Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 3. Design of the PCR chamber fluidic system. The DNA solution enters the chamber from the right and fills the chamber. When the liquid level reaches the constriction channel in the top left-hand side of the chamber, the pressure required to pass through this constriction is much greater than that required for the solution to traverse the bypass. The DNA solution therefore passes over the bridge, leaving a precise volume of DNA in the PCR chamber (chamber highlighted by red solution). Following PCR amplification, the reaction volume is recovered by closing the bridge at Y using valve V15 and flushing the formamide through the chamber, collecting the PCR product. chamber was filled until the solution reached the restriction channel. The pressure required to breach this channel was greater than that required for the solution to flow over the bypass and consequently the flow took that direction, leaving a precisely measured volume of PCR reaction (9.96 ± 0.21 µL (n ) 20)) in the PCR chamber. The amount of DNA in the 10 µL PCR chamber therefore approximated 5.2 ± 2.5 ng, based upon the ReaX bead volume of 4 µL. Samples recovered from the PCR chamber and run on the AB 3130xl gave similar peak heights to the control samples amplified in tube (data not shown). Following amplification, V15 is closed and the whole of the PCR sample is recovered from the PCR chamber and delivered with ILS 500 CC5 in formamide to the loading well of the electrophoresis chip. We rely on surface effects to smoothly remove the vast majority of the PCR reaction from the chamber. As the formamide is driven from its storage chamber, the air in the channel in front of the formamide is pushed through the PCR chamber and removes the bulk (80-90%) of the PCR reaction through the constriction. The formamide then fills the chamber, taking the remaining PCR solution with it. The surface tension at this small scale ensures that the bulk of the formamide is also flushed from the chamber leaving just one or two microlitres in the 90° corners of PCR chamber. The volume delivered to chamber D varied between cartridges with a mean volume of 25.1 ± 4.8 µL (n ) 25) with a range of 15-35 µL. The variation in volume was due to three things: (1) The wax in the valves 13 and 14 is molten as the liquid flows by, allowing some liquid to mix with the wax. This liquid is trapped in the system as the wax solidifies. (2) Liquid is occasionally lost in the valve structures of V13 and/or V14. (3) Our channels are machined rather than injection molded, creating a rough surface that can retain 1-2 µL per inch of channel. The volume of sample released from PCR, denatured in chamber D and delivered to the µCE is a variable that could have a significant effect on the final result: If only half of the PCR volume of a given PCR product was recovered we would expect to observe lower peak heights in the profile. The 6996 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 system was therefore designed to push the PCR mixture out of the PCR chamber prior to flushing the chamber with formamide and we believe that the majority of the PCR product is recovered, minimizing any variability from this function of the cartridge. Sample loss in the channels, wax and valve housings in the circuit before the PCR chamber will reduce the amount of formamide and size standard reaching the denaturation chamber while the inefficiencies in the circuitry post PCR chamber would reduce the volume of PCR product and formamide in proportion to their volumetric ratios. Current laboratory practice is that the sample is heat denatured at 95 °C for 3 min and snap-cooled on ice prior to loading onto the 3130xl platform for injection to the CE. While the sample was easily heat denatured on the cartridge, no facility for snap cooling was available and the sample was loaded directly into the CE. Evaluation of the profiles demonstrated that this process was acceptable because the DNA profiles produced with no snap cooling were found to be comparable to those that had been snap cooled. The electropherogram from the internal lane standard (GeneScan 500 LIZ) diluted to a volume ratio of 9:1 with Hi-Di formamide (Applied Biosystems) and heat-denatured at 95 °C for 5 min was used to assess the separation efficiency of the µCE module. The chromatographic resolution R was calculated by fitting the peaks with a Gaussian curve to obtain the migration time, t i, and the full-width at half-maximum, wi for peak i according to the following equations: 41 R ) (2 ln 2) 1/2 t 2 - t 1 w 1 + w 2 And subsequently: Rbp ) ∆bp/R expresses the resolution in base pairs, that is, the minimum base pair separation of two peaks that would still appear separated at the baseline in the electropherogram. Figure 4A displays an example of an electropherogram which represents the “binned” data prior to deconvolution into the target wavelengths, that is, the electropherogram represents the sum of photon counts over time, and represents the whole of the signal collected by the CCD by time. Figure 4B shows the evolution of the resolution Rbp on the µCE system as a function of the known peak size (red trace) and the comparison with an electropherogram obtained on a commercial ABI 310 capillary electrophoresis analyzer (Applied Biosystems) using the same polymer matrix at 65 °C with a cathode potential of 10.3 kV. The resolution obtained on the µCE module is very close to that of a commercial CE apparatus. Typically, the resolution values obtained for a GeneScan 500 LIZ varied between 1.02 and 1.24 bp for peaks within the 140-160 bp range. Ongoing work aims at further improving the resolution by (1) using a pinched injection and (2) tailoring the composition of the HEC/PVP mixture for use in a microchip rather than a capillary. A series of allelic ladder runs was recorded on the CE subsystem using a CC5-labeled ILS 500 size standard (Promega) to estimate the run-to-run stability of the system. Consecutive runs were acquired on three different microchips to determine the variability between microchips. Figure 5 shows a typical sized run processed with GeneMarker HID 1.76. Inserts B though E are (41) Manabe, T.; Chen, N.; Terabe, S.; Yohda, M.; Endo, I. Anal. Chem. 1994, 66, 4243–4252.
Figure 4. (A) Typical raw electropherogram obtained for a GeneScan 500 LIZ run on the CE subsystem using a 140 mm microchip at 50 °C. (B) Comparison of the resolution (in base pair) Rbp of the µCE system (red trace; 2) to that obtained on a 310 apparatus (Applied Biosystems) at 65 °C in a 36 cm capillary (blue trace; 1). close-ups of the [7-9] allele region (304-310 bp) on the D22S1045 marker, the [10-11] region (146-150 bp) on the D18S51 marker, the [9-11] region (97-105 bp) on the TH01 marker, and the [21.2-26] region (177-195 bp) on the FGA marker respectively. Table 3 summarizes the observed shifts in base pair sizing for five different allelic ladder runs using a number of alleles from Figure 5B-E. The maximum observed variation around the tabulated value in the positioning of an allele is only about 0.2 bp on the TH01 marker, 0.7 bp on the D18S51 marker, 0.4 bp on the FGA marker, and 0.7 bp on the D22S1045 marker. The variability increases with the fragment size as expected from the absolute chromatographic resolution of the CE subsystem given in Figure 4B. The allele sizing data gained from running a minimum of five allelic ladders of known designations was used to define the expected sizes of the unknown alleles in the amplified DNA samples. Integrated Sample Run. We were able to obtain STR profiles from lysed cells from buccal swabs using a disposable plastic cartridge attached to a reusable glass microchip device without any manual interruption of the programmed DNA extraction, purification, amplification, transfer and CE analysis process. A typical electropherogram including the ILS is shown in Figure 6 after applying GeneMarker HID 1.76 to size the migrated DNA fragments. The DNA profiles obtained as an integrated automated run were always similar to those obtained on an Applied Biosystems ABI 3130xl analyzer in terms of the number and position of the peaks and their relative intensities. Currently we are unable to routinely label all the peaks with a definitive allele designation as the migration of the peaks of some samples has given out of window peaks. We believe this may be due to a difference in the time base of the instrumentation and the NanoIdentity software: The software assumes an equal time base per data point while the instrumentation collects data on a variable time base which averages 0.21 s with a maximum of 0.35 s and a minimum of 0.07 s. An updated Figure 5. (A) Powerplex ESI16 allelic ladder run using CC5 ILS500 size standard (Promega, Madison, WI) sized using GeneMarker HID 1.76 (SoftGenetics LLC, State College, PA) with appropriate panels and bins provided by Promega. (B) Close-up of the [7-9] allele region (304-310 bp) on the D22S1045 marker. (C) Close-up of the [10-11] region (146-150 bp) on the D18S51 marker. (D) Close-up of the [9-11] region (97-105 bp) on the TH01 marker. (E) Close-up of the [21.2-26] region (177-195 bp) on the FGA marker. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6997
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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
Page 201 and 202: Figure 4. EID (a) and IRMPD (b) of
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Page 205 and 206: Figure 1. Schematic flowchart showi
Page 207 and 208: difference, ppm compound Table 1. I
Page 209 and 210: isoforms, its successful use, in th
Page 211 and 212: Figure 5. Extracted ion current ESI
Page 213 and 214: m/z 1172.935 was observed for Ser14
Page 215 and 216: linked products via affinity tags.
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
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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: Table 2. Capillary Electrophoresis
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 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
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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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