Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 8. Fluorescence imaging of Qdot-based FLFTS for (A) 100 ng/mL and (B) 10 ng/mL nitrated ceruloplasmin and (C) 10 µg/mL ceruloplasmin without nitration in human plasma. viewed by naked eyes, which is equivalent to 200 ng/mL for an undiluted plasma sample. Successfully detecting the spiked human plasma samples using a strip reader or fluorescence imaging displays the promise of Qdot-based FLFTS for various clinical applications in the near future. CONCLUSIONS In summary, Qdot as a promising alternative reporter was successfully integrated with lateral flow tests strip and first developed for rapid, sensitive, and one-step quantitative detection of a trace amount of nitrated ceruloplasmin. This portable 7014 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 biosensor takes advantage of the speed and low cost of conventional immunochromatographic strip as well as high sensitivity and photostability of Qdot-based fluorescent immunoassay. Under optimal conditions, this proposed Qdot-based FLFTS is capable of detecting a minimum of 1 ng/mL nitrated ceruloplasmina within 10 min. Furthermore, the linear relationship between peak area and the logarithm of target concentration was observed in the range of 1 ng/mL to 10 µg/mL with a detection limit of 0.4 ng/ mL in a spiked plasma sample, which is equivalent to 8 ng/mL for undiluted plasma. Moreover, the presence of non-nitrated ceruoloplasmin showed no effect on the biosensor response, illustrating the good selectivity. Overall, the Qdot-based FlFTS, considered as an advance in alternative immunosensors, has a great potential for rapid, sensitive, and portable analysis of other protein biomarkers in clinical diagnostics, basic discovery, and a variety of other biomedical applications. ACKNOWLEDGMENT The work was done at Pacific Northwest National Laboratory (PNNL) supported by Grant U54 ES16015 from the National Institute of Environmental Health Sciences (NIEHS), NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the federal government. PNNL is operated by Battelle for DOE under Contract DE-AC05- 76RL01830. Received for review May 28, 2010. Accepted July 1, 2010. AC101405A
Anal. Chem. 2010, 82, 7015–7020 Selection of Column Dimensions and Gradient Conditions to Maximize the Peak-Production Rate in Comprehensive Off-Line Two-Dimensional Liquid Chromatography Using Monolithic Columns Sebastiaan Eeltink,* ,† Sebastiaan Dolman, ‡,⊥ Gabriel Vivo-Truyols, § Peter Schoenmakers, § Remco Swart, ‡ Mario Ursem, ‡ and Gert Desmet † Department of Chemical Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium, Dionex Corporation, Abberdaan 114, 1046 AA Amsterdam, The Netherlands, and Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands The peak-production rate (peak capacity per unit time) in comprehensive off-line two-dimensional liquid chromatography (LC/×/LC) was optimized for the separation of peptides using poly(styrene-co-divinylbenzene) monolithic columns in the reversed-phase (RP) mode. A firstdimension ( 1 D) separation was performed on a monolithic column operating at a pH of 8, followed by sequential analysis of all the 1 D fractions on a monolithic column operating at a pH of 2. To obtain the highest peak-production rate, effects of column length, gradient duration, and sampling time were examined. RP/×/RP was performed at undersampling conditions using a short 10 min 1 D gradient. The peak-production rate was highest using a 50 mm long 2 D column applying an 8-10 min 2 D gradient time and was almost a factor of two higher than when a 250 mm monolithic column was used. The best way to obtain a higher peakproduction rate in off-line LC/×/LC proved to be an increase in the number of 1 D fractions collected. Increasing the 2 D gradient time was less effective. The potential of the optimized RP/×/RP method is demonstrated by analyzing proteomics samples of various complexities. Finally, the trade-off between peak capacity and analysis time is discussed in quantitative terms for both onedimensional RP gradient-elution chromatography and the off-line two-dimensional (RP/×/RP) approach. At the conditions applied, the RP/×/RP approach provided a higher peak-production rate than the 1 D-LC approach when collecting three 1 D fractions, which corresponds to a total analysis time of 60 min. Porous polymer monolithic columns have emerged in the 1990s 1,2 and have become a viable alternative for packed-column * Corresponding author. Tel.: +32 (0)2 629 3324. Fax: +32 (0)2 629 3248. E-mail: seeltink@vub.ac.be. † Vrije Universiteit Brussel. ‡ Dionex Corporation. § University of Amsterdam. ⊥ Present address: Bruker Biosciences, 1/28A Albert Street, Preston VIC 3072, Australia. (1) Hjertén, S; Liao, J.-L; Zhang, R. J. Chromatogr., A 1989, 473, 273–275. technology for the gradient-elution separation of peptides and proteins. 3-5 Macroporous polymer monolithic materials were initially developed by Svec et al. in large I.D. column formats via a molding process. 6,7 Macroporous poly(styrene-co-divinylbenzene) (PS-DVB) monolithic rods were operated at high flow rates (up to 25 mL/min) for the gradient separations of proteins. 7 This group also demonstrated that the porous properties could be controlled by optimizing the polymerization mixture, i.e., type and composition of the porogenic solvent, and the percentage of difunctional monomer (“cross-linker”) in the mixture. 8,9 Huber and co-workers developed styrene-based monoliths in situ in 200 µm I.D. capillary columns and applied these columns for liquidchromatographic mass-spectrometric (LC-MS) analysis of samples from proteomics and genomics studies, including peptides, proteins, single-stranded oligonucleotides, and double-stranded DNA fragments. 10-12 Excellent separation performance was, for example, demonstrated by a baseline separation of phosphorylated oligodeoxyadenylic acids, ranging in size from the 12-mer to the 60-mer, and yielded peak widths of only 5.7 s when applying a 7.5 min gradient, 13 resulting in a peak capacity of approximately 80. Recently, we reported the use of a1mlong poly(styrene-codivinylbenzene) monolithic column yielding a peak capacity in excess of 1000 for a peptide separation when applying a 600 min gradient. 14 (2) Peters, E. C.; Petro, M.; Svec, F; Frechet, J. M. J. Anal. Chem. 1997, 69, 3646–3649. (3) Ivanov, A. R.; Zang, L.; Karger, B. L. Anal. Chem. 2003, 75, 5306–5316. (4) Geiser, L.; Eeltink, S.; Svec, F.; Frechet, J. M. J. J. Chromatogr., A 2008, 1188, 88–96. (5) Levkin, P. A.; Eeltink, S.; Stratton, T. R.; Brennen, R.; Robotti, K.; Killeen, K.; Svec, F.; Frechet, M. J. M. J. Chromatogr., A 2008, 1200, 55–61. (6) Svec, F.; Frechet, J. M. J. Anal. Chem. 1992, 54, 820–822. (7) Wang, Q. C.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1993, 65, 2243–2248. (8) Viklund, C.; Svec, F.; Frechet, J. M. J. Chem. Mater. 1996, 8, 744–750. (9) Eeltink, S.; Geiser, L; Svec, F; Frechet, J. M. J. J. Sep. Sci. 2007, 30, 407–413. (10) Toll, H.; Wintringer, R.; Schweiger-Hufnagel, U.; Huber, C. G. J. Sep. Sci. 2005, 28, 1666–1674. (11) Holzl, G.; Oberacher, H.; Pitsch, S.; Stutz; Huber, C. G. Anal. Chem. 2005, 77, 673–680. (12) Walcher, W.; Toll, H.; Ingendoh, A.; Huber, C. G. J. Chromatogr., A 2004, 1053, 107–117. (13) Oberacher, H.; Mayr, B. M.; Huber, C. G. J. Am. Soc. Mass Spec. 2004, 15, 32–42. (14) Eeltink, S.; Dolman, S.; Detobel, F.; Swart, R.; Ursem, M.; Schoenmakers, P. J. J. Chromatogr., A 2010, in press. 10.1021/ac101514d © 2010 American Chemical Society 7015 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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Page 221 and 222: Figure 3. (A) ESI-LTQ-CID-MS 2 prod
Page 223 and 224: Figure 5. (A) MALDI-TOF/TOF product
Page 225 and 226: Anal. Chem. 2010, 82, 6969-6975 Ana
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
Page 247 and 248: Anal. Chem. 2010, 82, 6991-6999 Int
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
Page 255 and 256: field. DNA profiles were delivered
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: Figure 6. Fluorescence imaging of Q
Page 273 and 274: Table 1. Effect of 1 D-LC Condition
Page 275 and 276: Figure 3. Peak-production rate vers
Page 277 and 278: Anal. Chem. 2010, 82, 7021-7026 Cel
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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