Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 8. Details of the best binding conformations with the lowest binding free energies in clusters (A) 1 and (B) 2 (cf., Supporting Information, Figure S6). In each case, the BSPOTPE molecule is shown in a stick representation with a semitransparent van der Waals surface and with a color scheme of gray for carbon, blue for nitrogen, red for oxygen, and yellow for sulfur. The residues interacting with BSPOTPE are shown as sticks (with nonbinding apolar hydrogen atoms omitted for clarity) and labeled on R carbon. Yellow dashed lines denote hydrogen bonds. accommodate the aromatic core of BSPOTPE via cation-π interaction. A similar binding mode is found in clusters 2 (Figure 8B). Additionally, Pro447 contacts the hydrophobic part of the luminogen, while His440 interacts with its sulfonate group. All the aforesaid interactions rigidify the molecular conformation of the BSPOTPE luminogen and hamper its intramolecular rotation, hence making it highly emissive in the binding state. The lowest binding energies of the conformations in clusters 1 and 2 are -8.60 and -7.54 kcal/mol, corresponding to estimated inhibition constants (K i) of 0.49 and 2.98 µM, respectively. These small Gibbs free energies indicate that the interaction between BSPOTPE and HSA is highly spontaneous and energetically favorable. The detailed binding modes of the conformations from the other clusters in the hydrophobic cavity closely resemble those of clusters 1 and 2, although they are grouped into different clusters. This is not unexpected because the ligand is treated as fully flexible in the computational modeling process, and a small rmsd tolerance is used for clustering. Thus, any difference in the coordinates of an atom between two conformations will increase the rmsd value, even though the atom is not important for binding interactions. HSA is a physiological carrier in the human body, and its hydrophobic regions are capable of binding insoluble endogenous compounds like fatty acids and drugs. With close scrutinization of other proteins, such as trypsin, pepsin, and papain, one can hardly find a hydrophobic pocket as in HSA. This may explain why the BSPOTPE luminogen shows excellent selectivity toward the albumin proteins over the other types of proteins. GndHCl is known to work as a bulky ionic cosolvent. It not only weakens hydrophobic interaction but also interferes with association between charged solutes. 39 When HSA unfolds, a large number of nonpolar side chains are exposed to the aqueous medium and stabilized by GndHCl. GndHCl may also intrude into the hydrophobic pocket of HSA and break down the spatial architecture of the protein folds. CD spectral data indicate that no significant alternation in the secondary structure occurs during the first unfolding transition in the low concentration range of GndHCl (6.0 M). No secondary structure or strand helicity is retained in this region, as confirmed by the CD spectral data. The luminogen molecules are fully released from the protein, and no emission signals can be collected. The unfolding process discussed above is summarized in Scheme 1. Upon removal of the GndHCl denaturant by dilution, the protein returns to its native form and the emission of BSPOTPE is recovered, indicating that the interaction between HSA and BSPOTPE is fully reversible. CONCLUDING REMARKS In summary, in this work, we developed an environmentally stable and synthetically readily accessible FL probe for HSA detection and quantitation. The nonluminescent BSPOTPE becomes emissive in the presence of HSA. The AIE bioprobe shows a linear calibration curve at [HSA] ) 0-100 nM, enabling the protein quantitation over a wide concentration range. It enjoys a low detection limit (down to 1 nM) and a high selectively toward albumins. The FL bioassay is tolerant of the species in the artificial urine and is promising for applications in real-life urinary protein detection. Design and fabrication of simple bioassay kits for clinical diagnostics are ongoing in our laboratories. BSPOTPE is successfully employed as a protein staining reagent in the PAGE analysis. It offers a simple, rapid, effective, and economic way for visualization of protein bands in the gel assay. Studies on the interaction between BSPOTPE and HSA
Scheme 1. Proposed Mechanism for Fluorescent Probing of GndHCl-Induced HSA Unfolding Process a a BSPOTPE and HSA are denoted by stars and springs, respectively. reveal the essential role of the hydrophobic cavities of the protein folding structure. The change in the FL intensity reflects the global stability of the protein. The unfolding process of HSA induced by GndHCl monitored by the AIE luminogen reveals a three-step transition with the molten-globule intermediate involved. Studies of other biologically important processes by AIE luminogens are in progress in our laboratories. ACKNOWLEDGMENT This work reported in this paper was partially supported by the Nano Science and Technology Program at HKUST, the Research Grants Council of Hong Kong (603008 and 602706), the University Grants committee of Hong Kong (AoE/P-03/08), and the National Science Foundation of China (20974028). B.Z.T. thanks the support from Cao Guangbiao Foundation of Zhejiang University. SUPPORTING INFORMATION AVAILABLE Text describing the procedures for artificial urine preparation, cytotoxicity assay, FRET measurement, DSC analysis, and computational modeling. Figures showing viability of the HeLa cells incubated in the presence of BSPOTPE, spectral overlap between BSPOTPE absorption and HSA emission, FL spectra of BSPOTPE and DSC thermograms of HSA in the presence of GndHCl, FL intensities of BSPOTPE at 470 nm in the presence and absence of HSA at different pH, and summary of the docked conformations of BSPOTPE clustered with a rmsd tolerance of 2.0 Å. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 7, 2010. Accepted July 7, 2010. AC1018028 <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010 7043
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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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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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Page 249 and 250: Figure 1. Representation of the Car
Page 251 and 252: Table 2. Capillary Electrophoresis
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Page 263 and 264: CONCLUSIONS In this paper we have i
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Page 273 and 274: Table 1. Effect of 1 D-LC Condition
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Page 281 and 282: Figure 3. Inhibitory effects of luc
Page 283 and 284: Anal. Chem. 2010, 82, 7027-7034 Pat
Page 285 and 286: Electrochemistry. Electrochemical m
Page 287 and 288: Figure 2. SEM images of Au substrat
Page 289 and 290: Table 3. Film Thickness Measurement
Page 291 and 292: Anal. Chem. 2010, 82, 7035-7043 Qua
Page 293 and 294: Figure 1. (A) FL spectra of BSPOTPE
Page 295 and 296: Figure 4. (A) Variation in the FL i
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Page 303 and 304: Figure 2. Free fluorophores do not
Page 305 and 306: Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308: Figure 2. Comparative analysis of d
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