Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 6. Analysis of 2b by HPLC-MS/MS using ESI in the positive ion mode. The cone voltage was set to 50 V and the collision energy was set to 30 eV. Selected ion chromatogram for the ion at m/z 703 (A). Mass spectrum of the peak corresponding to 2b at 12 min (B). Fragment ion spectrum of m/z 703 (C). samples contain cholesterol 5R-OOH, which in the presence of acids is easily converted to 1a. 9 Moreover, the sensitivity of PBH and dansyl hydrazine methods were also compared (Figure S5 in the SI). PBH method conducted at neutral pH was almost 10 times more sensitive than dansyl hydrazine method. This difference was even larger when PBH reaction was carried out with acid, reaching a 90 times higher sensitivity. PBH was also used for the quantification of ChAld formed during cholesterol oxidation promoted by the HOCl/H 2O2 reaction system. This is a biologically relevant reaction system that is known to generate stoichiometric amounts of singlet molecular oxygen. The formation of 2a was analyzed by HPLC coupled with fluorescence detector (Figure 7). The complete reaction system containing cholesterol and HOCl/H2O2 showed the appearance of an intense fluorescent peak corresponding to 2a. The fluorescent adduct was quantified using the calibration curve and a value of 0.2 µM of1a was found. This corresponds to a yield of 0.2%. DISCUSSION Cholesterol is oxidized in the presence of reactive oxygen species generating aldehydes, hydroperoxides and epoxides. 6,7 Recently, two cholesterol aldehydes have attracted attention, the cholesterol carboxyaldehyde (1a) and cholesterol secoaldehyde (1b). The formation of 1a and 1b was first described in the oxidation of cholesterol with ozone. 11,12 Wentworth and co-workers reported the presence of 1a and 1b in atherosclerotic plaques and related the detection of these aldehydes as an evidence for ozone generation in human tissues. 8 On the other hand, two recent studies identified the generation of 1a in the reaction of cholesterol with singlet molecular oxygen. 9,10 Indeed, Brinkhorst and 6780 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 7. Analysis of cholesterol aldehydes generated by the reaction of cholesterol with H2O2 and HOCl using PBH. HPLC/ fluorescence chromatograms obtained for cholesterol (A), and cholesterol incubated with H2O2 (B), HOCl (C), and H2O2 + HOCl (D). Reactions were carried out with 100 µM cholesterol in the presence of 1 mM HOCl, 1 mM H2O2 and 100 µM PBHfor6hat37°C under continuous agitation. co-workers reported the formation of 1a from cholesterol 5Rhydroperoxide by Hock cleavage in acid media 9 and Uemi and co-workers reported the formation of 1a in the reaction of cholesterol with singlet molecular oxygen generated either by photooxidation or by the thermodecomposition of endoperoxides. 10 In this study, we have developed a new detection method for cholesterol carboxyaldehyde, 1a, using the fluorescent probe PBH, which contains the highly fluorescent pyrene group. The
eaction of 1a with PBH can be conducted in the absence of acids, which is important to avoid the formation of 1a from acid catalyzed cholesterol 5R-OOH decomposition. 9 Bosco and co-workers 14 used dansyl hydrazine as a fluorescent label to detected 1a and 1b in a brain tissue sample from Lewy body disease. However, the derivatization of cholesterol aldehydes with dansyl hydrazine is conducted in the presence of strong acids, such as, 5 mM sulfuric acid. 14 In fact, we have done a comparative analysis of the two fluorescent probes (PBH and dansyl hydrazine) to detect 1a in vitro and observed that the method using dansyl hydrazine can overestimate the basal level of 1a, especially in samples containing cholesterol 5R-OOH. The 1a yields in photooxidized cholesterol samples estimated by PBH (neutral pH) and dansyl hydrazine were 8.7% and 16.0%, respectively. This data indicates that the derivatization in the absence of acid is a critical point that should be considered when estimating 1a basal level in samples, especially when they were oxidized by singlet molecular oxygen. When comparing dansyl hydrazine and PBH methods, the latter was approximately 10 times more sensitive for 1a detection (Figure S5 in the SI). This difference was even larger when PBH derivatization was carried out at acid condition, clearly evidencing the superiority of PBH method compared to dansyl hydrazine in terms of sensitivity. A number of studies has detected the formation of cholesterol aldehydes in vitro, 9,10,21 as well as in vivo. 8,14-16 In this study, we have used PBH to analyze the formation of 1a during cholesterol oxidation mediated by ozone and singlet molecular oxygen. The reaction of cholesterol with ozone yielded 1a and 1b and also other minor unidentified products. We could not quantify exactly the amount of 1b due to the lack of an appropriate pure standard for it. Nonetheless, the relative peak intensities suggest that cholesterol ozonation yields 1b as a major product and a small amount of its aldolization product (Figure 5). The estimated ratio of 1a:1b in the ozonation was 1:10. Similar results were described for the ozonation of human LDL, where a relative yield of 1:4 was found. 13 On the other hand, cholesterol oxidation promoted by singlet molecular oxygen yielded 1a as the major product consistent with the data described by Brinkhorst et al. 9 and Uemi et al. 10 The estimated yield of 1a in the photooxidation of cholesterol was approximately 8.7% and the ratio of 1a:1b was 165:1. These values are in agreement with those reported for the photooxidation of human LDL using hematoporphyrin IX for 14 h where only 1a was detected. 13 The incubation of cholesterol in (26) Khan, A. U.; Kasha, M. J. Am. Chem. Soc. 1970, 92, 3293–3300. the presence of H2O2 and HOCl also gave 1a as the major product, consistent with the oxidation of cholesterol promoted by singlet molecular oxygen. It is known that the reaction of H2O2 with HOCl generate stoichiometric amounts of singlet molecular oxygen 26 and this reaction is suggested to play an important role during inflammatory conditions. 22 The predominant formation of 1a over 1b by this reaction was also reported by Tomono and co-workers. 21 They incubated cholesterol (100 µM) in the presence of H2O2 (100 µM) and NaOCl (100 µM) and detected five times more 1a than 1b using dansyl hydrazine as a fluorescent probe. 21 CONCLUSIONS We have developed a new sensitive method for the detection of cholesterol aldehydes using PBH as a fluorescent probe. This new methodology allows detecting and quantifying the relative amounts of 1a and 1b with high sensitivity, which is important to assess the relative contributions of ozone and singlet molecular oxygen to the oxidation of cholesterol in biological systems. Derivatization of cholesterol aldehydes using PBH can be conducted without the addition of strong acids, which is important for the determination of the basal level of cholesterol aldehydes present in biological tissues from different sources. In conclusion, the easy and reliable method developed in this study can help to investigate the formation cholesterol aldehydes in biological system, which is critical to clarify their relevance in disease progression. ACKNOWLEDGMENT This work was supported by the Brazilian research funding institutions FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), CNPq (Conselho Nacional para o Desenvolvimento Científico e Tecnológico), INCT de Processos Redox em Biomedicina - Redoxoma, and Pró-Reitoria de Pesquisa-USP. SUPPORTING INFORMATION AVAILABLE NMR analysis of 1a and 1b. HPLC-MS/MS detection of 2a and 2b. HPLC-fluorescence data showing the pH effect on 2a formation and the sensitivity of dansyl hydrazine and PBH methods. Dansyl hydrazine derivatization method. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 10, 2010. Accepted July 8, 2010. AC1006427 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6781
Page 1 and 2: Anal. Chem. 2010, 82, 6745-6750 Let
Page 3 and 4: purchased from Invitrogen-Molecular
Page 5 and 6: Figure 3. Electropherograms of TPP-
Page 7 and 8: Anal. Chem. 2010, 82, 6751-6755 Res
Page 9 and 10: (pH 8.0) with cysteine and cystamin
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
Page 15 and 16: coater at 2000 rpm for 20 s, and th
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Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
Page 23 and 24: Figure 1. GC/MS total ion chromatog
Page 25 and 26: Table 2. Concentrations and Stable
Page 27 and 28: on a substrate are preferred. 20-24
Page 29 and 30: Figure 4. SERS analysis of NAADP co
Page 31 and 32: Anal. Chem. 2010, 82, 6775-6781 Hig
Page 33 and 34: tion, 2 µL of proprionaldehyde wer
Page 35: Figure 4. Analysis of 2a by HPLC-MS
Page 39 and 40: Numerous references had demonstrate
Page 41 and 42: Figure 1. TEM images of the prepare
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Page 45 and 46: esult in a big SPR signal change wi
Page 47 and 48: also reduces chemical noise, which
Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
Page 53 and 54: Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56: Figure 1. Schematic view of the pre
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Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
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Page 63 and 64: Anal. Chem. 2010, 82, 6807-6813 Dir
Page 65 and 66: Polymerase (1 U per sample). Reacti
Page 67 and 68: of which was constant for all ampli
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Page 71 and 72: carbon black and RP-C18 for the ext
Page 73 and 74: solution in an equal volume, and 1
Page 75 and 76: Table 2. Concentrations and Ratios
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Page 79 and 80: mg/mL protein, followed by separati
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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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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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