Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 6. Trade-off between 1 D-LC performance using 50 mm (solid line) and 250 mm (dotted line) long monolithic columns and the optimized off-line 2 D-LC approach collecting g2 fractions (solid symbols). 1 D-LC conditions described in Figure 1; 2 D-LC conditions described in Figure 2. time, the dwell time, and the gradient time. The lines depict the 1 D-LC performance. The closed circles represent the RP(pH)8)/×/RP(pH)2) performance collecting discrete numbers of fractions (two and more). It is evident that below 45 min the highest � is obtained using 50 mm long monolithic columns in the 1 D-LC mode. This yields a maximum peak capacity of 320. At a total analysis time of 70 min, 50 mm and 250 mm long monolithic columns show comparable performance. For 1 D-LC analysis longer than 70 min, the 250 mm monolithic column provides the �. The maximum 1 D-LC peak capacity of 475 can be achieved within 3 h. However, the � is much smaller than what can be realized by LC/×/LC. A peak capacity of 480 7020 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 can already be achieved in 60 min using LC/×/LC when analyzing only three 1 D fractions. The peak capacity linearly increases with the number of fractions collected and approximately doubles every 60 min. CONCLUDING REMARKS An approach to select column dimensions and LC conditions in LC/×/LC to maximize � have been demonstrated. Although the LC/×/LC optimization strategy has been performed with monolithic columns, this approach can be used when applying other types of LC columns. At undersampling conditions, the 1 D separation performance is subsidiary of sampling rate. As a result, a short first-dimension analysis time is optimal. This can be achieved using short 1 D column length and applying a short 1 tG. The optimal 2 tG depends on 2 D column length and on sampling rate. The optimal 2 tG shift to lower values when shorter 2 D columns are used yields higher values for �. For separations requiring a maximum peak capacity up to 340, 1 D-LC using a 50 mm long monolithic column was found to be superior to LC/×/LC in terms of analysis time. For more demanding separations, 250 mm long monoliths can be used, applying longer gradient times (nc, max = 475). However, the � in LC/×/LC is superior for an analysis taking longer than 60 min (collecting three fractions and more), and the achievable peak capacity increases linearly with analysis time. ACKNOWLEDGMENT Support of this work by a grant of the Research Foundation Flanders (G.0919.09) is gratefully acknowledged. Received for review June 8, 2010. Accepted July 17, 2010. AC101514D
Anal. Chem. 2010, 82, 7021–7026 Cell-Free Expression of Soluble and Membrane Proteins in an Array Device for Drug Screening Ruba Khnouf, † Daniel Olivero, ‡ Shouguang Jin,* ,§ Matthew A. Coleman,* ,| and Z. Hugh Fan* ,†,‡ Department of Biomedical Engineering, University of Florida, P.O. Box 116131, Gainesville, Florida 32611, Department of Mechanical and Aerospace Engineering, University of Florida, P.O. Box 116250, Gainesville, Florida 32611, Department of Molecular Genetics and Microbiology, University of Florida, P.O. Box 100266, Gainesville, Florida 32610, and Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551 Enzymes and membrane protein receptors represent almost three-quarters of all current drug targets. As a result, it would be beneficial to have a platform to produce them in a high-throughput format for drug screening. We have developed a miniaturized fluid array device for cellfree protein synthesis, and the device was exploited to produce both soluble and membrane proteins. Two membrane-associated proteins, bacteriorhodopsin and ApoA lipoprotein, were coexpressed in an expression medium in the presence of lipids. Simultaneous expression of ApoA lipoprotein enhanced the solubility of bacteriorhodopsin and would facilitate functional studies. In addition, the device was employed to produce two enzymes, luciferase and �-lactamase, both of which were demonstrated to be compatible with enzyme inhibition assays. �-lactamase, a drug target associated with antibiotic resistance, was further used to show the capability of the device for drug screening. �-Lactamase was synthesized in the 96 units of the device and then assayed by a range of concentrations of four mock drug compounds without harvesting and purification. The inhibitory effects of these compounds on �-lactamase were measured in a parallel format, and the degree in their drug effectiveness agreed well with the data in the literature. This work demonstrated the feasibility of the use of the fluid array device and cell-free protein expression for drug screening, with advantages in less reagent consumption, shorter analysis time, and higher throughput. There is an escalating need to discover new drug targets for various diseases that impact the quality of life of countless individuals. Once a target is validated, it is necessary to find drug candidates by screening a large library of compounds. For both target discovery and drug screening, novel platforms such as highthroughput screening (HTS) are required in the modern pharmaceutical era. 1-3 * To whom correspondence should be addressed. E-mail: hfan@ufl.edu (Z.H.F.). Fax: 1-352-392-7303 (Z.H.F.). † Department of Biomedical Engineering, University of Florida. ‡ Department of Mechanical and Aerospace Engineering, University of Florida. § Department of Molecular Genetics and Microbiology, University of Florida. | Lawrence Livermore National Laboratory. (1) Drews, J. Science 2000, 287, 1960–1964. (2) Carnero, A. Clin. Transl. Oncol. 2006, 8, 482–490. Although cell-based HTS has become increasingly popular due to its ability to deliver phenotype information at the cellular level, there is still a considerable need to have innovative platforms that provide biological and chemical information at the molecular level. 4 Cell-based screening can result in “off target hits” because different portions of a cellular process other than the target could be affected by library compounds. 5 In addition, those compounds that cause cytotoxicity would result in reduced signal even though they do not inhibit the desired target. 5 Galarneau et al. showed a great example that both cell-free and cell-based assays were required to provide complementary and indispensible information for their study in protein-protein interactions. 4 Cell-free protein synthesis (CFPS) is a platform technology that addresses the shortcomings mentioned above for cell-based assays. CFPS utilizes an efficient, often-coupled transcription and translation reaction to produce recombinant proteins. 6-12 It eliminates the time-consuming steps of conventional cell-based protein production, including transformation, cell culture maintenance, and expression optimization. The proteins synthesized could be used for functional and structural studies, drug screening, and other applications. Since there is no cellular control mechanism, CFPS will not suffer from cytotoxicity or other challenges associated with cell-based protein expression (e.g., inclusion body). 6 A large number of soluble proteins have been synthesized using CFPS, and many of them are enzymes and functionally active. 13 More importantly, CFPS is an open system and, thus, can be modified by simply adding various components. This open nature has been used for producing membrane proteins. Lipids, liposome, and lipoprotein particles have been introduced into the cell-free (3) Pereira, D. A.; Williams, J. A. Br. J. Pharmacol. 2007, 152, 53–61. (4) Galarneau, A.; Primeau, M.; Trudeau, L. E.; Michnick, S. W. Nat. Biotechnol. 2002, 20, 619–622. (5) von Ahsen, O.; Bomer, U. ChemBioChem 2005, 6, 481–490. (6) Spirin, A. S.; Baranov, V. I.; Ryabova, L. A.; Ovodov, S. Y.; Alakhov, Y. B. Science 1988, 242, 1162–1164. (7) Kigawa, T.; Yabuki, T.; Yoshida, Y.; Tsutsui, M.; Ito, Y.; Shibata, T.; Yokoyama, S. FEBS Lett. 1999, 442, 15–19. (8) Madin, K.; Sawasaki, T.; Ogasawara, T.; Endo, Y. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 559–564. (9) Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T. Nat. Biotechnol. 2001, 19, 751–755. (10) Angenendt, P.; Nyarsik, L.; Szaflarski, W.; Glokler, J.; Nierhaus, K. H.; Lehrach, H.; Cahill, D. J.; Lueking, A. Anal. Chem. 2004, 76, 1844–1849. (11) Jewett, M. C.; Swartz, J. R. Biotechnol. Prog. 2004, 20, 102–109. (12) Katzen, F.; Chang, G.; Kudlicki, W. Trends Biotechnol. 2005, 23, 150–156. (13) Spirin, A. S. Trends Biotechnol. 2004, 22, 538–545. 10.1021/ac1015479 © 2010 American Chemical Society 7021 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Published on Web 07/28/2010
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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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Page 227 and 228: Figure 3. Equilibrium response as a
Page 229 and 230: Figure 6. The average measured resp
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 239 and 240: Anal. Chem. 2010, 82, 6983-6990 Imp
Page 241 and 242: Figure 2. Configuration editor wind
Page 243 and 244: Figure 4. Dependencies between volu
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
Page 253 and 254: Figure 4. (A) Typical raw electroph
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Page 257 and 258: Another approach to handle this lim
Page 259 and 260: (principal components, PCs) in whic
Page 261 and 262: Figure 5. Relevant score plots and
Page 263 and 264: CONCLUSIONS In this paper we have i
Page 265 and 266: electroactive-species loaded liposo
Page 267 and 268: Figure 2. Qdot-based FLFTS response
Page 269 and 270: Figure 6. Fluorescence imaging of Q
Page 271 and 272: Anal. Chem. 2010, 82, 7015-7020 Sel
Page 273 and 274: Table 1. Effect of 1 D-LC Condition
Page 275: Figure 3. Peak-production rate vers
Page 279 and 280: luciferase (T7 control vector) was
Page 281 and 282: Figure 3. Inhibitory effects of luc
Page 283 and 284: Anal. Chem. 2010, 82, 7027-7034 Pat
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
Page 291 and 292: Anal. Chem. 2010, 82, 7035-7043 Qua
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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