Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 2. (a) Gel electrophoresis of membrane proteins. Lane 1 is a protein ladder. Lanes 2-4 are for the negative control. Lanes 5-7 are for expression of bacteriorhodopsin. Lanes 8-10 are for expression of apolipoprotein. Lanes 11-13 are for coexpression of bacteriorhodopsin and apolipoprotein. The first lane in each group is for the reaction mixture; the second lane is for the pellet, and the third lane is for the supernatant. The arrow in lane 5 indicates the position of bacteriorhodopsin while the arrow in lane 8 indicates the position of apolipoprotein. (b) Western blotting of membrane proteins with the same lane designation as in (a). (c) Pictures of tubes containing expressed protein products (after centrifugation). They are in the same order as in (a) from the left to the right. consumption (10 µL versus 1 mL). The cost-savings would be substantial when a large number of arrays are required for highthroughput applications. The additional advantage of miniaturization is to enable a high-density array, which makes it possible to have high-throughput CFPS for applications such as drug screening. An alternative way to run high-throughput CFPS is in a conventional 96-well or 384-well microplate. Without a membrane in the conventional microplate, we found the synthesis yield was much lower. 20-22 For the membrane protein to be discussed below, coexpression of bacteriorhodopsin and apoA lipoprotein in the fluid array resulted in a mixture with the characteristic purple color of bacteriorhodopsin as shown in Figure 1c, demonstrating the functional production of bacteriorhodopsin. Note that the protein expression product in the device was transferred to a transparent tube for the purpose of taking a picture. In contrast, when the same expression was carried out in a conventional microplate, the amount of proteins expressed was too low to have an effect on the color of the mixture as shown in Figure 1d. The yellow color of the mixture is very similar to the negative control that contains no DNA vectors. Membrane Proteins. The fluid array device was used to produce two membrane-associated proteins, apoA lipoprotein and bacteriorhodopsin. ApoA lipoprotein can form nanoparticles due to the presence of lipid in the expression medium. 16 Its expression was confirmed by polyacrylamide gel electrophoresis (PAGE) as shown in Figure 2a (arrow in lane 8). Since the lipoprotein particle is soluble, it was in the supernatant after centrifugation (lane 10). Expression of bacteriorhodopsin in the fluid array was also confirmed by PAGE (arrow in lane 5). After centrifugation of the 7024 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 mixture, most bacteriorhodopsin molecules precipitated to form pellets since they were insoluble (lane 6). 15,16,30 Bacteriorhodopsin and ApoA lipoprotein could be coexpressed in a single reaction; the lipoprotein’s ability to form lipid bilayer patches from lipid micelles can be used to increase the solubility of other membrane-associated proteins such as bacteriorhodopsin. 15,16,30 The parallel expression of both proteins was confirmed by PAGE (lane 11). After centrifugation of the mixture, most bacteriorhodopsin molecules stayed in the supernatant due to the increased solubility in the presence of lipoprotein particles (lane 13). Since there are many protein bands belonging to the components of the protein expression system, we carried out Western blotting to verify the presence of bacteriorhodopsin and nanolipoprotein in the samples. The blot in Figure 2b shows much cleaner protein bands for the proteins of interest. Note that Western blotting in Figure 2b is more sensitive than Coomassie blue staining in Figure 2a; hence, a minute amount of bacteriorhodopsin undetectable in lane 7 in Figure 2a was shown as a band in Figure 2b. The effect of coexpressed lipoprotein on the solubility of bacteriorhodopsin is evident from the physical appearance of the protein product solutions at the end of expression, followed by centrifugation, as shown in Figure 2c. These proteins were expressed in the device, and the product mixtures were transferred to a tube, followed by centrifugation to separate soluble and insoluble materials for visualization. The negative control with no DNA vectors shows the color of the cell-free reaction mixture. The product mixture for expression of apoA lipoprotein is homogeneous since apoA lipoprotein is soluble. However, the product mixture for expression of bacteriorhodopsin contains a purple pellet and clear supernatant after centrifugation. This is in agreement of the result of gel electrophoresis in Figure 2a that bacteriorhodopsin is in the pellet. When two proteins were simultaneously coexpressed, the solubility of bacteriorhodopsin was increased in the presence of apoA lipoproteins. Importantly, the bacteriorhodopsin sample was purple, which indicates the formation of a functional protein in our device. Soluble Proteins. As we have reported previously, several soluble proteins can be expressed in the miniaturized fluid array device, including luciferase, green fluorescent protein, �-glucoronidase, alkaline phosphatase, �-lactamase, and �-galactosidase. 20-22 In this work, we chose two soluble proteins, luciferase and �-lactamase, to demonstrate the enzyme inhibition assay. Luciferase gene has been extensively used as a reporter gene for visualizing biomolecular processes in living animals, 27,31 and luciferase-based luminescence assays have been increasingly used for high-throughput drug screening. 32 As a result, we selected luciferase to verify if the protein expressed in the CFPS device can be used for enzyme inhibition assays without harvesting and purification from the protein expression mixture that contains a number of cofactors, enzymes, and others. We first studied the kinetics of luciferase assay reactions in the presence of an inhibitor, luciferin 6′-methyl ether (LME). LME is a known luciferase assay inhibitor and has been used by others in the (30) Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Arch. Biochem. Biophys. 2006, 450, 215–222. (31) Prescher, J. A.; Contag, C. H. Curr. Opin. Chem. Biol. 2010, 14, 80–89. (32) Fan, F.; Wood, K. V. Assay Drug Dev. Technol. 2007, 5, 127–136.
Figure 3. Inhibitory effects of luciferin 6′-methyl ether on the luciferase assay. (a) The luciferase assay reaction kinetics in the presence of various concentrations of inhibitors. (b) The inhibitory effects as a function of the inhibitor concentration. The decrease in percentage of the luminescence signal in the y-axis is relative to the positive control, in which no inhibitor was added. The lines are the best fit in the power relationship among the experimental data points. The error bars are invisible, as they are smaller than the data markers. literature. 27,28 The inhibitory assay was performed using luciferase synthesized in the miniaturized fluid array device; the expression product was directly used for the assay without any treatment. Figure 3a shows the reaction kinetic curves we obtained. The resemblance of these curves with those in the literature using purified luciferase 33 indicates that the enzyme synthesized in the cell-free medium can be used for the enzyme inhibition assay without harvesting and purification. As a result, we can use CFPS products directly for the enzyme inhibition assay. Figure 3b shows the inhibitory effects on the luciferase assay as a function of the LME concentration. In addition, we evaluated the specificity of each enzyme assay. For example, the UV absorbance-based enzyme assay of �-lactamase using m-{[(phenylacetyl)glycyl]oxy}benzoic acid (PBA) should be specific to �-lactamase. Figure 4 shows the assay kinetics, in which signal increased over a period of time when �-lactamase was cleaving the chromogenic substrate. It leveled off to form a plateau when the reactions reached equilibrium. However, when the same assay was applied to luciferase, there was negligible signal increase, except for the initial point that was due to the addition of a solution in a way similar to a negative control. These results suggest that the �-lactamase assay is specific. The data also indicate that reliable assay results for �-lactamase should be recorded at 5 min after the assay reagents were added into the expression products. Drug Screening. To demonstrate the utility of the miniaturized fluid array for drug screening, �-lactamase was chosen as a mock drug target. �-Lactamase is an enzyme that hydrolyzes the amide bond of �-lactam antibiotics, causing the antimicrobial agents to lose their effectiveness. Three known �-lactamase (33) DeLuca, M.; Wannlund, J.; McElroy, W. D. Anal. Biochem. 1979, 95, 194– 198. Figure 4. Enzyme assay kinetics of �-lactamase using a chromogenic agent, m-{[(phenylacetyl)glycyl]oxy}benzoic acid. The same assay was applied to luciferase, showing negligible increase over the background signal. Figure 5. Inhibition of clavulanate acid (open cirles), tazobactam (diamonds), and sulbactam (triangles) on the enzymatic activities of �-lactamase. The concentrations of these inhibitors in the x-axis are in the log scale. Also shown is negligible and concentrationindependent inhibition of cefotaxime (solid circles) on �-lactamase. Each data point represents an average obtained from three repeat experiments, and the error bars indicate one standard deviation. The chemical structures of all compounds tested are shown at the top; all of them have a �-lactam ring that consists of three carbon atoms and one nitrogen atom with a cyclic amide functional group. inhibitors (clavulanate acid, tazobactam, and sulbactam) and one noninhibitory chemical (cefotaxime) were used as mock compounds. All of these compounds contain a �-lactam ring that can be hydrolyzed by �-lactamase, as shown in Figure 5. However, cefotaxime is a very large molecule, difficult to be hydrolyzed by �-lactamase. �-Lactamase was produced in each unit of the array device, and a series of varying concentrations of each compound was added into several wells to obtain a response curve. The relationship between the concentrations of each compound and the level of �-lactamase inhibition is also shown in Figure 5. The percentage of inhibition is calculated against the one without any Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 7025
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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
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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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Page 231 and 232: for the fill time, and we find that
Page 233 and 234: nucleotide tails. 3-5 Thus, the amo
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Page 241 and 242: Figure 2. Configuration editor wind
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Page 245 and 246: Since the time for liquid expulsion
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Page 249 and 250: Figure 1. Representation of the Car
Page 251 and 252: Table 2. Capillary Electrophoresis
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Page 263 and 264: CONCLUSIONS In this paper we have i
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Page 273 and 274: Table 1. Effect of 1 D-LC Condition
Page 275 and 276: Figure 3. Peak-production rate vers
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Page 279: luciferase (T7 control vector) was
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Page 285 and 286: Electrochemistry. Electrochemical m
Page 287 and 288: Figure 2. SEM images of Au substrat
Page 289 and 290: Table 3. Film Thickness Measurement
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Page 293 and 294: Figure 1. (A) FL spectra of BSPOTPE
Page 295 and 296: Figure 4. (A) Variation in the FL i
Page 297 and 298: Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300: Scheme 1. Proposed Mechanism for Fl
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Page 303 and 304: Figure 2. Free fluorophores do not
Page 305 and 306: Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308: Figure 2. Comparative analysis of d
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