Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 6. List of ions used for the determination of pig kidney MT sequences. (a) MT-1g, (b) MT-1h, (c) N-Ac-MT-1g, (d) MT-1a and MT-2a, (e) N-Ac-MT-1h, (f) N-Ac-MT-2a, (g) N-Ac-MT-1a, (h) N-Ac-MT-2a. Bold characters highlight residues where modifications are observed. no mass fragment specific to MT-1e in MS/MS spectra, its presence or absence cannot be unambiguously confirmed. Even if present, its contribution to the MTs pool would be very low. By selecting appropriate and narrow retention time ranges in the MS/ MS chromatogram, y ions detected covered 74% and 87% of the MT-1a and MT-2a amino acid sequence (Figure 6d) confirming the identification of Cd7MT-1a and Cd7MT-2a. An amino acid sequence of MT-2a without the N-terminal was observed for the peak eluting at 40.5 min (Figure 6f). The mass difference with regard to MT-2a isoforms indicated N-acetylation on methionine residue leading to the identification of N-Ac-MT- 2a. This was also confirmed by two peaks of the MT2a specific fragments (acetylated and non acetylated) in Figure 3-ESI e. The sequence of the MT eluting at 41 min suggests strongly its 6956 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 identification as N-Ac-MT-1a (δ -0.3 ppm). Indeed, the presence of the sequence fragment 39 Ala -Gly-Cys-Ala 42 and the overall sequence coverage of 95% supported this identification. The molecular masses of the remaining four apo-MTs in Table 2 did not correspond to any of the reported pig MTs subisoforms. The 42-Da difference between the forms eluting at 33.8 and 37.4 min (peaks 1 and 3) and between the forms eluting at 35 and 38.5 min (peaks 2 and 5) indicated two pairs of acetylated and non-acetylated isoforms. The amino acid sequence of the MT subisoform eluting at 33.8 min (5940.124 Da, Figure 6a) was found to be as a mixture of MT-1a and MT-1e as m/z 1091.924 and m/z 940.374 (charge +2) ions were observed for, respectively, Ala39 and Lys43 residues and matched the theoretical y ions (m/z 1091.929 and m/z 940.376 (charge +2). Furthermore, a y ion at
m/z 1172.935 was observed for Ser14 residue (theoretical m/z 1172.939, z ) 4, δ 3.1 ppm) which differentiated this sequence from the one of MT-1f which contains a Thr14 residue. As the sequence matched 98% of pig MT-1 sequence and has never been reported before, it was denoted here as MT-1g. The sequence coverage with y ions was 88.5%. Consequently, the subisoform eluting at 37.4 min (5982.149 Da, Figure 6c) was referred to as N-Ac-MT-1g. Regarding the identification of the remaining pair of acetylated and non-acetylated isoforms eluting at 35.0 min (5944.149 Da) and 38.5 min (5986.179 Da), the HCD spectrum of the N-acetylated form (m/z 1198.243, charge +5) gives the sequence information. It was found identical to MT1c until Pro38 and then residues Ala39, Arg43, Gln46, Ile49 of MT1c were, respectively, replaced by Val39, Lys43, Lys46, Val49 as m/z 1098.4547 (theoretical m/z 1098.4550, δ 0.2 ppm), m/z 933.3862 (theoretical m/z 933.3869, δ 0.7 ppm), m/z 781.813 (theoretical m/z 781.816, δ 3.2 ppm) and m/z 1274.494 (theoretical m/z 1274.499, δ 4.1 ppm) were detected at +2 charge state and the last one at +1 charge state. An ExPASy database search indicated that these modifications on 46 and 49 amino acids residues were reported for MT of others organisms (pig (in MT3), sheep, bovine and human). The sequence coverage was 92%. As this MT has not been reported before, this pair of isoforms was denoted as MT-1h and N-Ac-MT-1h, respectively. The composition of the complexes formed indicates a release of Cd ions from the nanoparticles and their complexation by MTs formed. The identification of the last peak in the µHPLC-ICP MS chromatogram (peak 8 at 44.3 min (cf. Figure 4b) was less straightforward as no apo-isoform could be obtained. The presence of the fragment sequence 7 Cys-Ala-Ala 9 among +4 charge state fragments is characteristic for an MT-2 class isoforms. Another part of an MT sequence could be identified among +2 charge state fragments as 37 Cys-Pro-Val-Gly-Cys-Ala-Lys-Cys-Ala 45 (δ 3.1 ppm), confirming the identity of the MT subisoform as MT2a. The end of the MT sequence 46 Gln-Gly-Cys-Ile-Cys-Lys- Gly-Ala-Ser-Asp 55 is observed as +1 charge state (δ 2.3 ppm). The metallothionein is obviously metalated and the mass difference in comparison with the N-Ac-MT2a theoretical mass (6011.190 Da) corresponds to the addition of Cu2Cd2 (351.665 Da) with a loss of 8H + . This state of metalation is corroborated by the observation of three y ions corresponding to the 7 Cys- (47) Meloni, G.; Faller, P.; Vasak, M. J. Biol. Chem. 2007, 282, 16068–16078. (48) Salgado, M. T.; Stillman, M. J. Biochem. Biophys. Res. Commun. 2004, 318, 73–80. Ala-Ala 9 fragment (m/z 1417.393, m/z 1391.644 and m/z 1373.883 charge +4) differing by 85.89 mass unit from the corresponding apo subisoforms. The isotopic pattern was similar to the theoretical isotopic pattern of fragments with Cu2Cd2 adduct. Therefore peak 8 (Figure 4b) was identified as N-terminal acetylated Cu2Cd2-MT2a (6355.799 Da, theoretical 6355.792 Da, δ -1.0 ppm) under acidic conditions and as N-terminal acetylated Cu2Cd6-MT2a (6797.354 Da, theoretical 6797.344 Da, δ -1.4 ppm) under neutral conditions. The late elution of a similar complex from a rat kidney extract was reported elsewhere. 23 The comparison of the molecular mass of the complex with that of the apoMT indicates the presence of a disulfide bridge formed by oxidation of Cys by Cu(II) leading to the loss of additional two protons. 17,47 Indeed, the product ion mass spectra of Cd2Cu2MT2-a (cf. Figure 6), show a non-fragmented region (from Gly10 until Cys37) of the sequence suggesting the binding of four metal ions to Cys. This binding site is shared between the R and � domains of the MT2-a which is in agreement with the hypothesis of Vaher et al. 17 We can also notice that the unfragmented central region binding metals contains 11 Cys like the R domain of MT2a which would explain the stabilization of Cd2Cu2-MT2a complex. 48 CONCLUSIONS µHPLC with the combined elemental detection by ICP MS, subppm molecular mass determination by electrospray orbital trap MS in conditions favoring metalation and demetalation, and HCD fragmentation allowing online protein sequencing was demonstrated as the, to date, most comprehensive approach to the characterization of metallothioneins in a biological cytosol. The detection limits down to the low femtomole levels allowed the characterization of metallothionein subisoforms induced in cell cultures exposed to CdS nanoparticles confirming the presence of known and the identification of new MT subisoforms. ACKNOWLEDGMENT The contribution of the Region of Aquitaine and the FEDER funds via CPER A2E (31486/08011464) project is acknowledged. We thank ICMCB, Bordeaux, for providing the 10 nm CdS nanoparticles used in this study. SUPPORTING INFORMATION AVAILABLE Figure 1-ESI, Figure 2-ESI, and Figure 3-ESI. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 12, 2010. Accepted July 8, 2010. AC101245H Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6957
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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),
Page 161 and 162: the multielement capabilities, the
Page 163 and 164: RESULTS AND DISCUSSION Sulfur Detec
Page 165 and 166: Table 2. Molecular Properties and C
Page 167 and 168: Anal. Chem. 2010, 82, 6911-6918 Dir
Page 169 and 170: dilution and hybridization buffer.
Page 171 and 172: solution under appropriate incubati
Page 173 and 174: Figure 4. Standardization curve for
Page 175 and 176: Anal. Chem. 2010, 82, 6919-6925 Ele
Page 177 and 178: the ×10 objective, to have a large
Page 179 and 180: Figure 2. With a suitable removal o
Page 181 and 182: the NB signal in a much better foot
Page 183 and 184: Scheme 1. Reactions of Selenium Rea
Page 185 and 186: Figure 2. (a) ESI-MS spectrum showi
Page 187 and 188: Figure 4. (a) ESI-MS spectrum showi
Page 189 and 190: Anal. Chem. 2010, 82, 6933-6939 Dif
Page 191 and 192: trode 28 by a finite element using
Page 193 and 194: Figure 4. Comparison between simula
Page 195 and 196: Figure 6. Comparison between simula
Page 197 and 198: educed in the vicinity of double bo
Page 199 and 200: Figure 2. Normalized product ion ab
Page 201 and 202: Figure 4. EID (a) and IRMPD (b) of
Page 203 and 204: Anal. Chem. 2010, 82, 6947-6957 Ide
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: Figure 5. Extracted ion current ESI
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
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
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
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Anal. Chem. 2010, 82, 7015-7020 Sel
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Table 1. Effect of 1 D-LC Condition
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Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
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luciferase (T7 control vector) was
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Figure 3. Inhibitory effects of luc
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Anal. Chem. 2010, 82, 7027-7034 Pat
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Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
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Table 3. Film Thickness Measurement
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Anal. Chem. 2010, 82, 7035-7043 Qua
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Figure 1. (A) FL spectra of BSPOTPE
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Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
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Scheme 1. Proposed Mechanism for Fl
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one or more drawbacks including poo
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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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