Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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on sample size (0.06 ‰ ng C 1- ), and linearity was best over the range of 1-10 ng C. The method was sensitive, requiring >0.2 ng C into the ion source to produce accurate and precise results. The analysis of ambient samples required small sample volumes, with ∼1.0 L of gas providing sufficient carbon for analysis. Clear distinctions in δ 13 C were observed between emissions released from plants and automobiles. In particular, ethanol emissions from automotive exhaust and metropolitan Miami were significantly enriched in 13 C. This is related to ethanol’s C4 plant origin and use as a fuel additive. Ambient samples can be differentiated, but the variation in δ 13 C values was not as great as for the source samples. Ambient samples suffer from additional complexity with multiple sources and sinks affecting single sampling locations. Clearly, more studies of sources and ambient sampling are required to define and characterize OVOCs in the troposphere along with laboratory studies to determine the kinetic isotope effects associated with OVOCs’ in situ production and loss from reaction with OH and photolysis. As it stands now, this technique can be used to differentiate OVOC sources and to assess the carbon isotopic 6806 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 values for OVOCs in ambient air. It should serve as a useful way to investigate transformations of organic gases in the atmosphere. ACKNOWLEDGMENT We thank Tom Brenna and Herbert Tobias for helpful discussions in developing this method and Rich Iannone for providing a template for raw data calculations. We acknowledge John Mak and Zhihui Wang for the working reference gas interlab comparison. We appreciate the efforts of Kevin Polk and his 1972 International Scout. Finally, we gratefully acknowledge the helpful comments made by two anonymous reviewers and support provided by NSF Grant No. 0450939. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 23, 2010. Accepted July 6, 2010. AC1007442
Anal. Chem. 2010, 82, 6807–6813 Direct Voltammetric Analysis of DNA Modified with Enzymatically Incorporated 7-Deazapurines Hana Pivoňková, † Petra Horáková, †,‡ Miloslava Fojtová, †,§ and Miroslav Fojta* ,† Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Královopolská 135, CZ-612 65 Brno, Czech Republic, Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, CZ-532 10 Pardubice, Czech Republic, and Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlárˇská 2, CZ-611 37 Brno, Czech Republic Nucleic acids studies use 7-deazaguanine (G*) and 7-deazaadenine (A*) as analogues of natural purine bases incapable of forming Hoogsteen base pairs, which prevents them from being involved in DNA triplexes and tetraplexes. Reduced propensity of the G*- and/or A*modified DNA to form alternative DNA structures is utilized, for example, in PCR amplification of guanine-rich sequences. Both G* and A* exhibit significantly lower potentials of their oxidation, compared to the respective natural nucleobases. At carbon electrodes, A* yields an oxidation peak which is by about 200-250 mV less positive than the peak due to adenine, but coincides with oxidation peak produced by natural guanine residues. On the other hand, oxidation signal of G* occurs at a potential by about 300 mV less positive than the peak due to guanine, being well separated from electrochemical signals of any natural DNA component. We show that enzymatic incorporation of G* and A* can easily be monitored by simple ex situ voltammetric analysis of the modified DNA at carbon electrodes. Particularly G* is shown as an attractive electroactive marker for DNA, efficiently incorporable by PCR. While densely G*-modified DNA fragments exhibit strong quenching of fluorescence of SYBR dyes, commonly used as fluorescent indicators in both gel staining and real time PCR applications, the electrochemical detection provides G*-specific signal suitable for the quantitation of the amplified DNA as well as for the determination of the DNA modification extent. Determination of DNA amplicons based on the measurement of peak G* ox is not affected by signals produced by residual oligonucleotide primers or primary templates containing natural purines. Electrochemical techniques are increasingly applied in the area of nucleic acids sensing (reviewed in refs 1-4). Nucleic acids * To whom correspondence should be addressed. E-mail: fojta@ibp.cz . † Academy of Sciences of the Czech Republic. ‡ University of Pardubice. § Masaryk University. (1) Fojta, M. In Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics; Palecek, E., Scheller, F., Wang, J., Eds.; Elsevier: Amsterdam, 2005, pp 386-431. (2) Fojta, M.; Jelen, F.; Havran, L.; Palecek, E. Curr Anal Chem 2008, 4, 250– 262. possess intrinsic electroactivity due to the presence of electrochemically oxidizable or reducible nucleobases, 2,4 making it possible to analyze them electrochemically without any labeling. Indeed, various label-free electrochemical techniques were proposed for the detection of DNA damage 1 or DNA hybridization. 3 In spite of these efforts, application of various redox indicators and labels proved useful particularly in sequence-specific DNA sensing requiring reliable discrimination between two complementary strands (e.g., target DNA and hybridization probe 5,6 ), specific determination of newly synthesized pieces or fragments of DNA (in primer extension or PCR-based assays 7-9 ) or even identification of a single nucleobase incorporated at a specific position (in SNP typing 9-11 ). Generally, introducing electroactive tags producing “new” specific electrochemical responses (not yielded by natural DNA components) increases specificity of the assays considerably. Electrochemically active moieties can be incorporated into nucleic acids during chemical oligonucleotide synthesis, postsynthetically by chemical modification of “natural” nucleic acids 5,6 or using modified nucleoside triphosphates (dNTPs) and DNA polymerases. 7-12 The latter approach represents a versatile way to facile construction of labeled or otherwise functionalized nucleic acids and to efficient sequence-specific DNA sensing. 12 A critical prerequisite for these applications is the ability of a DNA polymerase to use modified dNTPs as substrates for efficient incorporation without losing sequence-specificity. C7substituted 7-deazapurines and C5-substituted pyrimidines are usually acceptable substrates for (at least some) DNA polymerases (3) Palecek, E.; Fojta, M. Talanta 2007, 74, 276–290. (4) Palecek, E.; Jelen, F. In Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics.; Palecek, E., Scheller, F., Wang, J., Eds.; Elsevier: Amsterdam, 2005, pp 74-174. (5) Flechsig, G. U.; Reske, T. Anal. Chem. 2007, 79, 2125–2130. (6) Fojta, M.; Kostecka, P.; Trefulka, M.; Havran, L.; Palecek, E. Anal. Chem. 2007, 79, 1022–1029. (7) Brazdilova, P.; Vrabel, M.; Pohl, R.; Pivonkova, H.; Havran, L.; Hocek, M.; Fojta, M. Chem.-Eur. J. 2007, 13, 9527–9533. (8) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770– 772. (9) Vrabel, M.; Horakova, P.; Pivonkova, H.; Kalachova, L.; Cernocka, H.; Cahova, H.; Pohl, R.; Sebest, P.; Havran, L.; Hocek, M.; Fojta, M. Chem.- Eur. J. 2009, 15, 1144–1154. (10) Cahova, H.; Havran, L.; Brazdilova, P.; Pivonkova, H.; Pohl, R.; Fojta, M.; Hocek, M. Angew. Chem., Int. Ed. 2008, 47, 2059–2062. (11) Horakova, P.; Simkova, E.; Vychodilova, Z.; Brazdova, M.; Fojta, M. Electroanalysis 2009, 21, 1723–1729. (12) Hocek, M.; Fojta, M. Org. Biomol. Chem. 2008, 6, 2233–2241. 10.1021/ac100757v © 2010 American Chemical Society 6807 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Published on Web 07/23/2010
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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
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
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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 and 36: Figure 4. Analysis of 2a by HPLC-MS
Page 37 and 38: eaction of 1a with PBH can be condu
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
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Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
Page 61: Table 3. Ambient Measurement Result
Page 65 and 66: Polymerase (1 U per sample). Reacti
Page 67 and 68: of which was constant for all ampli
Page 69 and 70: analytically useful signals at less
Page 71 and 72: carbon black and RP-C18 for the ext
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Page 75 and 76: Table 2. Concentrations and Ratios
Page 77 and 78: Anal. Chem. 2010, 82, 6821-6829 Mac
Page 79 and 80: mg/mL protein, followed by separati
Page 81 and 82: Figure 2. Productivity of SEQUST an
Page 83 and 84: Figure 4. High-resolution MS/MS spe
Page 85 and 86: Figure 6. Characterization of PSMs
Page 87 and 88: containing T-T mismatches. 23 Based
Page 89 and 90: Figure 1. Extinction spectra of sol
Page 91 and 92: Figure 3. (A) The value of Ex650 nm
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Page 95 and 96: wished to explore the dehydration o
Page 97 and 98: constant medium for separation; we
Page 99 and 100: Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102: Figure 5. Quantitative calibration
Page 103 and 104: Anal. Chem. 2010, 82, 6847-6853 Met
Page 105 and 106: 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
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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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