Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 1. (A) Schematic illustration of the test strip and (B1-B4) the detection of nitrated ceruloplasmin using fluorescent Qdot-based FLFTS. (B1) Aqueous sample containing nitrated ceruloplasmin is applied to sample pad. (B2) Nitrated ceruloplasmin combines with QD-antinitrotyrosine conjugate and also migrates along the porous membrane by capillary action. (B3) Nitrated ceruloplasmin is captured by anticeruloplasmin antibodies immobilized on the test line. The excess Qdot conjugates continue to migrate toward the absorption pad. (B4) Fluorescence signal of Qdot is detected using a test strip reader (solid line). As a control, ceruloplasmin without nitration cannot be recognized by Qdot-antinitrotyrosine conjugates, so no fluorescence signal can be seen on the test strip (dotted line). exclusion column followed by the addition of PBS. During the elution by gravity, the first ten drops only of colored conjugate (∼200 µL) was collected into a centrifuge tube and stored at 4 °C until use. Following the instructions from the protocol for the Qdot antibody conjugation kit from Invitrogen, the conjugate concentration was determined by measuring the absorbance density of the conjugate at 585 nm with a Ultrospec 2100 Pro UV/Visible spectrophotometer and, then, using the formula A ) εcL, where A is the absorbance, ε is the molar extinction coefficient, c is the molar concentration of Qdot conjugate, and L is the path length of the cuvette. Nitration of Ceruloplasmin. One mg/mL Human ceruloplasmin in phosphate buffered saline (pH 7.4) was nitrated by bolus addition of 1 mM authentic peroxynitrite (R&D Systems, Minneapolis, MN) according to the manufacturer’s recommendations. The volume of added peroxynitrite was
Figure 2. Qdot-based FLFTS response for (a) 100 ng/mL, (b) 10 ng/mL, and (d) 0 ng/mL nitrated human ceruloplasmin, (c) 10 µg/mL human ceruloplasmin served as a control. action. After about 10 min, the cassette containing the test strip was inserted into an ESE-Quant FLUO reader, followed by the recording of fluorescence intensity from Qdot on the test line to quantify the analytes. For the detection of nitrated ceruloplasmin in human plasma, 20 times diluted plasma spiked with different quantities of nitrated ceruloplasmin was added onto the sample pad. The results were obtained by reading the optical response with the strip reader after 10 min. Meanwhile, these strips after assay were put under a UV light, and the corresponding fluorescence images were captured directly by a Sony DSLR-A300 digital camera. RESULTS AND DISCUSSION Assay Principle of Qdot-Based FLFTS. Figure 1 schematically illustrates the configuration and measuring principle of Qdotbased FLFTS. This FLFTS is composed of sample application pad, conjugation pad, absorption pad, and nitrocellulose membrane (Figure 1A). All the components were assembled onto a plastic adhesive backing card. During the assay, aqueous sample containing nitrated ceruloplasmin was applied onto the sample application pad as shown in Figure 1B1. Subsequently, the analyte migrated along the porous membrane by capillary action and then bound with Qdot-antinitrotyrosine on the conjugation zone according to the specific antibody-antigen interaction (Figure 1B2). The formed complexes continued to migrate along the membrane and were captured by anticeruloplasmin antibodies, which resulted in the accumulation of Qdot on the test line (Figure 1B3). The excess Qdot conjugates continue to flow into the absorption pad to the end of the strip. Quantitative analysis was realized by reading the fluorescence intensities of test line with a portable strip reader (Figure 1B4). The more analyte in the sample, the more Qdot conjugates would be captured to the test line, which leads to the increase of fluorescence intensity. According to the principle described above, the fluorescence intensity on the test line would be proportional to the concentration of nitrated ceruloplasmin in the samples. Figure 2 shows the typical corresponding responses of Qdotbased FLFTS to different concentrations of nitrated ceruloplasmin. Here, human ceruloplasmin without nitration served as a control. As shown in this figure, well-defined curves were observed in the Figure 3. Effect of Qdot-antinitrotyrosine conjugates concentration on the signal-to-noise ratio (S/N) of Qdot-based FLFTS for 1 µg/mL nitrated human ceruloplasmin. presence of nitrated ceruloplasmin and the peak area was getting larger as the target concentration increased from 10 to 100 ng/ mL because more Qdot was captured on the test line based on the mechanism of Qdot-based FLFTS. In contrast, the presence of ceruloplasmin without nitration did not contribute to the signal and exhibited a very low background. The results indicate the great possibility of Qdot-based FLFTS for sensitive protein detections. Determination of Qdot Conjugate Concentration. Following the instructions for the Qdot antibody conjugation kit from Invitrogen, the Qdot conjugate eluting from the final column could be determined according to the Beer-Lambert law 42 by measuring the absorbance density of the conjugate at 585 nm: A ) εcL Where A is the absorbance, ε is the molar extinction coefficient (as 400 000 M -1 cm -1 provided by Invitrogen for Qdot 585), c is the molar concentration, and L is the path length of the cuvette. The result shows that the Qdot conjugate has A ) 0.6 measured in a cuvette with 1 cm path length, so c ) A/ε ) 0.6/400 000 ) 1.5 × 10 -6 M, which is the original concentration of as-prepared Qdot conjugate stock solution. ParameterOptimization.TheamountofQdot-antinitrotyrosine, loaded on the glass fiber by physical absorption, directly affects the fluorescence response of Qdot-based FLFTS since the signal mainly depends on the amount of Qdot-antinitrotyrosine conjugates captured on the test line. To probe the optimal amount of Qdot conjugates for the assay, we diluted them into various concentrations and investigated the influence on signal-to-noise (S/N) ratio of the biosensor for 1 µg/mL nitrated human ceruloplasmin. Ten µg/mL non-nitrated ceruloplasmin served here as a control. As can be seen from Figure 3, the S/N ratio was found to be highest for dispensing 150 nM Qdot-antinitrotyrosine. However, the decrease of S/N at a higher concentration is resulted from the increase of background signal due to too high of a concentration of Qdot-antinitrotyrosine conjugate while that at (42) Ingle, J. D. J.; Crouch, S. R. Spectrochemical Analysis; Prentice Hall: Upper Saddle Rive, NJ, 1988. <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010 7011
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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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Page 217 and 218: Scheme 2. Fragmentation Mechanism o
Page 219 and 220: Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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
Page 235 and 236: allow the use of higher aptamer con
Page 237 and 238: quent ligation of the aptamers afte
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: electroactive-species loaded liposo
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 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
Page 301 and 302: one or more drawbacks including poo
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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