Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Switzerland), was placed inside the chromatographic oven to bypass the combustion furnace when required. The valve has an extension between the body and the actuator which allows the valve to be mounted in the heated zone while the handle remains outside at ambient temperature. This valve prevented the solvent from entering the combustion unit and therefore enlarged the catalytic activity of the Cu and Pt wires. Additionally, the valve allowed the direct connection of the column with the EI source for conventional GC-MS work in order to identify the different species under analysis by their fragmentation pattern. Moreover, such qualitative analysis is essential to assess peak purity for each compound. For postcolumn isotope dilution analysis the valve was initially in the “load” position (combustion oven bypassed) and the valve was manually switched to the “inject” position at the same time that the EI filament switched on. All connections between the valve and the rest of the components of the system were performed by means of 0.32 mm i.d. deactivated fused silica capillaries and fixed to the valve with appropriate polyimide coated fused silica adapters (VICI AG International, Schenkon, Switzerland). Combustion Furnace. The laboratory-made combustion furnace consisted of a 60 cm long ceramic tube (3 mm O.D., 0.5 mm I.D.) (Elemental microanalysis, Devon, U. K.) filled with copper and platinum wires and heated by a Nichrome wire. Proper thermal isolation of the combustion furnace was provided with glass wool. The combustion furnace was set vertically on top of the GC with the lower end of the ceramic tube inside the chromatographic oven to avoid cold spots after the separation. High temperatures were accurately controlled using a temperature sensor and an external controller, allowing temperature settings inside the tube to be within ±1 °C over a wide temperature range (50-1200 °C). The copper wires were previously oxidized by passing an oxygen flow (1 mL/min) at 450 °C during 4-5 h and this procedure was performed on a weekly basis to preserve its oxidizing capabilities. Fused silica capillaries (0,32 mm i.d.) were used to connect the furnace to the valve and to the ion source respectively. For this purpose a length (∼1 cm) at one end of each capillary was uncoated, to prevent the polyimide coating to be burnt, and introduced into the ceramic tube. Reducing unions 1/8′′ to 1/16′′ (SGE, Victoria, Australia) and appropriate graphite ferrules (0.5 mm i.d.) were used to hold the capillaries in place at both sides of the ceramic tube. For oxidation and combustion, the furnace was operated at 850 °C. Gas Cylinder and Mass Flow Controller. The 13 CO2 container was a dual inlet 5 L high pressure stainless steel gas cylinder (Iberfluid, Barcelona, Spain). The cylinder was equipped on one end with, in this order, an opening valve, a Swagelok tee where helium could be introduced from a high pressure cylinder, and a second opening valve connected to the other end of the tee. This second valve was connected to a septum for the manual injection of gases into the cylinder. Before the cylinder was pressurized, 13 CO2 was injected into the container by means of a gastight syringe (Hamilton, Reno, U. S. A.) through this valve. After the injection of the tracer, the valve was closed and the container was pressurized up to 6 bar with helium using the connection in the Swagelok tee. When the set pressure was reached the filling valve was also closed. At the other end of the cylinder a pressure gauge, an opening 6864 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 valve and a mass-flow controller (Bronkhorst, Ruurlo, Netherlands) calibrated for He were coupled for the accurate control of the tracer flow. The opening valve was closed during the filling of the cylinder, remaining open the rest of the time. The flow rate for the postcolumn spike was set at 0.5 mL/min. Effluent and spike flows were mixed after combustion by means of a 0.25 mm bore stainless steel microvolume “Y” connector (VICI AG International, Schenkon, Switzerland). The whole instrumental setup is shown in Figure 1. As can be observed, the instrument can be operated in the standard GC- MS configuration (qualitative) or in the combustion-postcolumn configuration (quantitative) depending on the position of the switching valve. Procedures. Preparation of 13 CO2. The spike was prepared from 13 C enriched Na2CO3 (99%). An accurate weighed amount (∼200 mg) was placed in a 25 mL three-necked round-bottomed flask, previously purged with He to avoid natural abundances CO2 contamination from ambient air. A small quantity (300 µL) of concentrated H3PO4, was injected into the flask through a septum cap. After the acid-base reaction, 4 mL of the gaseous phase, containing 13 CO2 diluted in He, were removed using a gastight syringe and injected into the container shown in Figure 1. Calibration of the 13 CO2 Postcolumn Flow. The flow rate of the spike could be accurately controlled by the mass flow controller between 0.1 and 5 mL min -1 . In our experiments, it was set at 0.5 mL min -1 . The exact mass flow (ng of 13 CO2 per min) being mixed with the natural CO2 coming from the column and combustion furnace was determined by adding internal standards of known concentration spiked to the sample as described before. 11,15 Quantification using Postcolumn Isotope Dilution. In our case, the isotope ratio 12 C/ 13 C was measured as the signal ratio at masses 44 and 45 (I44/I45) corresponding to the continuous blend of natural abundance 12 CO2 present in the chromatographic eluent and the enriched 13 CO2, added postcolumn. Selected Ion Monitoring (SIM) at masses 44.0 and 45.0 was performed for the duration of the chromatogram with 70 ms integration time per mass. The mass window was ∼0.1 mass units. Then, the isotope ratio in the blend, Rb ) I44/I45, was calculated to build the isotope ratio chromatogram (Rb vs time). The postcolumn isotope dilution equation, 7 shown as equation 1 below, was then applied to every point in the chromatogram to obtain the mass-flow chromatogram (ng of C/min vs time). The integration of the mass flow chromatogram directly provided the amount of carbon (in ng) eluted in each chromatographic peak. MF n ) MF t AW n 13 At AWt An 12( Rb - Rt 1 - RbRn) In this equation MFn corresponds to the mass flow of carbon from the natural abundance sample injected, whereas MFt corresponds to the mass flow of carbon from the postcolumn spike or tracer. AWn and AWt correspond to the atomic weight of carbon in the sample and tracer, respectively. The isotope abundances At 13 and An 12 correspond to the isotopic composition of 13 C in the tracer and 12 C in the sample. Finally, Rt is the (1)
Figure 1. Schematic illustration of the setup showing the modification made in the GC-MS instrument. The two possible valve configurations are shown in the inset: Left, for structural identification by conventional GC-EI-MS analysis, and right, for absolute quantification using GC- Combustion-MS and postcolumn addition of 13 CO2. Adapted from reference. 15 isotope ratio (12/13) in the tracer and Rn is the natural isotope ratio (13/12) in the sample, whereas Rb is the measured isotope ratio in the blend. Additionally, eq 1 can be used to quantify the mass flow of enriched carbon dioxide when an internal standard of known concentration is spiked to the sample. Head-Space SPME Procedure. Test solutions (∼10 ng/g) were prepared by diluting appropriately the BTEX standards in Milli Q water. Samples of ∼7 g were pipetted into 10 mL glass vials with open-top phenolic closures and PTFE/silicone septa (Supelco, Bellefonte, USA). Three fiber coatings, 100 µm polydimethylsiloxane (PDMS), 70/30 µm polydimethylsiloxane/divinylbenzene (PDMS/DVB), and 50/30 µm carboxen/polydimethylsiloxane/ divinylbenzene (CAR/PDMS/DVB) were tested for comparative purposes. Samples were saturated in all cases with NaCl to improve extraction efficiency and stirred at 900 rpm at 30 °C during 30 min to allow equilibration between the liquid and gas phases. Then the fibers were inserted in the headspace and extracted during 15 min to ensure equilibrium conditions between the headspace and the fiber. After extraction, the fibers were removed and thermal desorption was carried out in the injection port of the GC at 250 °C. RESULTS AND DISCUSSION Online Continuous Measurement of the 12 C/ 13 C Isotope Ratios. The optimum conditions for the continuous measurement of 12 C/ 13 C isotope ratios were established by connecting directly the reservoir that contained the 13 CO2 spike to the mass spectrometer. 15 The mass spectrum obtained for the mass range 43-47 when such postcolumn flow was set at 1 mL/ min is provided in the Supporting Information (Figure SI-1). The 13 C isotope enrichment in the postcolumn spike (∼97%) can be clearly seen from the spectrum. Resolution was optimized while still preserving a good sensitivity. The mass resolution finally obtained allowed baseline separation even for isotope ratios very far from 1. The experiments performed for the selection of the optimum integration time are given in the Supporting Information (Figure SI-2). An integration time of 70 ms was finally selected considering both the precision in the ratio measurement and the number of points per chromatographic peak (∼15) to obtain well-defined peak profiles. Spike Enrichment and Stability of the 12 CO2/ 13 CO2 Isotope Ratio. Again, to estimate the 13 C enrichment obtained after the preparation of the 13 CO2 spike, its reservoir was connected directly to the mass spectrometer. A constant flow of1mLmin -1 was set and the signals at m/z ratios 44 and 45 were continuously monitored during 2 min for each independent isotope ratio measurement under the measurement conditions described in the procedures. The isotope ratio was calculated obtaining an average value of 0.034 ± 0.0003, which corresponds to a 13 C abundance of 96.70 ± 0.02% (n ) 5) (see the mass spectrum shown in the Figure SI-1). The slight difference between the experimentally obtained enrichment and that provided for the precursor Na2 13 CO3 by the supplier (99%), can be ascribed to small contaminations by natural abundance CO2 from the ambient air during the generation of the tracer Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6865
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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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Page 75 and 76: Table 2. Concentrations and Ratios
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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
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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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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
Page 119: corresponding compound if its chemi
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
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
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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
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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.
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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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