electropherograms of TPP-HE oxidation products and R123 in the whole cell lysate (∼2 × 10 6 myoblasts) and the blank buffer. Separations were performed in a 42 cm-long capillary at -400 V/cm in MEKC running buffer. The 488 nm line of an argon-ion laser was used for excitation, and a 607-663 nm bandpass filter was used for detection. Traces have been offset vertically for clarity. The respective electrophoretic mobilities of OH-TPP-E + and R123 were -(6.89 ( 0.11) × 10 -4 and -(6.41 ( 0.09) × 10 -4 cm 2 V -1 s -1 (n ) 3). Separation efficiencies were calculated to be ∼260 000 for OH- TPP-E + and ∼31 000 for R123. equations were used to carry out calculations described by the eqs 10 and 11. The limits of detection (LOD at signal/noise ) 3) were ∼2 amol for OH-TPP-E + and ∼0.2 amol for R123. It would be possible to obtain a better LOD (e.g., zeptomole levels) for R123 by using a different bandpass filter (e.g., centered at 530 nm). This alternative was not pursued here. Data Analysis. The MEKC electropherograms were analyzed using Igor Pro 5.0 software (Wavemetrics, Lake Oswego, OR). Superoxide concentrations in single myoblasts under each condition were reported as mean ± standard deviation (SD). A student’s t test was used to determine statistical significance of the data, with p values of
Page 1: Anal. Chem. 2010, 82, 6745-6750 Let
Page 5 and 6: Figure 3. Electropherograms of TPP-
Page 7 and 8: Anal. Chem. 2010, 82, 6751-6755 Res
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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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
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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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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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