Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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that of the glycosylated IgG (Figure S1b, Supporting Information). This indicates that the glycosylated and deglycosylated antibodies have nearly identical gas-phase collision cross sections. The relative abundance of each gas-phase conformer can be roughly estimated by fitting the arrival time distribution to a sum of log-normal functions and integrating the relative area of each peak. The log-normal function is an asymmetric Gaussian function whose logarithm is normally distributed. 33 The log-normal function was used because it was empirically found to best fit the experimental peak shape of this ion mobility data. For the 26+ charge state, the normalized area of peaks 1 and 2 from the best fits are 42% and 58%, respectively. Similar areas are observed for other charge states. These abundances are similar to the relative peak areas observed in electropherograms of IgG2s separated with capillary electrophoresis. We and others have shown CE-SDS to be a resolving technique for the separation of IgG2 structural isoforms. 3,5,35 However, this correlation alone does not signify that the resolved gas-phase conformers are definitively due to disulfide variants. To elucidate whether the gas-phase conformers observed for IgG2 molecules are related to their disulfide connectivity, we analyzed an IgG1 antibody, mAb#2 (theoretical MW for the most abundant glycoform (G1F/G0F):148408.0 Da), as a control (Figure 2c). The most significant difference between human IgG1 and IgG2 subclasses is the primary structure of the hinge region, resulting in the absence of disulfide related isoforms in the IgG1. 3,4 In contrast to the IgG2 mobility data, for each charge state of mAb#2, the arrival time profile is relatively narrow and consists of a single uniform distribution (Figure 2c). For example, the arrival time profile for the 26+ charge state of mAb#2 (Figure 2d) shows a single peak at 8.7 ms. This arrival time profile is representative of the distribution observed for each of the charge states. This suggests that the multiple conformers observed of mAb#1 are due to disulfide variants in the antibody. To further demonstrate that the observed gas-phase conformers are indeed IgG2 disulfide variants, individual disulfide isoforms were selectively enriched in a refolding experiment using redox chemistry employing cysteine/cystamine. Dillon et al. previously demonstrated that isoform IgG2-A and IgG2-B can be redoxenriched by refolding with and without 1 M GuHCl, respectively. 4 Under redox conditions in buffer alone, IgG2-A is refolded to IgG2- B. Liu et al. demonstrated that a slow conversion of IgG2-A to IgG2-B also occurs in vivo. 34 Isoform conversion toward IgG2-A requires in vitro refolding in presence of low levels of chaotropic reagents. 4 Figure 3a,b shows the arrival time distributions for the +26 charge state for redox enriched mAb#1 in the presence of guanidine (isoform A) and redox enriched mAb#1 in the absence of guanidine (isoform B), respectively. In contrast to IMMS for the untreated Mab#1 antibody (Figure 3, dashed line), a single abundant conformer is observed for each of these enriched isoforms. As a control, the IgG1 antibody, mAb#2, was also subjected to the same redox refolding protocol with or without guanidine. All treated IgG1 samples have identical arrival time (33) Brown, R. Personal Eng. Instrum. News 1991, 8, 51–54. (34) Liu, Y. D.; Chen, X.; Enk, J. Z.; Plant, M.; Dillon, T. M.; Flynn, G. C. J. Biol. Chem. 2008, 283, 29266–29272. (35) Lacher, N. A.; Wang, Q.; Roberts, R. K.; Holovics, H. J.; Aykent, S.; Schlittler, M. R.; Thompson, M. R.; Demarest, C. W. Electrophoresis 2010, 31, 448– 458. 6754 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 3. Arrival time distributions for the 26+ charge states of IgGs treated with redox reagents (cystamine, cysteine). (a,b) mAb#1 and (c,d) mAb#2 (control). Samples represented in (a) and (c) had 1 M GuHCl added to the buffer. The dashed line shows the arrival time distribution for the 26 + charge state of untreated mAb#1. distribution profiles as the untreated IgG1 molecule (Figure 3c,d), demonstrating that this refolding protocol does not affect the overall tertiary structure of the antibody. Comparing the untreated IgG2 IMMS trace with the enriched isoform distributions (Figure 3a,b) identifies peaks 1 (9.3 ms) and 2 (10.4 ms) in the mobility spectra as isoform A and isoform B, respectively. The relatively late arrival time of isoform B indicates that this form of the antibody has a larger gas-phase collision cross section compared to isoform A. The IMMS resolution of the isoforms correlates with the previously observed capillary electrophoresis separation. 5,8,35 With CE, IgG2 isoforms were resolved into two peaks, with IgG2-A migrating more rapidly than the IgG2-B isoform. The intermediate IgG2-A/B forms were split between the two peaks, IgG2-A/B1 migrating with -A and IgG2-A/B2 with -B. 8 While the disulfide connectivities of IgG2 isoforms have been well characterized, 3,4,8,9 only limited information is available describing the overall tertiary structure of human IgGs. To investigate if the relative ordering of gas-phase collision cross sections (IgG1 ≈ IgG2-A < IgG2-B) correlates with IgG solution structures, collision cross sections were calculated for two IgG structures using Waters’ CCS software. 36 The calculated cross section of an IgG antibody, with a disulfide bonding pattern consistent with the B isoform, is 8385 Å 2 (unpublished results). This value is 3% smaller than the calculated cross section of an IgG1 antibody (protein data bank code 1HZH, 8653 Å 2 ). 37 (36) Williams, J. P.; Lough, J. A.; Campuzano, I.; Richardson, K.; Sadle, P. J. Rapid Commun. Mass Spectrom. 2009, 23, 3563. (37) Saphire, E. O.; Parren, P. W.; Pantophlet, R.; Zwick, M. B.; Morris, G. M.; Rudd, P. M.; Dwek, R. A.; Stanfield, R. L.; Burton, D. R.; Wilson, I. A. Science 2001, 293, 1155–1159.
Figure 4. Ion mobility mass spectra of (a) mAb#3 and (b) Cys f Ser Mab#3 mutant. The extracted arrival time distributions for the 25+ charge states of these molecules are shown in (c) and (d), respectively. These limited calculations using individual static structures do not explain the relatively late arrival time of the IgG2-B isoform and suggest that the overall three-dimensional structure of these gas-phase ions may be quite different than the solution structure. While the IMMS results demonstrate that ion mobility separates covalent disulfide mediated IgG2 structural isoforms, the overall three-dimensional structure of these gasphase ions is presently not clear. To investigate the generality of these ion mobility results, measurements were also performed on a different IgG2 antibody (mAb#3) which also exists as an ensemble of disulfide mediated isoforms (Figure 4a). Ion mobility separation of mAb#3 reveals two abundant gas-phase conformer populations and one minor conformer, for each charge state. These three populations are readily apparent in the arrival time distribution for the 25+ charge state (Figure 4c) which shows three distinct peaks with drift times of 9.5, 10.3, and 11.9 ms. The relative abundance is determined by fitting the arrival time distribution to a sum of log-normal functions of each conformer and is roughly estimated to be 46%, 49%, and 5%. Figure 4b, shows the ion mobility mass spectrum for a mutant form of this antibody with a single point mutation introduced at amino acid 232 (Cys f Ser) by site-directed mutagenesis. A single narrow peak is observed for the arrival time distribution of each charge state. The arrival time profile of the 25+ charge state is shown in Figure 4d as an illustrative example demonstrating a homogeneous population, with a single peak at 9.3 ms. This peak corresponds to the gas-phase conformer with the more compact structure (isoform A, vide supra). This is consistent with previous studies demonstrating that this mutant is homogeneous with respect to disulfide bonding and of the IgG2-A type. 10,38 This result also unambiguously confirms our assertion that the multiple IMMS peaks observed in this study of selected IgG2s can be correlated with the disulfide bonding patterns in these molecules. (38) Lightle, S.; Aykent, S.; Lacher, N.; Mitaksov, V.; Wells, K.; Zobel, J.; Oliphant, T. Protein Sci. 2010, 19, 753–762. CONCLUSIONS The results of the present study demonstrate that ion mobility as a shape-selective separation methodology can be used to detect disulfide heterogeneity in large (150 kDa) intact IgG2 antibodies. Two to three gas-phase conformers are observed by ion mobility for IgG2 antibodies. These gas-phase conformers were maintained with deglycosylated IgG2s. Analysis of redox refolded IgG2s as well as an IgG2 with a Cys f Ser single point mutation clearly demonstrates that the observed gas-phase conformers are related to disulfide variants. Ion mobility is fast (millisecond measurements), sensitive (nanomole), and amenable to high throughput automation. IMMS is a powerful new methodology for the characterization of intact antibodies and may be useful to routinely fingerprint higher order structure of these protein biopharmaceuticals in the near future. We are currently extending these measurements to investigate the utility of IMMS for the analysis of IgG2s containing lambda light chains as well as to directly characterize the binding of antigen targets to individual disulfide isoforms of IgG2 antibodies. ACKNOWLEDGMENT We are grateful to Allen Sickmier and Leszek Poppe for insightful discussions, Keith Richardson (Waters Inc.) for providing the Waters’ CCS software, Mike Berke, Rick Stanton, and Mikhail Toupikov for help with the data analysis software, and Mike Treuheit, Dean Pettit, Peter Grandsard, and Philip Tagari for championing and supporting this work. We also thank our Amgen colleagues, whose names are listed in the references, for their expert contributions to the collective knowledge built recently on IgG2 isoforms. 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 May 19, 2010. Accepted July 6, 2010. AC1013139 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6755
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: (pH 8.0) with cysteine and cystamin
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
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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
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Page 45 and 46: esult in a big SPR signal change wi
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Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
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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
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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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