Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 5. Analysis of urine sample using a running buffer consisting of 50 mM H3PO4-Tris, pH 4. Separation voltage: -10 kV with an injection time of 5sat-10 kV. Detection: BDD at +1.0 V vs Ag/ AgCl, 3 M NaCl. The urine samples were filtered using a 0.22 µm Millipore filter and diluted in the injection buffer with different dilution. (A) 10-fold dilution, pristine and spiked urine samples were presented as the lower and upper curve, respectively. Peak identification: (1) IXS; (2) VMA; (3) AA; (4) HVA; (5) UA; and (6) TRP. The diluted urine sample was spiked with 20 µM IXS, 20 µM VMA, 100 µM AA, 20 µM HVA, 100 µM UA, and 20 µM TRP. (B) 15-fold dilution, pristine and spiked urine samples were presented as the lower and upper curve, respectively. Peak identification: (1) IXS; (2) VMA; (3) AA; (4) HVA; (5) UA; and (6) TRP. The diluted urine sample was spiked with 20 µM IXS, 20 µM VMA, 100 µM AA, 20 µM HVA, 100 µM UA, and 20 µM TRP. For further LOD improvement, a series of experiments based on field-amplified sample stacking (FASS) 30 was performed by exploiting the conductivity difference between the sample zone and the running buffer to effect preconcentration. A standard solution of 6 analytes including ascorbic and uric acids, two endogenous urinary compounds, was prepared in 10 mM H3PO4 pH 2 and injected at -10 kVfor 10 s with the separation performed at -10 kV using 50 mM H3PO4 pH 4 as the running buffer. It should be noted that at pH 3, ascorbic acid migrated very closely with HVA, whereas uric acid was not baseline separated from TRP. In principle, the amount of stacking is proportional to the conductivity difference between the running (30) Weng, Q.-F.; Xu, G.-W.; Yuan, K.-L; Tang, P J. Chromatogr. B 2006, 835, 55–61. 6902 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 6. Analysis of an urine sample using a running buffer consisting of 50 mM H3PO4-Tris, pH 4. Separation voltage: -10 kV with an injection time of 5sat-10 kV. Detection: BDD at +1.0Vvs Ag/AgCl, 3 M NaCl. The urine sample was filtered using a 0.22 µm Millipore filter and diluted 8-fold in the injection buffer. (A) with sample stacking and (B) without sample stacking. buffer and the sample solution. A preconcentration factor of 4 was observed for IXS, AA, and HVA compared to 3 for VMA and UA and only 1.25 for TRP (Figure 4, curve b). With sample stacking, the detection limit of IXS, AA, and HVA was 75 nM compared with nonstacking (300 nM, Figure 4, curve a), significantly lower than the physiological levels of these biomarkers in urine as discussed later. Notice that peak-width was broadened when the injection time was greater than 10 s (data not shown). In another attempt, the capillary was first filled with 50 mM H3PO4 pH 4 followed by a sample injection as described above. The cathodic running buffer was changed to 10 mM H3PO4, pH2 instead of 50 mM H3PO4, pH 4. In this case, the separation window was narrower, resulting in the comigration of three pairs: IXS/VMA, AA/HVA, and UA/TRP (Figure 4, curve c). No further improvement in separation resolution was attained by adding several organic modifiers including 10% methanol, 10% acetonitrile, 5 mM methyl �-cyclodextrin, and 5 mM cetyl trimethylammonium bromide in the running buffer. Analysis of Biomarkers in Urine. The electropherogram of an authentic urine sample obtained from a healthy female shows 5 low peaks and one very high peak. All biomarkers were detected even if the urine sample was diluted to 10- and 15-fold, respectively in the injection buffer (Figure 5). Standard IXS, HVA, VMA,
ascorbic acid (AA), uric acid (UA), and TRP were spiked into the urine sample for peak identification. Dilution of the urine sample also improved peak to peak separation and peak identification. All peaks were identified and the sequence was assigned as IXS, VMA, AA, HVA, UA, and TRP. The highest peak was attributed to UA as expected since its normal concentration in urine ranges from 1.23 to 3.7 mM (1.48-4.43 mmol/day with an average urine volume of 1200 mL). 31 The UA peak was confirmed by matching the migration time and spiking the urine sample with 30 µM uric acid. Peak deconvolution and peak area were obtained using WinPLOTR (Version: May, 2009, http://www.cdifx.univ-rennes1.fr/ winplotr/winplotr.htm). The concentration for each analyte was estimated by comparing the peak areas of the authentic and spiked urine samples. The average concentration of IXS, VMA, HVA, AA, and UA for two different urine samples obtained from the same healthy female was determined to be 170, 55, 87, 142, and 1,075 µM, respectively. Normal levels of such analytes in urine are 100-1000 µM for IXS, 32 50-1000 µM for VMA, 10 14-125 µM for HVA, 10 and 150-200 µM for AA, 33 i.e., the values estimated by CE-ECD fall in the normal range for healthy subjects. Therefore, sample staking was not mandatory for the simultaneous analysis of IXS, HVA, and VMA in the presence of UA, AA, and TRP since the LOD of such analytes was significantly lower than their normal physiological levels. Sample stacking of the urine samples at low dilution (below 5-fold) did not improve the detection limits (figure not shown), very likely due to a high level of salts in the urine sample. With an 8-fold dilution, sample stacking provided a significant improvement in the LOD with a sharper corresponding peak for each analyte as shown in Figure 6A. In particular, sample stacking also allowed for the detection of TRP, the last peak that migrated very (31) Tietz, N. W. Fundamentals of Clinical Chemistry: W.B. Saunders Co.: Philadelphia, 1987. (32) Harlit, H. J. Biol. Chem. 1933, 537–545. (33) Ridi, E.; Moubasher, M. S. R.; Hassan, Z. F. Biochem. J. 1951, 49, 246– 251. (34) Matsuo, M.; Tasaki, R.; Kodama, H.; Hamasaki, Y. J. Inherit. Metab. Dis. 2005, 28 (1), 89–93. (35) Luo, D.; Wu, L.; Zhi, J. ACS Nano 2009, 3 (8), 2121–2128. closely to the uric acid peak. In addition, both HVA and VMA were positively detected and identified compared with nonsample stacking (Figure 6B). Thus, the method became useful for estimation of the urine HVA/VMA ratio, a useful screening method for Menkes disease. This ratio could range from 4.1 to 69.7 owing to impaired activity of dopamine �-hydroxylase, a copper-dependent enzyme. 34 CONCLUSIONS In brief, a novel scheme was described for electrophoretic separation and detection of several important biomarkers in urine. A fused silica capillary with a coating layer of PDDA and AuNPs reversed the electroosmotic flow and served as a stable layer for resolving the analytes. No electrode fouling was observed during repeated analysis and even with the urine sample. The detection limit obtained for IXS, HVA, and VMA was considerably below their normal physiological levels in biological samples. The method was simple and capable of measuring several important biomarkers in urine samples without sample pretreatment with excellent selectivity and detection sensitivity. Sample stacking could be easily performed to improve detection limits of these analytes in urine samples provided such samples were diluted properly to reduce the level of uric acid and salts. Furthermore, many other basic neurotransmitters and their acidic metabolites could also be detected. Notice also that a BDD nanoforest electrode (BDDNF) can be fabricated by hot filament chemical vapor deposition. 35 This type of electrode exhibits improved detection sensitivity compared to conventional planar BDD electrodes. Integration of BDDSNF with capillary electrophoresis is a subject of future endeavor. ACKNOWLEDGMENT The authors thank the Science Foundation Ireland (SFI) for an SFI Walton Fellowship (JHTL), an IRCSET Embark Award (LZ), and an SFI-SRC Grant for the Irish Separation Science Cluster (ISSC). Received for review April 27, 2010. Accepted July 5, 2010. AC101105Q Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6903
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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
Page 107 and 108: Table 1. Counting Properties and De
Page 109 and 110: Table 3. Analysis of 226 Ra in Sedi
Page 111 and 112: mercial microarray scanner and fabr
Page 113 and 114: Figure 2. Optical transmission meas
Page 115 and 116: Figure 5. Volcano plots detailing t
Page 117 and 118: data as well. The 41 genes in Table
Page 119 and 120: corresponding compound if its chemi
Page 121 and 122: Figure 1. Schematic illustration of
Page 123 and 124: Table 1. Absolute Quantification Re
Page 125 and 126: ment. Therefore, the long-time drea
Page 127 and 128: Figure 1. Chemically actuated micro
Page 129 and 130: Figure 2. Influence of a surfactant
Page 131 and 132: Figure 5. Device to eject and mix s
Page 133 and 134: 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: Table 2. Linearity and Detection Li
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
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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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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Figure 5. (A) MALDI-TOF/TOF product
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
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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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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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