Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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of two immiscible electrolyte solutions (ITIES) and has allowed researchers to design reversibly operating polyion sensors, 19-21 flash chronopotentiometric electrodes for rapid determination of kinetically labile components, 22 ion channel mimetic systems, 23 thin layer coulometric sensors, 24 thin layer electrodes for stripping voltammetry, 25 as well as controlled current coulometric sensors. 26 The requirements for the realization of all solid state voltammetric ion sensors are more difficult to satisfy than the potentiometric sensors mentioned above, since the required redox capacity must be sufficiently high to sustain the ion transfer current. Shvarev showed that PEDOT is a promising conducting polymer underlayer for this purpose, 27 Amemiya 25,28 and our group 29 studied conducting polymers for thin layer voltammetric applications. With these materials, the redox properties are typically conducive to anion extraction from the sample solution. To allow for cation extraction, the polymer may sometimes be doped to obtain its stable oxidized form. 29 An alternative strategy was suggested by Samec et al. 30 by the incorporation of lipophilic ferrocene derivatives. This appears to be an attractive alternative to conducting polymer based systems, owing to the well-defined reversible redox couple and a potentially high redox capacity of the resulting membranes. Freely dissolving such redox active species may, however, result in eventual leaching from the membrane (especially in its oxidized, positively charged form) and in interferences from redox active species in the sample. To address these limitations and to introduce an alternative material, the chemical attachment of ferrocene groups to polymer chains is explored here. From a chemical point of view, poly(vinyl chloride) is easily modifiable due to the susceptibility for nucleophilic substitution of the chlorine atoms. 31,32 The most commonly used method for introducing new functional groups into the PVC chain is the reaction with sodium azide. 33,34 This modification was used for example in the cross-linking of the polymer. 35 Recently, the Huisgen cycloaddition, known also as “click chemistry”, has become a very popular method of transforming azide groups. 36 This type of reaction is attractive because of its high selectivity, mild reaction conditions, very high yields, and lack of byproducts. It was successfully applied for modification of a wide range of (19) Shvarev, A.; Bakker, E. J. Am. Chem. Soc. 2003, 125, 11192–11193. (20) Samec, Z.; Trojanek, A.; Langmaier, J.; Samcova, E. Electrochem. Commun. 2003, 5, 867–870. (21) Yuan, Y.; Amemiya, S. Anal. Chem. 2004, 76, 6877–6886. (22) Gemene, K. L.; Bakker, E. Anal. Chem. 2008, 80, 3743–3750. (23) Xu, Y.; Bakker, E. Langmuir 2009, 26, 568–573. (24) Yoshizumi, A.; Uehara, A.; Kasuno, M.; Kitatsuhi, Y.; Yoshida, Z.; Kihara, S. J. Electroanal. Chem. 2005, 581, 275. (25) Kim, Y.; Amemiya, S. Anal. Chem. 2008, 80, 6056–6065. (26) Bhakthavatsalam, V.; Shvarev, A.; Bakker, E. Analyst 2006, 131, 895–900. (27) Perera, H.; Fordyce, K.; Shvarev, A. Anal. Chem. 2007, 79, 4564–4573. (28) Guo, J.; Amemiya, S. Anal. Chem. 2006, 78, 6893–6902. (29) Si, P.; Bakker, E. Chem. Commun. 2009, 35, 5260–5262. (30) Langmaier, J.; Olsak, J.; Samcova, E.; Samec, Z.; Trojanek, A. Electroanalysis 2006, 18, 1329–1338. (31) Kameda, T.; Ono, M.; Grause, G.; Mizoguchi, T.; Yoshioka, T. Polym. Degrad. Stab. 2009, 94, 107–112. (32) Kameda, T.; Fukuda, Y.; Grause, G.; Yoshioka, T. J. Appl. Polym. Sci. 2010, 116, 36–44. (33) Sacristan, J.; Mijangos, C.; Reinecke, H.; Spells, S.; Yarwood, J. Macromolecules 2000, 33, 6134–6139. (34) Martinez, G. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 2476–2486. (35) Jayakrishnan, A.; Sunny, M. C. Polymer 1996, 37, 5213–5218. (36) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596–2599. 6888 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 different polymers, 37 but it has not yet become popular for the modification of PVC. Considering that the modified polymer is intended to be applied for electrochemical electrode preparation, any redox activity is expected to be related to the ferrocene groups only. The redox active properties of a triazol linker obtained by applying the Huisgen cycloaddition has been studied by Kirrs and co-workers, 38 revealing that it is indeed electrochemically inert. In this paper we present studies on properties of membranes containing poly(vinyl chloride) modified with ferrocene groups via the click-chemistry reaction. EXPERIMENTAL SECTION General Methods and Materials. High molecular weight poly(vinyl chloride) (PVC), sodium azide, ethynylferrocene, Lascorbic acid, copper sulfate pentahydrate, tetradodecylammonium tetrakis(4-chlorophenyl)borate (ETH 500), bis(2-ethylhexyl) sebacate (DOS), potassium hexacyanoferrate (III), potassium chloride, sodium perchlorate, sodium nitrate, sodium thiocyanate, anhydrous tetrahydrofuran (THF), and dimethylformamide (DMF) were purchased from Sigma Aldrich and used without further purification. Aqueous solutions were prepared by dissolving the appropriate salts in Milli-Q-purified distilled water. IR spectra were taken using a Perkin-Elmer Spectrum 100 FT- IR spectrometer with attenuated total reflectance (ATR) sampling accessory and ATR corrected. UV-vis spectra were recorded in the 900-350 nm wavelength range on a GBC UV-vis 916 spectrophotometer using 1 cm thick quartz cuvettes. The UV-vis calibration curve was obtained for three samples containing 2, 6, and 12 µmol/mL of ferrocene dissolved in a matrix containing 33 mg of PVC per mL of THF. The first order derivative of the spectrum was used to determine the content of ferrocene attached to the PVC. Glassy carbon rods of 5 mm diameter were purchased from SPI Supplies/Structure Probe, Inc. (West Chester, PA) and assembled into an epoxy resin body (EpoFix Kit, Struers Australia, Milton QLD, Australia). Synthesis. The substitution reaction of PVC with NaN 3 was carried out using 1 g (16 mmol based on monomeric unit) of PVC and 1.04 g (16 mmol) of NaN3 in 42 mL of DMF-water mixture (5:1 volume ratio) and heated at 50 °C under nitrogen for various times from 24 to 168 h. When the desired degree of modification was achieved, the reaction mixture was cooled to room temperature, the product was filtered, washed with distilled water and methanol, and subsequently dried under reduced pressure. In the click-chemistry reaction between azide-modified PVC and ethynylferrocene, 300 mg of N3PVC, 60 mg (0.29 mmol) of ethynylferrocene, 140 mg (0.56 mmol) of CuSO4 · 5H2O, and 500 mg (2.84 mmol) of ascorbic acid were added to 28 mL of a THF-water mixture (6:1 volume ratio). After 4hofreaction time, the product was precipitated with water. Ferrocene modified PVC was filtered, washed thoroughly with methanol, and then dissolved in 20 mL of THF. The THF solution was then filtered again to remove insoluble impurities, and the product was precipitated with methanol, filtered, and dried under reduced pressure. Composition of Membrane Cocktails. Membrane cocktail was prepared by dissolving 7.5 mg of FcPVC, 37.5 mg of DOS, (37) Lutz, J.-F. Angew. Chem., Int. Ed. 2007, 46, 1018–1025. (38) Verschoor-Kirss, M.; Kreisz, J.; Feighery, W.; Reiff, W. M.; Frommen, C. M.; Kirss, R. U. Organomet. Chem. 2009, 694, 3262–3269.
Figure 1. Infrared spectra of (A) unmodified PVC, (B) azide-modified PVC, and (C) ferrocene-modified PVC. Insets: structures of azide modified PVC and FcPVC. and 5 mg of ETH 500 in 1 mL of THF. DOS is a plasticizer commonly used as a solvent in PVC-based (liquid) membranes. The lipophilic electrolyte ETH 500 is added to provide counterions for the extracted analyte in the membrane and is also used to keep the electrical resistance of the membrane low. Electrodes were prepared by drop casting 40 µL of cocktail and letting the THF evaporate overnight under ambient conditions. Electrochemical Experiments. All cyclic voltammetry experiments were performed at a 50 mV s -1 scan rate. Normal pulse voltammograms were performed in a -0.6 to +0.8 V potential range using 10 mM solutions of NaCl, NaSCN, NaNO3, and NaClO4. Potential pulses (1 s) with a 20 mV potential increase were followed by a5sregeneration pulse (baseline potential) at open circuit potential (as measured before each voltammetric experiment). Membrane doping was performed with a single pulse of 10 min duration in a potential range of 400-800 mV in 10 mM NaClO4. A time of 10 min was chosen experimentally as the shortest time allowing the observed current to sufficiently decay. In the sandwiched electrode experiment, a three pulse procedure was applied. The first 30 s pulse at 0 mV was to confirm the steady state of the system before a 300 s long pulse at 200 mV was applied to induce electrochemical processes, while the last, carried out for 300 s at 0 mV, was to allow the system to return to its original state. Cyclic voltammetry in the presence of hexacyanoferrate(III) ion was performed in 1 mM K3Fe(CN)6 solution with 0.1 M KCl as a background, and the potential was scanned between a 0.8 and -0.5 V potential range starting from 0.4 V and going to negative potentials first. The stability of the signal generated by the FcPVC modified electrode was examined by application ofa1spulse at 0.5 V and followed by 20 s regeneration at the applied open circuit potential, using a three electrode configuration and 10 mM sodium perchlorate. The procedure was repeated 40 times. The redox capacity of the FcPVC was tested in the same solution by applying a 0.8 V potential for 30, 60, 120, 300, and 600 s, each time followed by a discharge pulse at 0 V for 200 s. Table 1. Reaction Time of Azidation and Resulting Degree of Ferrocene Modification of PVC, Estimated by Spectrophotometry reaction time [h] degree of modification [mol %] 24 1.9 60 3.2 120 5.3 168 6.1 RESULTS AND DISCUSSION In an initial step, PVC was modified with azide groups using NaN3 according to Sacristan et al. 33 The efficiency of the reaction was confirmed by comparing the IR spectrum for commercial, not modified PVC, and the same polymer after modification with azide groups. As shown in Figure 1A,B, the distinctive azide band at 2100 cm -1 , not visible for unmodified PVC, gives a well developed peak after reaction, confirming the effective partial modification of the polymeric backbone with azide groups. Further modification of azide-modified PVC with ferrocene groups required the use of a large excess of catalyst (200 mol % relative to the ethynylferrocene) to allow a high yield of reaction. As seen in Figure 1C, the IR spectrum for the reaction product did not reveal any signal from azide groups, indicating full conversion of the substrate. Since the ferrocene groups absorb strongly at 440 nm, the content of the ferrocene groups in the obtained FcPVC could be conveniently estimated from a calibration curve prepared for mixtures of nonmodified PVC with various ethynylferrocene contents dissolved in THF. Since the baselines for a THF solution of the ferrocene-modified PVC were significantly different, the ferrocene content was calculated based on spectral derivatives. On the basis of the spectrophotometric results and considering complete conversion of azide to ferrocene in the polymer backbone (as indicated by FT-IR) it may be concluded that the content of the ferrocene groups in PVC can be easily controlled by adjusting the time of azidation reaction. As shown in Table 1 for the polymer that has been modified with azide Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6889
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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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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
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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
Page 111 and 112: mercial microarray scanner and fabr
Page 113 and 114: Figure 2. Optical transmission meas
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Page 117 and 118: data as well. The 41 genes in Table
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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
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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
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Page 147 and 148: Figure 3. Cyclic voltammograms obta
Page 149 and 150: Figure 6. Calculated charge from ch
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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
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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
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Page 191 and 192: trode 28 by a finite element using
Page 193 and 194: 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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