Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 3. ESI-MS spectrum showing the recovery of unmodified GSH 30 min after adding DTT to the reaction mixture of GSH and ebselen. is completed in seconds at room temperature. 41,42 This is an advantage for shortening analysis time in real-world applications, particularly for high-throughput proteomics analysis. Reactions with Peptide Thiols. In addition to reacting with amino acid cysteine, both ebselen and the selenium reagent 2 were also tested with thiol peptides. Figure 2a displays the ESI- MS spectrum showing the reaction of GSH (0.1 mM, the structure of this tripeptide is shown in the figure inset) with ebselen (0.2 mM) in acetonitrile/water (1:1 by volume) containing 1% acetic acid. As expected, the reaction product resulting from the reaction of ebselen with GSH (m/z 583) was observed. As shown in the CID spectrum of m/z 583 (Figure 2b), water loss (the formation of m/z 565), backbone cleavages (the formation of m/z 508 and m/z 454), and the protonated GSH (m/z 308) and ebselen (m/z 276) were seen, confirming the product ion structure and the addition of peptide thiol onto ebselen (step a of eq 1 in Scheme 1). It can be seen that, besides backbone cleavages, Se-S bond cleavage was also observed leading to the formation of the fragment ion of m/z 276 and 308, which may be driven by the reformation of the five-membered ring of ebselen as assisted via the nucleophilic attack of selenium by the adjacent amide nitrogen of the ebselen tag (shown in the inset of Figure 2b). On the basis of this hypothesis, the CID behavior of the derivatized peptide ions could be tuned, simply by removing the adjacent amide group of the derivatizing selenium reagent. We thus tested the reagent 2. Figure 2c illustrates ESI-MS spectrum showing the reaction of GSH (0.1 mM) with reagent 2 (0.5 mM) in acetonitrile/water (1:1 by volume) containing 1% acetic acid, in which the reaction product (m/z 464) is seen. As shown in eq 2 in Scheme 1, the derivatized thiol tag does not contain amide group as the reagent 2 is split during the reaction. Indeed, the CID of the product ion at m/z 464 (Figure 2d) does not lead to the cleavage of the Se-S bond. Instead, it shows the loss of one water molecule (the formation of m/z 446), the backbone cleavages (the formation of m/z 389, 335, and 318), and the cleavage of the S-C bond (the formation of m/z 189). In this case, the selenium tag is stable, surviving the CID process, which would be valuable in top-down proteomics (41) Cotgreave, I. A.; Mogenstern, R.; Engman, L.; Ahokas, J. Chem.-Biol. Interact. 1992, 84, 69–76. (42) Haenen, G. A. M. M.; Rooij, B. M. D.; Vermeulen, N. P. E.; Bast, A. Mol. Pharm. 1990, 37, 412–422. 6930 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 studies (e.g., for pinpointing thiol location). In Figure 2c, one peak at m/z 191 corresponding to the protonated PhSe(dO)OH probably arose from the impurity of the reagent 2 used, which was technical grade. In addition, it appears that ebselen is more reactive than reagent 2, probably due to the tension of its five-membered ring. The calculated Se-N bond length of ebselen of 1.916 Å is longer than that of reagent 2 (1.855 Å, Figure 2-S in the Supporting Information), so the Se-N bond of ebselen is easier to break by thiol than that of reagent 2. Further support for this conclusion comes from the fact that nearly all of the GSH was reacted with ebselen in the mixing ratio of 1:2 (GSH to ebselen, Figure 2a), while a small amount of GSH remained when the ratio of GSH to reagent 2 used was 1:5 (Figure 2c). In the case of labeling by ebselen, the facile cleavage of Se-S could be useful in selective detection of cysteine containing peptides from mixtures, based on the resulting characteristic fragment ion of m/z 276. In this experiment, selective reaction of ebselen (0.1 mM) with GSH (50 µM) in the presence of other peptides of angiotensin II, bradykinin, and MRFA (50 µM for each) in methanol/water (1:1 by volume) containing 1% acetic acid was carried out. As illustrated in Figure 2e, the ESI-MS spectrum displays that the reaction of ebselen with GSH is highly selective, only a product (m/z 583) resulting from the reaction of ebselen with GSH was detected. However, the spectrum has multiple peaks and appears complicated. Precursor ion scanning (PIS) was applied to ions generated by ESI of the same reaction mixture and a much clearer spectrum containing only m/z 276 (the protonated ebselen) and 583 (the protonated ion of ebselenderivatized GSH) are seen (Figure 2f), in which cysteinecontaining peptide GSH can be rapidly identified. Reversibility of a derivatization reaction is very important as it allows enrichment and purification of analyte compounds from complex matrices. 43 However, as already mentioned above, commonly used thiol derivatization reactions such as nucleophilic substitution and Michael-addition are irreversible, limiting their analytical utilities. In this study, the reversibility of the thiol derivatization reaction was investigated by using dithiothreitol (DTT) as a reductant. As shown in Figure 3, the ESI-MS spectrum reveals the recovery of GSH (m/z 308) 30 min after adding DTT (0.5 mM) to the reaction mixture containing GSH (5 µM) and ebselen (10 µM). In the spectrum, one can see that m/z 583, the protonated molecule of the ebselen-derivatized GSH, disappears upon reaction with DTT. In addition, m/z 553 arose, probably indicating that the resulting reduction product, ebselen selenol (compound 5, Scheme 1), was air sensitive and readily oxidized by O 2 in the ambient environment into a more stable diselenide Se-Se product. 41,44 The CID spectrum of m/z 553 shows the loss of PhNH2 and the formation of the protonated ebselen (m/z 276, Figure 3-Sa in the Supporting Information), in agreement with the peak assignment. The m/z 575 corresponds to the sodiated molecule of the diselenide (see its CID in Figure 3-Sb in the Supporting Information). These results clearly demonstrate the excellent reversibility of the thiol derivatization. In combination with the extraordinary selectivity of the reaction, this reactivity (43) Smith, M. E. B.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L. M.; Papaioannou, D.; Waksman, G.; Caddick, S.; Baker, J. R. J. Am. Chem. Soc. 2010, 132, 1960–1965. (44) Engman, L.; Hallberg, A. J. Org. Chem. 1989, 54, 2964–2966.
Figure 4. (a) ESI-MS spectrum showing �-lactoglobulin A; (b) ESI-MS spectrum showing the �-lactoglobulin A fully derivatized by ebselen (charge numbers are labeled with primes). The insets show the corresponding deconvoluted spectra; (c) high-resolution Orbitrap MS spectrum showing the exact match of the isotopic peak distribution between the simulated (shown in blue discrete line) and detected peak (shown in black solid line) of 16+ derivatized �-lactoglobulin A ions; (d) ESI-MS spectrum showing the partially derivatized �-lactoglobulin A (charge numbers are labeled with double primes) 30 s after mixing the protein (5 µM) with 1,4-benzoquinone (10 µM); in this spectrum, the major peaks correspond to intact protein ions. can be used in a variety of applications focused on enrichment and purification of biological thiols from cells and tissues. Reaction with Protein Thiols. Selenium derivatizing reagents were used for identification of free cysteine residues in proteins due to the high selectivity for thiol groups. �-Lactoglobulin A used as a model system in this study (162 amino acid residues) contains two disulfide bridges (Cys 66 -Cys 160 and Cys 106 -Cys 119 ) and one free Cys 121 . 45 Figure 4a shows the ESI-MS spectrum of �-lactoglobulin A (5 µM) in methanol/water (1:1 by volume) containing 1% acetic acid. Multiply charged ions of �-lactoglobulin A are detected, and deconvolution of the mass spectrum provides the protein mass of 18 364 Da (shown in the inset of Figure 4a). In Figure 4b, the ESI-MS spectrum shows the reaction of �-lactoglobulin A (5 µM) and ebselen (10 µM) in methanol/water (1:1 by volume) containing 1% acetic acid. Deconvolution of the mass spectrum of ebselen-derivatized �-lactoglobulin A provides a mass of 18 639 Da (shown in the inset of Figure 4b). After derivatization of �-lactoglobulin A by ebselen, a mass gain of 275 Da was observed, indicating addition of one ebselen (MW 275 Da) to the sole free cysteine residue of the protein. Furthermore, only the ions of the ebselen-derivatized �-lactoglobulin A were seen in the spectrum shown in Figure 4b, suggesting that all of the protein was reacted with ebselen and a quantitative conversion yield was obtained. High-resolution mass analysis using Orbitrap MS was used for unambiguous identification of the derivatization product. (45) Surroca, Y.; Haverkamp, J.; Heck, A. J. R. J. Chromatogr., A 2002, 970, 275–285. Figure 4c displays the Orbitrap MS spectrum showing the exact match between the simulated (shown in blue discrete line) and experimentally observed (shown in black solid line) isotope distribution of the 16+ charge state of the derivatized �-lactoglobulin A ions. Furthermore, we performed tandem MS analysis using the LTQ-Orbitrap. High-resolution Orbitrap CID MS 2 spectrum of the 16+ derivatized �-lactoglobulin A ions (m/z 1166, in Figure 4-S in the Supporting Information) contains fragment ions y′139 12+ and y′130 11+ that carry the selenium tags. This suggests that the derivatization site is located on the last 130 amino acid residues of the protein, in agreement with the position of the free cysteine residue in �-lactoglobulin A (Cys 121 ). These results indicate the potential use of the selenium derivatization strategy in top-down proteomics studies. In the case of �-lactoglobulin A derivatization, ebselen was compared with one commonly used thiol tagging reagent, 1,4benzoquinone. It was found by ESI-MS that the protein can be fully reacted with ebselen 30 s after mixing (The spectrum is the same as shown in Figure 4b). By contrast, under the same conditions (e.g., concentrations and solvents used for the reaction were kept the same), only ∼30% protein was derivatized with 1,4benzoquinone 30 s after mixing the protein and the reagent. This indicates that the labeling using selenium reagents is much faster and more efficient than using the Michael-addition reagents such as 1,4-benzoquinone. The result suggests that the selenium chemistry would be quite useful in the high-throughput analysis of thiol containing proteins. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6931
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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
Page 135 and 136: NaCl, phosphate buffer saline (PBS)
Page 137 and 138: Figure 2. Product ion mass spectra
Page 139 and 140: -70 °C resolved the problem, givin
Page 141 and 142: Table 1. Intraday Precision and Acc
Page 143 and 144: Anal. Chem. 2010, 82, 6887-6894 Fer
Page 145 and 146: Figure 1. Infrared spectra of (A) u
Page 147 and 148: Figure 3. Cyclic voltammograms obta
Page 149 and 150: Figure 6. Calculated charge from ch
Page 151 and 152: Anal. Chem. 2010, 82, 6895-6903 Ele
Page 153 and 154: Table 1. Chemical Structure, pKa Va
Page 155 and 156: pH with a tilted baseline (Figure 1
Page 157 and 158: Table 2. Linearity and Detection Li
Page 159 and 160: 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: Figure 2. (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 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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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
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Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
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Table 2. Capillary Electrophoresis
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Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
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Another approach to handle this lim
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(principal components, PCs) in whic
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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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